阅读说明：本技术 拉沙洛菌素和盐霉素单链抗体和双特异性单链抗体及其应用 (Lasacosin and salinomycin single-chain antibody and bispecific single-chain antibody and application thereof ) 是由 李建成 马月姣 陈莹娴 黄婧洁 李苗 于 2021-09-29 设计创作，主要内容包括：本发明公开了拉沙洛菌素和盐霉素单链抗体和双特异性单链抗体及其应用。该双特异性单链抗体含有名称为LAS-VH的LAS重链可变区、名称为SAL-VH的SAL重链可变区、名称为LAS-VL的LAS轻链可变区和名称为SAL-VL的SAL轻链可变区,所述LAS-VH、SAL-VH、LAS-VL和SAL-VL均由决定簇互补区和框架区组成；所述决定簇互补区均由CDR1、CDR2和CDR3组成。该双特异性单链抗体可用于检测盐霉素和拉沙洛菌素残留。(The invention discloses a single-chain antibody and a bispecific single-chain antibody of lasalomycin and salinomycin and application thereof. The bispecific single chain antibody comprises a LAS heavy chain variable region named as LAS-VH, an SAL heavy chain variable region named as SAL-VH, a LAS light chain variable region named as LAS-VL and an SAL light chain variable region named as SAL-VL, wherein the LAS-VH, the SAL-VH, the LAS-VL and the SAL-VL are all composed of determinant complementary regions and framework regions; the determinant complementarity region is composed of CDR1, CDR2 and CDR 3. The bispecific single chain antibody can be used for detecting salinomycin and lasalomycin residues.)

1. A bispecific single chain antibody or antigen-binding portion thereof of lasalomycin and salinomycin, characterized in that: the bispecific single chain antibody or antigen-binding portion thereof comprises a LAS heavy chain variable region designated as LAS-VH, an SAL heavy chain variable region designated as SAL-VH, a LAS light chain variable region designated as LAS-VL, and an SAL light chain variable region designated as SAL-VL, each of which consists of a determinant complementary region and a framework region; the determinant complementarity region is composed of CDR1, CDR2 and CDR 3;

the amino acid sequence of the CDR1 of the LAS-VH is shown in SEQ ID No.4 from position 25 to position 34;

the amino acid sequence of the CDR2 of the LAS-VH is shown in the 50 th to 58 th positions in SEQ ID No. 4;

the amino acid sequence of the CDR3 of the LAS-VH is shown in SEQ ID No.4 from position 95 to position 108;

the amino acid sequence of CDR1 of SAL-VH is shown in SEQ ID No.4 from 274 th position to 282 th position;

the amino acid sequence of CDR2 of SAL-VH is shown in SEQ ID No.4 from position 299 to position 308;

the amino acid sequence of CDR3 of SAL-VH is shown in SEQ ID No.4 from position 345 to position 358;

the amino acid sequence of the CDR1 of the LAS-VL is shown in SEQ ID No.4 from position 401 to position 413;

the amino acid sequence of CDR2 of LAS-VL is shown in SEQ ID No.4 from position 429 to position 433;

the amino acid sequence of the CDR3 of the LAS-VL is shown in the 468 th to 478 th positions of SEQ ID No. 4;

the amino acid sequence of CDR1 of SAL-VL is shown in SEQ ID No.4 from position 151 to position 158;

the amino acid sequence of CDR2 of SAL-VL is shown in SEQ ID No.4 from position 174 to position 178;

the amino acid sequence of CDR3 of SAL-VL is shown in SEQ ID No.4 at positions 213-223.

2. The bispecific single chain antibody or antigen-binding portion thereof according to claim 1, characterized in that: the amino acid sequence of the LAS-VH is shown as 1 st to 118 th in SEQ ID No. 4; the amino acid sequence of the LAS-VL is shown as 376 th to 489 th in SEQ ID No. 4; the amino acid sequence of the SAL-VH is shown as 250 th-368 nd positions in SEQ ID No. 4; the amino acid sequence of the SAL-VL is shown in 126 th to 234 th positions in SEQ ID No. 4.

3. The bispecific single chain antibody or antigen binding portion thereof according to claim 1 or 2, characterized in that: the amino acid sequence of the bispecific single chain antibody or the antigen binding part thereof is shown as SEQ ID No. 4.

4. A biomaterial associated with the bispecific single chain antibody or antigen binding portion thereof of any one of claims 1 to 3, said biomaterial being any one of:

B1) a nucleic acid molecule encoding the bispecific single chain antibody or antigen binding portion thereof of any one of claims 1 to 3;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector comprising the nucleic acid molecule of B1);

B4) a recombinant vector comprising the expression cassette of B2);

B5) a recombinant microorganism comprising the nucleic acid molecule of B1);

B6) a recombinant microorganism comprising the expression cassette of B2);

B7) a recombinant microorganism containing the recombinant vector of B3);

B8) a recombinant microorganism comprising the recombinant vector of B4).

5. The biomaterial of claim 3, wherein: B1) the nucleic acid molecule is a gene encoding the bispecific single chain antibody or antigen-binding portion thereof according to any one of claims 1 to 3, said gene being a DNA molecule according to A) or B) below:

A) the coding gene sequence of the CDR1 of the LAS-VH is shown as 73 rd position to 102 th position in SEQ ID No. 3; the coding gene sequence of the CDR2 of the LAS-VH is shown as 148 th to 174 th in SEQ ID No. 3; the coding gene sequence of the CDR3 of the LAS-VH is shown as 283 th to 324 th in SEQ ID No. 3; the coding gene sequence of CDR1 of SAL-VH is shown in 820 th site to 846 th site in SEQ ID No. 3; the coding gene sequence of CDR2 of SAL-VH is shown in 895 th-924 th position in SEQ ID No. 3; the coding gene sequence of CDR3 of SAL-VH is shown in 1033 th site to 1074 th site of SEQ ID No. 3; the coding gene sequence of the CDR1 of the LAS-VL is shown as 451 th to 474 th in SEQ ID No. 3; the coding gene sequence of the CDR2 of the LAS-VL is shown as 520 th to 534 th in SEQ ID No. 3; the coding gene sequence of the CDR3 of the LAS-VL is shown as 637-669 in SEQ ID No. 3; the coding gene sequence of CDR1 of SAL-VL is shown in 1201-1239 in SEQ ID No. 3; the coding gene sequence of CDR2 of SAL-VL is shown in 1285 th position to 1299 th position in SEQ ID No. 3; the coding gene sequence of CDR3 of SAL-VL is shown in position 1402-1434 in SEQ ID No. 3;

B) a DNA having 90% or more identity to the DNA molecule defined in A) and encoding said bispecific single chain antibody or antigen binding portion thereof.

6. A latanomycin single chain antibody or an antigen binding portion thereof or a biological material thereof characterized in that: a lasalomycin single chain antibody or antigen-binding portion thereof comprising a LAS heavy chain variable region, designated as LAS-VH, and a LAS light chain variable region, designated as LAS-VL, both of which consist of determinant complementary regions and framework regions; the determinant complementarity region is composed of CDR1, CDR2 and CDR 3; the amino acid sequence of the CDR1 of the LAS-VH is shown in the 154 th to 163 th positions of SEQ ID No. 5; the amino acid sequence of the CDR2 of the LAS-VH is shown in SEQ ID No.5 from position 179 to position 187; the amino acid sequence of the CDR3 of the LAS-VL is shown in the 224 th to 237 th positions in SEQ ID No.5, and the amino acid sequence of the CDR1 of the LAS-VL is shown in the 26 th to 38 th positions in SEQ ID No. 5; the amino acid sequence of the CDR2 of the LAS-VL is shown in SEQ ID No.5 from position 54 to position 58; the amino acid sequence of CDR3 of LAS-VL is shown in SEQ ID No.5 from position 93 to position 103;

the biological material is any one of the following materials:

C1) a nucleic acid molecule encoding said latanomycin single chain antibody or antigen binding portion thereof;

C2) an expression cassette comprising the nucleic acid molecule of C1);

C3) a recombinant vector comprising the nucleic acid molecule of C1);

C4) a recombinant vector comprising the expression cassette of C2);

C5) a recombinant microorganism comprising the nucleic acid molecule of C1);

C6) a recombinant microorganism comprising the expression cassette of C2);

C7) a recombinant microorganism comprising the recombinant vector of C3);

C8) a recombinant microorganism comprising the recombinant vector of C4).

7. Salinomycin single chain antibody or an antigen binding portion thereof or a biological material thereof, characterized in that: a salinomycin single chain antibody or antigen binding portion thereof comprises an SAL heavy chain variable region designated SAL-VH and an SAL light chain variable region designated SAL-VL, both of which consist of determinant complementarity regions and framework regions; the determinant complementarity region is composed of CDR1, CDR2 and CDR 3; the amino acid sequence of CDR1 of SAL-VH is shown in SEQ ID No.6 from position 149 to position 157; the amino acid sequence of CDR2 of SAL-VH is shown in SEQ ID No.6 from position 174 to position 183; the amino acid sequence of the CDR3 of the SAL-VL is shown in the 220 th to 233 th positions in SEQ ID No.6, and the amino acid sequence of the CDR1 of the SAL-VL is shown in the 26 th to 33 th positions in SEQ ID No. 6; the amino acid sequence of CDR2 of SAL-VL is shown in SEQ ID No.6 from position 49 to position 53; the amino acid sequence of CDR3 of SAL-VL is shown in positions 88-98 of SEQ ID No. 6;

the biological material is any one of the following materials:

D1) a nucleic acid molecule encoding the salinomycin single chain antibody or antigen binding portion thereof;

D2) an expression cassette comprising the nucleic acid molecule of D1);

D3) a recombinant vector comprising the nucleic acid molecule of D1);

D4) a recombinant vector comprising the expression cassette of D2);

D5) a recombinant microorganism comprising the nucleic acid molecule of D1);

D6) a recombinant microorganism comprising the expression cassette of D2);

D7) a recombinant microorganism comprising the recombinant vector of D3);

D8) a recombinant microorganism comprising the recombinant vector of D4).

8. Product for the detection of lasalomycin and/or salinomycin, characterized in that: a bispecific single chain antibody or antigen-binding portion thereof comprising a rasalomycin according to any one of claims 1 to 3 and salinomycin, a rasalomycin single chain antibody or antigen-binding portion thereof according to claim 6 or a salinomycin single chain antibody or antigen-binding portion thereof according to claim 7.

9. The use of a bispecific single chain antibody or antigen-binding portion thereof for rasalomycin and salinomycin as claimed in any one of claims 1 to 3, a biomaterial as claimed in claim 4 or 5, a single chain antibody or antigen-binding portion thereof or biomaterial for rasalomycin as claimed in claim 6, or a single chain antibody or antigen-binding portion thereof or biomaterial for salinomycin as claimed in claim 7 in the preparation of a product for detecting rasalomycin and/or salinomycin or in the detection of rasalomycin and/or salinomycin.

10. The product according to claim 8 or the use according to claim 9, characterized in that: the product is a kit.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a single-chain antibody and a bispecific single-chain antibody of lasalomycin and salinomycin and application thereof.

Background

Lasalocidin (LAS) belongs to a bivalent polyether ionophore antibiotic, the chemical structural formula of which is shown in figure 1, and the Lasalocidin can be used for preventing and treating chicken coccidiosis and also can be used for preventing and treating coccidiosis of turkeys, lambs and calves. The anticoccidial mechanism is to capture or release bivalent cations to influence the ion balance of the larvae and cause the larvae to break and die, and the lasalocid has obvious inhibiting and killing effects on coccidian sporozoites and merozoites in asexual periods of the 1 st generation and the 2 nd generation. Salinomycin (Salinomycin, SAL) is a monocarboxylic acid polyether type antibiotic special for animals, has a chemical structural formula shown in figure 2, has strong inhibiting and killing effects on most gram-positive bacteria and various coccidia, can be used for preventing and treating diarrhea of pigs, promoting growth and improving survival rate, and is mainly used for preventing coccidia of poultry. The two medicines have the advantages of small dosage, broad spectrum, high efficiency, low drug resistance, growth promotion and the like, are widely used as feed additives for preventing and treating coccidiosis of poultry, but have potential toxic effects and narrow safety range, and have incompatibility with some antibiotics such as tiamulin, tetracycline and sulfonamides. Therefore, when using LAS and SAL, attention should be paid to their own toxicity and incompatibility, and the problem of their residual should be paid to them. The maximum residual limit of the lasalocid in the approved animal food in China is as follows: bovine liver 0.7 μ g/mL, chicken fat 1.2 μ g/mL, chicken and turkey liver 0.4 μ g/mL, turkey fat 0.4 μ g/mL, sheep liver 1 μ g/mL, rabbit liver 0.7 μ g/mL; the maximum residual limit of salinomycin is as follows: chicken meat 0.6 mug/mL, chicken fat 1.2 mug/mL and chicken liver 1.8 mug/mL.

At present, a plurality of methods for detecting residual of the lasalomycin and the salinomycin exist, the most mature and common residual analysis method is still an instrument method, but the veterinary drug residual instrument analysis has certain restriction conditions, such as expensive instruments, high requirements for pretreatment methods, time and labor consumption, small sample treatment amount and the like, so that the development of a novel veterinary drug residual rapid detection technology is urgent, and an immunoassay kit, a test strip and the like become the key directions for rapid detection.

Disclosure of Invention

It is an object of the present invention to provide a bispecific single chain antibody or antigen-binding portion thereof, of rasalomectin and salinomycin.

The invention provides a bispecific single chain antibody or an antigen binding part thereof of lasalomycin and salinomycin, wherein the bispecific single chain antibody or the antigen binding part thereof contains a LAS heavy chain variable region named as LAS-VH, an SAL heavy chain variable region named as SAL-VH, a LAS light chain variable region named as LAS-VL and an SAL light chain variable region named as SAL-VL, and the LAS-VH, the SAL-VH, the LAS-VL and the SAL-VL are all composed of a determinant complementary region and a framework region; the determinant complementarity region is composed of CDR1, CDR2 and CDR 3;

the amino acid sequence of the CDR1 of the LAS-VH is shown in SEQ ID No.4 from position 25 to position 34;

the amino acid sequence of the CDR2 of the LAS-VH is shown in the 50 th to 58 th positions in SEQ ID No. 4;

the amino acid sequence of the CDR3 of the LAS-VH is shown in SEQ ID No.4 from position 95 to position 108;

the amino acid sequence of CDR1 of SAL-VH is shown in SEQ ID No.4 from 274 th position to 282 th position;

the amino acid sequence of CDR2 of SAL-VH is shown in SEQ ID No.4 from position 299 to position 308;

the amino acid sequence of CDR3 of SAL-VH is shown in SEQ ID No.4 from position 345 to position 358;

the amino acid sequence of the CDR1 of the LAS-VL is shown in SEQ ID No.4 from position 401 to position 413;

the amino acid sequence of CDR2 of LAS-VL is shown in SEQ ID No.4 from position 429 to position 433;

the amino acid sequence of the CDR3 of the LAS-VL is shown in the 468 th to 478 th positions of SEQ ID No. 4;

the amino acid sequence of CDR1 of SAL-VL is shown in SEQ ID No.4 from position 151 to position 158;

the amino acid sequence of CDR2 of SAL-VL is shown in SEQ ID No.4 from position 174 to position 178;

the amino acid sequence of CDR3 of SAL-VL is shown in SEQ ID No.4 at positions 213-223.

Alternatively, the amino acid sequence of the LAS-VH is as shown in SEQ ID No.4 from position 1 to position 118, according to the bispecific single chain antibody or antigen binding portion thereof described above; the amino acid sequence of the LAS-VL is shown as 376 th to 489 th in SEQ ID No. 4; the amino acid sequence of the SAL-VH is shown as 250 th-368 nd positions in SEQ ID No. 4; the amino acid sequence of the SAL-VL is shown in 126 th to 234 th positions in SEQ ID No. 4.

Alternatively, the amino acid sequence of the bispecific single chain antibody or antigen-binding portion thereof according to the above is as shown in SEQ ID No. 4.

The above-mentioned bispecific single chain antibody gene can be synthesized by the overlapping chain reaction (SOE-PCR). RNA is extracted from hybridoma cells capable of secreting SAL antibody and LAS antibody, heavy chain variable region (VH) gene and light chain variable region (VL) gene of the antibody are obtained through reverse transcription and amplification, and the dual specificity single chain antibody gene capable of simultaneously recognizing SAL and LAS can be obtained through three times of SOE-PCR. The first time, the heavy chain variable region gene of SAL and the light chain variable region gene of LAS are hybridized to obtain VH (SAL) -Linker A-VL (LAS) hybrid chain, the second time, the light chain variable region gene of SAL and the heavy chain variable region gene of LAS are hybridized to obtain VH (LAS) -Linker A-VL (SAL) hybrid chain, and the third time, the two hybrid chains are connected to obtain the bispecific single chain antibody gene (ScDb gene) of VH (LAS) -Linker A-VL (SAL) -Linker B-VH (SAL) -Linker A-VL (LAS). The ScDb gene is inserted into a plasmid and then introduced into a competent cell for expression, and a large amount of ScDb can be obtained without animal experiments.

The present invention also provides a biomaterial related to the bispecific single chain antibody or the antigen-binding portion thereof as described above, which is any one of:

B1) a nucleic acid molecule encoding the bispecific single chain antibody or antigen binding portion thereof described above;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector comprising the nucleic acid molecule of B1);

B4) a recombinant vector comprising the expression cassette of B2);

B5) a recombinant microorganism comprising the nucleic acid molecule of B1);

B6) a recombinant microorganism comprising the expression cassette of B2);

B7) a recombinant microorganism containing the recombinant vector of B3);

B8) a recombinant microorganism comprising the recombinant vector of B4).

Alternatively, according to the above-mentioned biological material, B1) the nucleic acid molecule is a gene encoding the above-mentioned bispecific single chain antibody or an antigen-binding portion thereof, said gene being a DNA molecule as described in a) or B) below:

A) the coding gene sequence of the CDR1 of the LAS-VH is shown as 73 rd position to 102 th position in SEQ ID No. 3; the coding gene sequence of the CDR2 of the LAS-VH is shown as 148 th to 174 th in SEQ ID No. 3; the coding gene sequence of the CDR3 of the LAS-VH is shown as 283 th to 324 th in SEQ ID No. 3; the coding gene sequence of CDR1 of SAL-VH is shown in 820 th site to 846 th site in SEQ ID No. 3; the coding gene sequence of CDR2 of SAL-VH is shown in 895 th-924 th position in SEQ ID No. 3; the coding gene sequence of CDR3 of SAL-VH is shown in 1033 th site to 1074 th site of SEQ ID No. 3; the coding gene sequence of the CDR1 of the LAS-VL is shown as 451 th to 474 th in SEQ ID No. 3; the coding gene sequence of the CDR2 of the LAS-VL is shown as 520 th to 534 th in SEQ ID No. 3; the coding gene sequence of the CDR3 of the LAS-VL is shown as 637-669 in SEQ ID No. 3; the coding gene sequence of CDR1 of SAL-VL is shown in 1201-1239 in SEQ ID No. 3; the coding gene sequence of CDR2 of SAL-VL is shown in 1285 th position to 1299 th position in SEQ ID No. 3; the coding gene sequence of CDR3 of SAL-VL is shown in position 1402-1434 in SEQ ID No. 3;

B) a DNA having 90% or more identity to the DNA molecule defined in A) and encoding said bispecific single chain antibody or antigen binding portion thereof.

In the above, identity refers to the identity of the sequences. Sequence identity can be determined using homology search sites on the internet, such as the BLAST web page of the NCBI home website.

The 90% or greater identity can be at least 90%, 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

B1) The nucleotide sequence of the nucleic acid molecule can be shown as SEQ ID No. 3.

The recombinant vector can be the recombinant plasmid ScDb-pJB33 recombinant plasmid prepared in the examples. The recombinant microorganism may be strain RV308-ScDb prepared in the examples.

The invention also provides a lasalomycin single-chain antibody or an antigen binding part thereof or a biological material thereof, wherein the lasalomycin single-chain antibody or the antigen binding part thereof contains a LAS heavy chain variable region named as LAS-VH and a LAS light chain variable region named as LAS-VL, and the LAS-VH and the LAS-VL are both composed of a determinant complementary region and a framework region; the determinant complementarity region is composed of CDR1, CDR2 and CDR 3; the amino acid sequence of the CDR1 of the LAS-VH is shown in the 154 th to 163 th positions of SEQ ID No. 5; the amino acid sequence of the CDR2 of the LAS-VH is shown in SEQ ID No.5 from position 179 to position 187; the amino acid sequence of the CDR3 of the LAS-VL is shown in the 224 th to 237 th positions in SEQ ID No.5, and the amino acid sequence of the CDR1 of the LAS-VL is shown in the 26 th to 38 th positions in SEQ ID No. 5; the amino acid sequence of the CDR2 of the LAS-VL is shown in SEQ ID No.5 from position 54 to position 58; the amino acid sequence of CDR3 of LAS-VL is shown in SEQ ID No.5 at positions 93-103. The amino acid sequence of the lassaxacin single chain antibody can be shown as SEQ ID No. 5.

The biological material is any one of the following materials:

C1) a nucleic acid molecule encoding said latanomycin single chain antibody or antigen binding portion thereof, e.g. having the nucleotide sequence shown in SEQ ID No. 1;

C2) an expression cassette comprising the nucleic acid molecule of C1);

C3) a recombinant vector comprising the nucleic acid molecule of C1);

C4) a recombinant vector comprising the expression cassette of C2);

C5) a recombinant microorganism comprising the nucleic acid molecule of C1);

C6) a recombinant microorganism comprising the expression cassette of C2);

C7) a recombinant microorganism comprising the recombinant vector of C3);

C8) a recombinant microorganism comprising the recombinant vector of C4).

The recombinant vector is, for example, the LAS-ScFv-pJB33 recombinant plasmid prepared in the examples. The recombinant microorganism is, for example, strain RV308-LAS-ScFv prepared in the examples.

The invention also provides a salinomycin single-chain antibody or an antigen binding part thereof or a biological material thereof, wherein the salinomycin single-chain antibody or the antigen binding part thereof contains an SAL heavy chain variable region named SAL-VH and an SAL light chain variable region named SAL-VL, and the SAL-VH and the SAL-VL both consist of a determinant complementary region and a framework region; the determinant complementarity region is composed of CDR1, CDR2 and CDR 3; the amino acid sequence of CDR1 of SAL-VH is shown in SEQ ID No.6 from position 149 to position 157; the amino acid sequence of CDR2 of SAL-VH is shown in SEQ ID No.6 from position 174 to position 183; the amino acid sequence of the CDR3 of the SAL-VL is shown in the 220 th to 233 th positions in SEQ ID No.6, and the amino acid sequence of the CDR1 of the SAL-VL is shown in the 26 th to 33 th positions in SEQ ID No. 6; the amino acid sequence of CDR2 of SAL-VL is shown in SEQ ID No.6 from position 49 to position 53; the amino acid sequence of CDR3 of SAL-VL is shown in SEQ ID No.6 at positions 88-98. The amino acid sequence of the salinomycin single-chain antibody is shown as SEQ ID No. 6.

The biological material is any one of the following materials:

D1) the nucleotide sequence of the nucleic acid molecule for coding the salinomycin single-chain antibody or the antigen binding part thereof is shown as SEQ ID No. 2;

D2) an expression cassette comprising the nucleic acid molecule of D1);

D3) a recombinant vector comprising the nucleic acid molecule of D1);

D4) a recombinant vector comprising the expression cassette of D2);

D5) a recombinant microorganism comprising the nucleic acid molecule of D1);

D6) a recombinant microorganism comprising the expression cassette of D2);

D7) a recombinant microorganism comprising the recombinant vector of D3);

D8) a recombinant microorganism comprising the recombinant vector of D4).

The recombinant vector is, for example, the SAL-ScFv-pJB33 recombinant plasmid prepared in the examples. The recombinant microorganism is, for example, the strain RV308-SAL-ScFv prepared in the examples.

The invention also provides a product for detecting the lassamycin and/or salinomycin, which comprises the dual-specificity single-chain antibody or the antigen binding part thereof of the lassamycin and the salinomycin, the lassamycin single-chain antibody or the antigen binding part thereof or the salinomycin single-chain antibody or the antigen binding part thereof.

The application of the dual-specificity single-chain antibody of the lasalomycin and the salinomycin or the antigen binding part thereof, the biological material, the single-chain antibody of the lasalomycin or the antigen binding part thereof or the biological material, or the single-chain antibody of the salinomycin or the antigen binding part thereof or the biological material thereof also belongs to the protection scope of the invention. The application is the application in the preparation of products for detecting the lasalocid and/or salinomycin or the detection of the lasalocid and/or salinomycin.

Herein, the product may be a kit.

The invention provides a preparation method of the single-chain antibody or the bispecific single-chain antibody, which comprises the steps of connecting a single-chain antibody gene or the bispecific single-chain antibody gene to a plasmid vector through double enzyme digestion, transferring the plasmid vector into an escherichia coli competent cell in an electrotransformation mode, and optimizing the concentration of an inducer and the induction time to ensure that the single-chain antibody or the bispecific single-chain antibody is expressed in a large quantity. The cell wall is broken by ultrasonic cracking method, so as to obtain a large amount of single-chain antibody or bispecific single-chain antibody. Finally, the antibody is purified in a nickel column by adopting imidazole with different concentration gradients.

The bispecific single chain antibody provided by the invention can be used for detecting salinomycin and lasalomycin residues. The detection method can comprise the steps of extracting and degreasing a sample to be detected to prepare a concentrated sample to be detected, and detecting the concentrated sample to be detected by indirect competition ELISA (enzyme-linked immunosorbent assay) by adopting the bispecific single-chain antibody. The bispecific single-chain antibody prepared by the invention is simple and convenient to prepare, can be expressed in a large amount, can realize the detection of two drugs, and has high sensitivity and good stability.

Drawings

FIG. 1 is a chemical structural formula of lasalomectin.

FIG. 2 is the chemical structural formula of salinomycin.

FIG. 3 is a schematic diagram of the construction route of the single-chain antibody gene of example 1.

FIG. 4 is a standard curve prepared in example 3.

FIG. 5 is a standard curve for substrate calibration in example 6.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

Coli RV308 and pJB33 plasmid vectors were both stored in the laboratory (described in Wen K,G，Schillberg S，et al.Improved fluoroquinolone detection in ELISA through engineering of a broad-specific single-chain variable fragment binding simultaneously to 20 fluoroquinolones[J].Analytical and bioanalytical chemistry，2012，403(9)：2771-2783.)

the reagents used in the examples described below are specifically as follows.

(1)2 × Fast Pfu mix: beijing Quanjin Biotechnology Ltd./AS 221-01

(2)2 × YT liquid medium:

tryptone 17g

Yeast extract 10g

NaCl 5g

Adding distilled water to a constant volume of 1000mL, sterilizing at high temperature, and storing in a constant temperature box at 37 deg.C

(3) LB solid medium: adding 3g of bacterial agar powder into 200mL of LB liquid for culture, sterilizing at high temperature, and storing in a constant temperature box at 37 ℃.

(4) LB liquid medium:

tryptone 10g

Yeast extract 5g

NaCl 10g

Adding distilled water to 1000mL, and sterilizing at high temperature for 20 min.

(5)Binding buffer：

50mM NaH₂PO₄·H₂O 6.90g

300mMNaCl 17.54g

10mM imidazole 0.68g

The volume was adjusted to 1000mL with ultrapure water, and the pH was adjusted to 8.0 with 1M NaOH.

(6) Lacloxacin coatingen: beijing Weideweikang Biotech Co., Ltd

(7) Salinomycin envelope antigen: beijing Weideweikang Biotech Co., Ltd

(8) Washing solution (20X):

adding ultrapure water to the solution until the volume is 1000 mL.

(9) LAS standard: sigma corporation/73996

(10) SAL standard: sigma corporation/S4526

(11) anti-His tag antibody: beijing Quanjin Biotechnology Ltd

(12) HRP-labeled goat anti-mouse IgG: beijing Quanjin Biotechnology Ltd

(13) Restriction enzyme Sfi I: NeB Corp USA

Example 1 splicing of Single chain antibody Gene fragments

1. Determination of the Gene sequences of the VH and VL of Single-chain antibodies

(1) Respectively recovering and culturing a hybridoma cell capable of secreting an SAL antibody and a hybridoma cell capable of secreting an LAS antibody, carrying out continuous passage twice, taking a cell culture solution to carry out antibody subtype identification, wherein the heavy chains of the SAL antibody and the LAS antibody are IgG1 types, and the light chains of the SAL antibody and the LAS antibody are Kappa types.

(2) And (2) extracting total RNA of the cells cultured in the step (1), carrying out reverse transcription on the total RNA to obtain cDNA, and connecting the cDNA with a known single-stranded DNA sequence (AS Linker, the sequence of which is shown in Table 2) to obtain cDNA-AS Linker.

(3) According to the antibody typing result, cDNA-AS Linker is taken AS a template, corresponding primers are adopted to respectively amplify the heavy chain and the light chain of the antibody, and the primers used by the heavy chain of the antibody are AS-F and IgG₁R, and the primers used for the light chain of the antibody are AS-F and Kappa-R.

(4) And (4) carrying out 1% agarose gel determination on the heavy chain and the light chain of the antibody amplified in the step (3), and recovering the gel to obtain a recovered product.

(5) And (4) respectively connecting the heavy chain and the light chain of the antibody recovered in the step (4) to plasmids, and transferring the plasmids into escherichia coli competent cells for TA cloning. White single colonies were picked for sequencing. Analyzing the sequencing result by using a gene translation tool and abYsis to obtain the gene sequences of the heavy chain variable region and the light chain variable region of the SAL and LAS antibodies.

2. Splicing of antibody VH and VL gene fragments

A schematic diagram of a construction route of a single-chain antibody gene (ScFv gene) is shown in FIG. 3, and an SOE PCR is adopted to connect a heavy chain variable region (VH) and a light chain variable region (VL) through a Linker to obtain the ScFv gene.

First round SOE-PCR: amplifying and culturing the bacterial liquid with the correct sequencing, extracting plasmids, and performing PCR amplification by taking a plasmid extracting solution as a template, wherein primers for amplifying a heavy chain variable region (SLA-VH) of the SAL antibody are SAL-VH-F1 and SAL-VH-R1, primers for amplifying a light chain variable region (SAL-VL) of the SAL antibody are SAL-VL-F1 and SAL-VL-R1, primers for amplifying a heavy chain variable region (LAS-VH) of the LAS antibody are LAS-VH-F1 and LAS-VH-R1, and primers for amplifying a light chain variable region (LAS-VL) of the LAS antibody are LAS-VL-F1 and LAS-VL-R1. The PCR reaction system is as follows:

and (3) point-throwing centrifugation after vortex mixing, wherein the PCR reaction conditions are as follows: 5min at 95 ℃; 1min at 95 ℃, 50s at 55 ℃ and 1min at 72 ℃; 30 cycles; 10min at 72 ℃. After electrophoresis on 1% agarose gel, the gel was cut and recovered.

The PCR reaction system and PCR reaction conditions of the second round and the third round of SOE-PCR are the same as those of the first round of SOE-PCR. Except that the template for each subsequent round of PCR amplification was the recovered product from the previous round of PCR amplification, VL was amplified twice, VH was amplified three times, and VH was amplified in the third round using the recovered product from the second round of VH amplification as template, and the primers in Table 1 were used.

TABLE 1 primers required for SOE-PCR reaction

The PCR products of VH and VL of the second round were recovered by purification. LAS-VH-R3 and LAS-VL-F2 were used as primers to link LAS-VH and LAS-VL to obtain the complete LAS-ScFv gene fragment. SAL-VH-R3 and SAL-VL-F2 are used as primers to link SAL-VH and SAL-VL to obtain a complete SAL-ScFv gene fragment. The reaction system is as follows:

the PCR reaction conditions were the same as the first round of SOE-PCR reaction conditions. The reaction product is identified by 1% agarose gel electrophoresis, and the complete scFv gene fragment is recovered by cutting gel.

The nucleotide sequence of the LAS-scFv gene fragment is shown as SEQ ID No.1, wherein, the 1 st to 341 th sites are VL sequence, the 342 nd and 387 th sites are Linker sequence, the 388 nd and 741 th sites are VH sequence, the 76 th to 114 th sites are CDR1 sequence of LAS-VL, the 159 th and 174 th sites are CDR2 sequence of LAS-VL, the 276 th and 309 th sites are CDR3 sequence of LAS-VL, the 459th and 489 th sites are CDR1 sequence of LAS-VH, the 534 th and 561 th sites are CDR2 sequence of LAS-VH, and the 669 th and 711 sites are CDR3 sequence of LAS-VH.

The nucleotide sequence of the SAL-scFv gene fragment is shown as SEQ ID No.2, wherein, the 1 st to 326 th sites are VL sequences, the 327 nd and 372 nd sites are Linker sequences, the 373 nd and 729 th sites are VH sequences, the 75 th to 99 th sites are CDR1 sequences of the SAL-VL, the 144 nd and 159 sites are CDR2 sequences of the SAL-VL, the 261 nd and 294 th sites are CDR3 sequences of the SAL-VL, the 444 nd and 471 th sites are CDR1 sequences of the SAL-VH, the 519 and 549 th sites are CDR2 sequences of the SAL-VH, and the 657 th and 699 th sites are CDR3 sequences of the SAL-VH.

Example 2 Glycerol preparation of RV308 competent cells

To the frozen Escherichia coli RV308 cells were added 500. mu.L of 2 XYT liquid medium, and cultured at 37 ℃ and 200rpm for 2 hours with shaking to recover the strain. After the strains are recovered, dipping micro-bacterial liquid and carrying out inverted culture on an LB solid culture medium until the size of bacterial colonies is uniform and the bacterial colonies are distributed on a flat plate; picking single colony to inoculate in 2mL LB liquid culture medium for transient recovery culture, and then carrying out amplification culture in 200mL LB liquid culture medium until OD600 is about 0.6; putting the required solution and reagent in an ice bath for at least 30min, uniformly distributing 200mL of culture solution comprising sterile water, 10% glycerol and a colony culture solution, putting the culture solution into a 50mL centrifuge tube, centrifuging for 15min at 4 ℃ and 3000rpm, removing the supernatant, and keeping the lower-layer precipitate; after the suspended bacteria are suspended by 10mL of ice-bath sterile water, supplementing 40mL of ice-bath water, centrifuging for 20min at the same rotating speed again, discarding the supernatant, and keeping the precipitate; adding 30mL of 10% ice-bath glycerol to wash and precipitate, centrifuging and then removing supernatant to obtain RV308 competent cells; adding 10% ice-bath glycerol 1.5mL, subpackaging, and quickly freezing and storing at-70 ℃ in 100 mu L each tube.

Example 3 transformation and expression of Single chain antibodies

1. Electric shock transformation of ScFv-pJB33 recombinant plasmid

The LAS-ScFv gene fragment and the SAL-ScFv gene fragment obtained in the above example 1 were respectively connected to pJB33 plasmid vector by double enzyme digestion to obtain ScFv-pJB33 recombinant plasmid, which was identified by agarose gel electrophoresis and then gel-cut and recovered. The LAS-ScFv-pJB33 recombinant plasmid is a vector which replaces a fragment (small fragment) inserted between pelB and (His)6tag recognition sites of a pJB33 plasmid vector with the LAS-ScFv gene fragment and keeps other nucleotide sequences of the pJB33 plasmid vector unchanged. The SAL-ScFv-pJB33 recombinant plasmid is a vector which replaces a fragment (small fragment) between pelB and (His)6tag recognition sites of a pJB33 plasmid vector with the SAL-ScFv gene fragment and keeps other nucleotide sequences of the pJB33 plasmid vector unchanged.

The recovered products (ScFv-pJB33 recombinant plasmids) were transformed into RV308 competent cells prepared in example 2 by electric shock, which was as follows:

irradiating the electric rotating cup with ultraviolet light for half an hour in advance, carefully placing the dialysis membrane on the liquid level of deionized water, and dripping 20 μ L of ScFv-pJB33 recombinant plasmid onto the dialysis membrane for at least 30min under ice bath condition in the whole dialysis process; taking out a tube of 100 mu L of competent cells RV308 from-80 ℃, adding a dialysis product when the competent cells are just thawed, and slightly shaking the tube to uniformly mix the competent cells; transferring to a dry electric rotor cup, loading into an electric rotor instrument, and electrically rotating for 5ms under the conditions of 2250v, 25uF and 150 omega; after the electrotransformation is finished, the electrotransformation products are all sucked into a test tube containing 900 mu L of 2 XYT liquid culture medium, and are subjected to shaking culture at the temperature of 37 ℃ for 1 h; 100. mu.L of the above culture solution was applied to 2 XYT agar plates (containing chloramphenicol at 34. mu.g/mL) and cultured in an inverted state at 37 ℃ for 12 hours.

And (3) positive screening:

and (2) selecting white colonies in the plate, carrying out amplification culture, carrying out shake culture in a 2 XYT liquid culture medium containing CAP at 37 ℃ until the OD value is about 0.6, carrying out bacteria liquid PCR reaction by taking pJB 33F and pJB 33R as upstream and downstream primers, and identifying positive colonies, wherein the reaction system is as follows:

the PCR conditions were the same as in example 1. After the reaction, the positive colonies were identified by agarose gel electrophoresis, and 1.5mL of the positive bacterial solution was sequenced by Biomed. The correctly sequenced colonies were kept at-80 ℃.

The strain RV308-LAS-ScFv expresses an LAS-ScFv gene fragment, an expression product is a single-chain antibody LAS-ScFv, the amino acid sequence of the single-chain antibody LAS-ScFv is shown as SEQ ID No.5, the 1 st to 114 th sites are VL sequences, the 115 th site 129 th site is a Linker sequence, the 130 th site 247 site is a VH sequence, the 26 th to 38 th sites are CDR1 sequences of LAS-VL, the 54 th to 58 th sites are CDR2 sequences of LAS-VL, the 93 th to 103 th sites are CDR3 sequences of LAS-VL, the 154 th site 163 is a CDR1 sequence of LAS-VH, the 179 th site 187 is a CDR2 sequence of LAS-VH, and the 224 th site 237 is a CDR3 sequence of LAS-VH.

The strain RV308-SAL-ScFv expresses SAL-ScFv gene fragments, the expression product is a single-chain antibody SAL-ScFv, the amino acid sequence of the single-chain antibody SAL-ScFv is shown as SEQ ID No.6, the 1 st to 109 th sites are VL sequences, the 110 th and 124 th sites are Linker sequences, the 125 th and 243 th sites are VH sequences, the 26 th to 33 th sites are CDR1 sequences of LAS-VL, the 49 th to 53 th sites are CDR2 sequences of LAS-VL, the 88 th to 98 th sites are CDR3 sequences of LAS-VL, the 149 th and 157 th sites are CDR1 sequences of LAS-VH, the 174 th and 183 are CDR2 sequences of LAS-VH, and the 220 th and 233 are CDR3 sequences of LAS-VH.

2. Expression and purification of ScFv

(1) When the expression was induced in a large amount, the colonies sequenced correctly as described above were picked and cultured in 100mL of 2 XYT liquid medium containing chloramphenicol (34. mu.g/mL). After shaking culture at 37 ℃ overnight (16h), 20mL of the culture broth was inoculated into 4L2 XYT liquid medium (containing chloramphenicol at 34. mu.g/mL) for scale-up culture. After the scale-up culture to OD600 of about 0.6 to 0.8, the inducer IPTG was added to 0.1mM, followed by induction at 16 ℃ and shaking culture overnight. Collecting bacterial liquid on the next day, centrifuging the collected bacterial liquid at 10000rpm at 4 ℃ for 10min, and collecting thalli. After the PBS is cooled by ice bath after high pressure, the thalli are washed, and the thalli can be stably stored at-80 ℃ after centrifugation so as to carry out subsequent tests.

When an ScFv gene fragment (LAS-ScFv gene fragment or SAL-ScFv gene fragment) is expressed, induction conditions are important for soluble expression and inclusion. Expression was carried out overnight (16h) at an induction temperature of 16 ℃ and an IPTG concentration of 0.1 mM. And (3) breaking cell walls of a large amount of induction-expressed thalli by using an ultrasonic cracker to obtain a cell expression product. Resuspending the thallus with a Binding buffer with a proper volume, carrying out ice bath ultrasonic treatment under the power of 200W by using an ultrasonic crusher to break cell walls (ultrasonic treatment is carried out for 3s, and stopping for 2s) to obtain cell suspension with uniform texture, centrifuging the cell suspension at the temperature of 4 ℃ and 10000rpm for 20min, and transferring supernatant into a new centrifugal tube, wherein the supernatant is ScFv extracting solution.

(2) The expressed ScFv protein has 6 × His label, can be combined with nickel column, and can be eluted by high-concentration imidazole so as to achieve the purpose of purification. Firstly, according to the protein expression amount, a purification column is filled with Ni-NTA with a proper volume for about 1h, the Ni-NTA is uniformly filled, then the nickel column is washed by ultrapure water, and the column is balanced by Binding buffer (10mM imidazole) with a proper volume.

(3) Filtering ScFv extract with 0.45 μm filter membrane, mixing with nickel filler, shaking at 4 deg.C for 2 hr, slowly passing through column, and collecting the flow-through solution.

(4) After all the supernatant had flowed out, the column was washed with Washing buffer (50mM imidazole) to remove the contaminating proteins adhered to the column, and the protein concentration was measured by Bradford while Washing until no protein flowed out.

(5) Finally, the target protein bound to the column was eluted with Elutionbuffer (300mM imidazole), and the eluate was collected and the protein concentration was measured with Bradford until no protein flowed out.

(6) Dialyzing the eluted protein with PBS for 48h, replacing PBS every 12h, collecting the target protein (i.e. purified protein), adding glycerol with the same volume during storage to protect the protein from denaturation, and standing at-20 ℃.

(7) Regenerating the nickel column: washed with 10 column volumes of ultrapure water, then blocked with 20% ethanol and placed at 4 ℃.

And identifying the target protein by using a protein electrophoresis combined Western-blotting method, and analyzing the purity of the target protein.

SDS-PAGE identification of ScFvs:

preparing needed gel and solution, cleaning the electrophoresis tank and the glass plate, starting glue pouring after the glass plate is fixed, wherein the glue pouring is performed first quickly and then slowly, and bubbles are prevented from occurring. After the rubber block is completely solidified, carefully pulling out the comb in the vertical direction to form a sample adding hole; taking 20 mu L of the purified ScFv sample to be heated and denatured at 100 ℃; adding electrophoresis liquid to submerge the glass plate, preparing for sample loading after determining that no liquid leakage exists, connecting a power supply to start electrophoresis after sample loading is finished, firstly performing low-voltage about 80V electrophoresis, adjusting the voltage to 120V when the sample is electrophoresed to separation gel after about 30min, and closing the power supply after electrophoresis is finished for about 1 h; carefully taking out the glass plate, prying the glass plate to take out the rubber block, soaking the glass plate in 0.25% Coomassie brilliant blue staining solution for oscillation dyeing for 2h, and if the dyeing is not uniform, properly prolonging the dyeing time; and after dyeing is finished, taking out the rubber block, washing off the dyeing liquid by using distilled water, then soaking the rubber block into a decoloring liquid for diffusion decoloring, and replacing the decoloring liquid at any time during decoloring until the protein strips are clear.

Western-blotting identification of ScFvs:

(1) when protein bands are clearly visible, cutting off glue holes and concentrated glue, soaking separation glue in a transfer buffer solution, cutting a PVDF membrane and 6 pieces of filter paper with the same size, and soaking the PVDF membrane and the 6 pieces of filter paper in the transfer buffer solution to fully wet the PVDF membrane and the 6 pieces of filter paper; the cellulose pad, the filter paper, the glue, the membrane, the filter paper and the cellulose pad are placed in sequence from the negative electrode to the positive electrode. Bubbles are removed at any time in each step, and the bubbles can affect the electric rotation.

(2) After placing, fixing the film transferring clamp in an electrophoresis tank, transferring for 2h at 110V voltage after finishing installation, performing the whole film transferring process under the conditions, adding ice at any time to cool, and taking the film after the transfer is finished.

(3) The membrane was washed right side up, with shaking in 1 XPBST solution 3 times for 10min each. Transfer to 5% skim milk, shake for 2h at room temperature.

(4) After blocking, the membrane was removed, washed with 1 XPBST buffer and washed 3 times for 10min each.

(5) anti-His antibody (diluted 1: 5000 with PBST) was added and the binding was shaken at room temperature for about 1 h. And (4) washing the membrane, and carrying out the same step as the step (4).

(6) HRP-labeled goat anti-mouse IgG (diluted 1: 8000 with PBST) was added and the binding was shaken at room temperature for 1 h. After the liquid is discarded, the membrane is washed as in step (4).

(7) And (3) dropwise adding a developing solution into the NC film with the front side facing upwards, setting different exposure times for imaging in a gel imager after shading and developing, and storing clear and bright pictures.

And (3) identification standard: the size of a specific protein band which appears in the experimental result is consistent with that of the target protein, and the impurity band near the target band is less, so that the purified protein is proved to be represented as the target protein.

The identification result shows that the RV308-LAS-ScFv induces and expresses LAS-ScFv gene fragments in a large quantity, and a single-chain antibody LAS-ScFv is obtained after a product is purified; the RV308-SAL-ScFv induces and expresses SAL-ScFv gene fragments in a large quantity, and a single-chain antibody SAL-ScFv is obtained after a product is purified.

The concentration of the single-chain antibody LAS-ScFv in the purified protein was 1.5mg/mL and the concentration of the single-chain antibody SAL-ScFv in the purified protein was 2.0mg/mL, as determined by the BCA kit.

icELISA detection of ScFvs:

(1) coating: the lasalomycin coatingen and the salinomycin coatingen are diluted by 1000 times by PBS, respectively added into a 96-well plate, each well is 100 mu L, and incubated for 2h in an incubator at 37 ℃ in the absence of light.

(2) Washing: the liquid in the wells was spun off, 270. mu.L of wash solution per well, washed 3 times, and patted dry after spinning off the liquid in the wells.

(3) And (3) sealing: sealing 150 μ L of 5% skimmed milk per well, incubating in a 37 deg.C incubator in dark for 2h, throwing out the liquid in the well, and patting to dry.

(4) Sample adding: mu.L of LAS or SAL standard substance diluted with PBS gradient at a concentration of 0ng/mL, 0.1ng/mL, 0.3ng/mL, 0.9ng/mL, 2.7ng/mL, 8.1ng/mL and the above-mentioned purified protein diluted 1: 1000 were added to each well, incubated at 37 ℃ in the dark for 30min, and washed as in (2).

(5) Adding a His antibody: mu.L of anti-His tag antibody diluted 1: 3000 per well was incubated at 37 ℃ in a dark room for 30min, and washed as in (2).

(6) Adding a secondary antibody: mu.L of HRP-labeled goat anti-mouse IgG (diluted 1: 3000) was added to each well, incubated for 30min, and washed as in (2).

(7) Color development: adding the prepared developing solution, each well is 100 μ L, and developing in a 37 deg.C incubator for 15min in dark.

(8) And (4) terminating: adding 50 μ L of stop solution into each well, and reading OD of each well in an enzyme-linked immunosorbent assay₄₅₀The value is obtained.

(9) Establishing a standard curve: OD corresponding to gradient concentration of standard₄₅₀The value is ordinate, the logarithm of the standard substance concentration is abscissa, and a standard curve is obtained through the treatment of Origin 8.5 software, and finally the purified protein IC50 value and the cross reaction rate (CR) are obtained.

The standard curve prepared using the single chain antibody LAS-ScFv is shown in the left panel of FIG. 4, with an IC50 of 12.9ng/mL and a linear range of 6.9-24.0 ng/mL. The standard curve generated using the single-chain antibody SAL-ScFv is shown in the right panel of FIG. 4, with an IC50 of 8.6ng/mL and a linear range of 4.7-16.0 ng/mL. The specificity of the recombinant antibody was evaluated by performing an iciELISA test on 8 common polyether antibiotics shown in Table 2, and calculating the cross-reactivity of the two single-chain antibodies to the polyether drugs in 8. The results are shown in table 2, the cross reaction rate of LAS-ScFv to 7 commonly used polyether antibiotics is less than 0.5%, no cross reaction exists, only the LAS can be used for detecting the drug, and the specificity is good; the cross-reactivity of SAL-ScFv to narasin is up to 89%, which is probably because narasin and narasin only differ by one methyl group, the structures are very similar, so that the antigen binding sites are basically the same, and the cross-reactivity to the other 6 commonly used polyether antibiotics is less than 0.5%.

TABLE 2 scFvs sensitivity and specificity

The detection of the ScFvs by the ICELISA showed that the purified single-chain antibody LAS-ScFv can be used for detecting LAS, and the purified single-chain antibody SAL-ScFc can be used for detecting SAL.

Example 4 splicing of the complete Gene of bispecific Single chain antibody

(1) The positive bacterial suspension of RV308-LAS-ScFv and RV308-SAL-ScFv obtained in example 3 was cultured overnight at 37 ℃ under 200rpm, and LAS-ScFv-pJB33 recombinant plasmid and SAL-ScFv-pJB33 recombinant plasmid were extracted from the culture and amplified as amplification templates for VH and VL, respectively. And (3) taking the LAS-ScFv-pJB33 recombinant plasmid as a template, and amplifying by using primers P1 and P2 to obtain the LAS-VH. LAS-ScFv-pJB33 recombinant plasmid is used as a template, and primers P3 and P4 are adopted for amplification to obtain LAS-VL. SAL-ScFv-pJB33 recombinant plasmid is used as a template, and primers P5 and P6 are adopted for amplification to obtain SAL-VH. SAL-ScFv-pJB33 recombinant plasmid is used as a template, and primers P7 and P8 are adopted for amplification to obtain SAL-VL. The reaction system is as follows:

adding the reaction system at low temperature as far as possible, slightly oscillating, uniformly mixing, centrifuging, and carrying out PCR amplification reaction under the following PCR reaction conditions: pre-denaturation at 95 ℃ for 5 min; 1min at 95 ℃, 50s at 55 ℃ and 1min at 72 ℃; 30 cycles; extension at 72 ℃ for 10 min. And after gel electrophoresis identification, cutting and recovering.

(2) Further amplifying the VH and VL products purified and recovered in the step (1), wherein the reaction system is as follows:

the PCR reaction conditions were as above.

(3) And (3) amplifying two hybridized ScFv gene fragments by using the PCR product in the step (2) as a template. Amplifying by using LAS-VH and SAL-VL as templates and P1 and P9 as primers to obtain VH_(LAS)-LinkerA-VL_(SAL)A hybrid chain. Amplifying by using LAS-VL and SAL-VH as templates and P10 and P11 as primers to obtain VH_(SAL)-LinkerA-VL_(LAS)A hybrid chain. The reaction system is as follows:

adding the reaction system at low temperature, whirling at low speed, mixing uniformly, centrifuging, and performing PCR amplification reaction under the same conditions. After the reaction is finished, carrying out electrophoresis identification, and then carrying out gel cutting and recovery.

(4) Recovering the product from step (3) (i.e., VH obtained in step (3))_(LAS)-Linker A-VL_(SAL)Hybrid chains and VH_(SAL)-LinkerA-VL_(LAS)Product of equal volume mixing of hybrid chains) as template to splice complete VH_(LAS)-Linker A-VL_(SAL)-Linker B-VH_(SAL)-Linker A-VL_(LAs)The bispecific single chain antibody gene (ScDb gene) of (4). The reaction system is as follows:

adding the reaction system under the condition of low temperature, slightly oscillating, uniformly mixing, centrifuging, and carrying out PCR amplification reaction under the same PCR reaction conditions. The product (i.e., ScDb gene) was also recovered by cutting the gel after identification.

The ScDb gene structure is VH_(LAS)-LinkerA-VL_(SAL)-Linker B-VH(sAL)-LinkerA-VL_(LAs)The sequence of the ScDb gene is shown as SEQ ID No.3, and the 1 st to 354 th sites are VH_(LAS)The 355-_(sAL)The 703 th and the 747 th positions are Linker B, and the 748 nd and the 1104 th positions are VH_(SAL)The 1105-1125 bit is LinkerA, the 1126-1467 bit is VL_(LAS)。

The ScDb gene expression antibody ScDb, the amino acid sequence of the antibody ScDb is shown in SEQ ID No.4, the 1-118 th sites are VH_(LAS)The 119 th-125 th position is Linker A, and the 126 th-234 th position is VL_(SAL)The 235 th-249 bit is Linker B, the 250 th-368 bit is VH_(SAL)369-VL at position 376-489_(LAS)。

Example 5 transformation and expression of antibody ScDb

1. Electric shock transformation of ScFv-pJB33 recombinant plasmid

The ScDb gene obtained in the example 4 is double-digested and connected to a pJB33 plasmid vector by a restriction enzyme Sfi I to obtain a ScDb-pJB33 recombinant plasmid, and the ScDb-pJB33 recombinant plasmid is identified by agarose gel electrophoresis and then cut into gel for recovery. The recombinant plasmid ScDb-pJB33 is a vector which replaces a fragment (small fragment) inserted between pelB and (His)6tag recognition sites of the pJB33 plasmid vector with the ScDb gene and keeps other nucleotide sequences of the pJB33 plasmid vector unchanged.

The recovered product (ScFv-pJB33 recombinant plasmid) was transformed into RV308 competent cells prepared in example 2 by electric shock, which was performed as follows:

irradiating the electric rotating cup with ultraviolet light for half an hour in advance, carefully placing the dialysis membrane on the liquid level of deionized water, and dripping 20 μ L of ScDb-pJB33 recombinant plasmid onto the dialysis membrane for at least 30min under ice bath condition in the whole dialysis process; taking out a tube of 100 mu L of competent cells RV308 from-80 ℃, adding a dialysis product when the competent cells are just thawed, and slightly shaking the tube to uniformly mix the competent cells; transferring to a dry electric rotor cup, loading into an electric rotor instrument, and electrically rotating for 5ms under the conditions of 2250v, 25uF and 150 omega; after the electrotransformation is finished, the electrotransformation products are all sucked into a test tube containing 900 mu L of 2 XYT liquid culture medium, and are subjected to shaking culture at the temperature of 37 ℃ for 1 h; 100. mu.L of the above culture solution was applied to 2 XYT agar plates (containing chloramphenicol at 34. mu.g/mL) and cultured in an inverted state at 37 ℃ for 12 hours.

Positive screening ScFv positive screening procedure as in example 3 was performed.

The strain RV308-ScDb expresses ScDb gene, the expression product is antibody ScDb, and the amino acid sequence of the antibody ScDb is shown in SEQ ID No. 4.

The mass induction expression method of the strain RV308-ScDb is the same as that of the example 3.

2. Expression and purification of ScDb

(1) When the ScDb gene is expressed, the induction conditions are important for the soluble expression and inclusion body. Expression was carried out overnight (16h) at an induction temperature of 16 ℃ and an IPTG concentration of 0.1 mM. And (3) breaking cell walls of a large amount of induction-expressed thalli by using an ultrasonic cracker to obtain a cell expression product. And (3) resuspending the thallus by using a Binding buffer with a proper volume, carrying out ice bath ultrasonic treatment by using an ultrasonic crusher under the power of 200W to break cell walls (ultrasonic treatment is carried out for 3s, and is stopped for 2s) to obtain cell suspension with uniform texture, centrifuging the cell suspension at the temperature of 4 ℃ and the rpm of 10000 for 20min, and transferring supernatant into a new centrifugal tube, wherein the supernatant is the ScDb extracting solution.

(2) The expressed ScDb protein has a 6 XHis tag, can be combined with a nickel column, and is eluted by high-concentration imidazole to achieve the aim of purification. Firstly, according to the protein expression amount, a purification column is filled with Ni-NTA with a proper volume for about 1h, the Ni-NTA is uniformly filled, then the nickel column is washed by ultrapure water, and the column is balanced by Binding buffer (10mM imidazole) with a proper volume.

(3) Filtering the ScDb extract obtained in the step (1) by using a 0.45-micron filter membrane, uniformly mixing with a nickel filler, shaking and combining for 2h at 4 ℃, slowly passing through a column, and collecting a flow-through liquid.

(4) After all the supernatant had flowed out, the column was washed with Washing buffer (50mM imidazole) to remove the contaminating proteins adhered to the column, and the protein concentration was measured by Bradford while Washing until no protein flowed out.

(5) Finally, the target protein bound to the column was eluted with Elutionbuffer (300mM imidazole), and the eluate was collected and the protein concentration was measured with Bradford until no protein flowed out.

(6) Dialyzing the eluted protein with PBS for 48h, replacing PBS every 12h, collecting target protein, adding glycerol with the same volume during storage to protect the protein from denaturation, and placing at-20 ℃.

(7) Regenerating the nickel column: washed with 10 column volumes of ultrapure water, then blocked with 20% ethanol and placed at 4 ℃.

The identification steps of SDS-PAGE and Western-blotting of the target protein are the same as those of ScFvs in example 3.

The identification result shows that RV308-ScDb induces and expresses ScDb gene fragments in a large quantity, and a single-chain antibody ScDb is obtained after a product is purified.

And (3) measuring the concentration of the single-chain antibody in the purified protein prepared by the BCA kit, wherein the concentration of the single-chain antibody scDb in the purified protein is 3.8 mg/mL.

Example 6 preliminary application of bispecific Single chain antibodies

1. Application of single-chain antibody ScDb

Diluting the Laxarotene coating antigen and the salinomycin coating antigen by 9000 times by using PBS, respectively adding the diluted Laxarotene coating antigen and the salinomycin coating antigen into an enzyme label plate, wherein each well is 100 mu L, and incubating for 2 hours in a 37 ℃ incubator in a dark place; throwing out liquid in the holes, washing the plate for 3 times by 270 mu L of washing liquid in each hole, and then patting dry after throwing out the liquid in the holes; sealing 150 mu L of 5% skimmed milk in each hole, incubating for 2h under the same condition, throwing out liquid in the hole, and patting to dry; 50 microliter LAS and SAL standard substance diluted by PBS and with gradient concentration of 0ng/mL, 0.1ng/mL, 0.3ng/mL, 0.9ng/mL, 2.7ng/mL and 8.1ng/mL and 9000 times diluted antibody ScDb prepared in example 5 are respectively added into each hole, and are incubated in an incubator at 37 ℃ for 30min under the condition of avoiding light, washed and dried; adding 50 μ L of anti-His tag antibody (diluted 1: 3000) into each well, incubating for 30min under the same conditions, washing, and patting dry; adding 50 μ L of HRP-labeled goat anti-mouse IgG (diluted 1: 3000) into each well, incubating in a incubator at 37 ℃ in the dark for 30min, washing, and patting dry; adding 100 μ L of the prepared color developing solution into each well, and developing in a dark place at 37 deg.C for 15 min; finally, 50 mu L of stop solution is added into each hole, and OD of each hole is read in an enzyme-linked immunosorbent assay₄₅₀The value is obtained. Gradient concentration and OD according to LAS or SAL Standard₄₅₀Values establish LAS or SAL standard curves.

The results show that the antibody ScDb can be used for detecting LAS and also can be used for detecting SAL.

2. Establishment of method for detecting SAL and LAS in sample by indirect competition ELISA

1) Establishing a matrix standard curve

Weighing 2g of chicken liver without LAS and SAL residues, placing the chicken liver into a 50mL centrifugal tube, whirling and uniformly mixing, adding 5mL of methanol for extraction, transferring supernatant into another 50mL centrifugal tube, repeatedly extracting the residual matrix in the original centrifugal tube, combining the supernatants obtained by two times of extraction, adding 10mL of n-hexane, slightly shaking for 20s, centrifuging at 5000r/min for 3min, discarding the n-hexane layer, drying the lower layer solution at 40 ℃ with nitrogen, finally re-dissolving with 2mL of 10% methanol water, shaking for 10min to obtain a chicken liver matrix extracting solution, and carrying out subsequent detection tests.

And (3) replacing PBS buffer solution with the prepared chicken liver matrix extracting solution, diluting LAS and SAL standard products in a gradient manner, and establishing a matrix labeling standard curve. The standard curve method is established, and the application of the single-chain antibody ScDb is the same as 1. FIG. 4 shows the matrix-normalized curve, the LAS matrix-normalized curve on the left side, the SAL matrix-normalized curve on the right side, in which the X-axis represents the LAS and SAL concentrations and the Y-axis represents the OD_450nmThe IC50 value for ScDb versus LAS was 0.7ng/mn, and the IC50 value for ScDb versus SAL was 0.4 ng/mL. The results show that the antibody ScDb can be used for detecting LAS in a sample and also can be used for detecting SAL in the sample.

2) Preparing extracting solution of sample to be detected

Weighing 2g of sample, placing the sample in a 50mL centrifugal tube, whirling and uniformly mixing, adding 5mL of methanol for extraction, transferring supernatant into another 50mL centrifugal tube, repeatedly extracting the residual matrix in the original centrifugal tube, combining the supernatant obtained in the two-time extraction, adding 10mL of n-hexane, slightly shaking for 20s, centrifuging at 5000r/min for 3min, discarding an n-hexane layer, drying the lower layer solution at 40 ℃ by nitrogen, finally redissolving by 2mL of 10% methanol water, oscillating for 10min to obtain a composite solution (sample extracting solution to be detected), and carrying out subsequent detection tests.

3) Detection of a sample to be tested by ScDb

The lasalocid coating antigen and salinomycin coating antigen are diluted by 9000 times and are respectively added to an enzyme label plate. Incubating 100 mu L of the suspension in an incubator at 37 ℃ for 2h in a dark place; throwing out liquid in the holes, washing the plate for 3 times by 270 mu L of washing liquid in each hole, and then patting dry after throwing out the liquid in the holes; sealing 150 mu L of 5% skimmed milk in each hole, incubating for 2h under the same condition, throwing out liquid in the hole, and patting to dry; adding 50 μ L of sample extract to be tested and the antibody ScDb prepared in example 5 (1: 9000 dilution) into each well, incubating at 37 deg.C in a dark condition for 30min, washing, and patting to dry; adding 50 μ L of anti-His tag antibody (diluted 1: 3000) into each well, incubating for 30min under the same conditions, washing, and patting dry; adding 50 μ L of HRP-labeled goat anti-mouse IgG (diluted 1: 3000) into each well, incubating in a incubator at 37 ℃ in the dark for 30min, washing, and patting dry; adding 100 μ L of the ready-to-use color developing solution into each wellDeveloping the solution in a 37 ℃ incubator for 15min in a dark place; finally, 50 mu L of stop solution is added into each hole, and OD of each hole is read in an enzyme-linked immunosorbent assay₄₅₀The value is obtained.

OD of the extract of the sample to be tested₄₅₀Values were taken into the matrix calibration curve to obtain residual amounts of SAL and LAS.

4) Method accuracy and precision determination

According to the standard curve, the accuracy and precision of the three concentration calculation methods, namely low, medium and high, are selected as shown in table 3, and 6 parallels are made for each concentration, namely 3 batches.

The specific method is as follows.

LAS and SAL standards were added to the chicken livers without LAS and SAL residues, respectively, at the concentrations in Table 3 and mixed well as the samples. The measured concentrations of LAS, SAL in the sample are obtained according to steps 3) and 4) above. The recovery and coefficient of variation were calculated as follows: the recovery rate (100%) is measured concentration/added concentration x 100%; coefficient of variation is standard deviation/average of recovery.

The results are shown in table 4, the substrate addition recovery rate of the LAS by the ScDb is between 81.8% and 98.3%, the intra-batch variation coefficient is less than 6.6%, and the inter-batch variation coefficient is less than 4.1%; the addition recovery rate of the ScDb to the SAL substrate is 82.3-99.0%, the intra-batch variation coefficient is less than 7.3%, and the inter-batch variation coefficient is less than 6.8%, so that the requirements of the veterinary drug residue test technical specification on accuracy and precision are met, and the method can be used for residue detection of actual samples.

TABLE 3 recovery and coefficient of variation of LAS and SAL additions in chicken livers

The primers used in the above examples are shown in tables 4 and 5.

TABLE 4 primers required for scFv amplification

TABLE 5 primers required for amplification of scDb

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Sequence listing

<110> university of agriculture in China

<120> lasalomycin and salinomycin single chain antibody and bispecific single chain antibody and application thereof

<130> 212198

<160> 6

<170> SIPOSequenceListing 1.0

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<211> 741

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

gatattttga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60

atctcttgca gatctagtca gagcattgta catagtaatg gaaacaccta tttagaatgg 120

tacctgcaga aaccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180

tctggggtcc cagacaggtt cagtgtcagt ggatcaggga cagatttcac actcaagatc 240

agcagagtgg aggctgagga tctgggagtt tattactgct ttcaaggttc acatgttcct 300

ccgacgttcg gtggaggcac caagctggaa atcaaacggg ctggtggtgg tggttctggc 360

ggcggcggct ccggtggtgg tggatccgaa gtgaagctgg tggagtctgg gggaggctta 420

gtgaagcctg gagggtccct gaagctctcc tgtgcagcct ctggattcac tttcagtagc 480

tatcccatgt cttgggttcg ccagactcca gaaaagaggc tggagtgggt cgcatccatt 540

agtagtggtg gtagtaccta ctatctagac aatgtggagg gccgattcac catctccaga 600

gatagtgcca ggaacatcct gtacctgcaa atgaccagtc tgaggtctga ggacacggcc 660

atgtattact gtgcaagagg ccgaggaggt tacgactggt ttgcttactg gggccaaggg 720

actctggtca ctgtctctgc a 765

<210> 2

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

gacattgtga tgacccagtc tcaaaaattc atgtccacat cagtaggaga cagggtcagc 60

gtcacctgca aggccagtca gaatgtgggt actaatgtag cctggtctca acagaaacca 120

gggcaatctc ctaaagcact gatttactcg gcatcccacc ggtacagtgg agtccctgat 180

cgtttcatag ggagtggtta tgggacagat ttcactctca ccattagcaa tgtgcagtct 240

gaagacttgg cagagtattt ctgtcagcaa tataacaact atccgtacac gttcggaggg 300

gggaccaagc tggaaataaa acgggctggt ggtggtggtt ctggcggcgg cggctccggt 360

ggtggtggat ccgaggtcca gctgcaacag tctggacctg agctggtgaa gcctggggct 420

tcagtgaaga tttcctgcag gacttctgga tacacattca ctgaatacac cattcactgg 480

gtgaagcgga gccatggaaa gagccttgag tggattggac atatttatcc taacaatggt 540

ggtgctaact acaaccagaa gttcaagggc aaggccactt tgactgtaga caggtcctcc 600

agcacagcct acatggaact ccgcagcctg acatctgagg attctgcggt ctattactgt 660

gctacttctg tttacgacgg gggctggtct gtttactggg gccaagggac tctggtcact 720

gtctctgca 753

<210> 3

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gaagtgaagc tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaagctc 60

tcctgtgcag cctctggatt cactttcagt agctatccca tgtcttgggt tcgccagact 120

ccagaaaaga ggctggagtg ggtcgcatcc attagtagtg gtggtagtac ctactatcta 180

gacaatgtgg agggccgatt caccatctcc agagatagtg ccaggaacat cctgtacctg 240

caaatgacca gtctgaggtc tgaggacacg gccatgtatt actgtgcaag aggccgagga 300

ggttacgact ggtttgctta ctggggccaa gggactctgg tcactgtctc tgcatctggc 360

ggtggcggta gtcaagacat tgtgatgacc cagtctcaaa aattcatgtc cacatcagta 420

ggagacaggg tcagcgtcac ctgcaaggcc agtcagaatg tgggtactaa tgtagcctgg 480

tctcaacaga aaccagggca atctcctaaa gcactgattt actcggcatc ccaccggtac 540

agtggagtcc ctgatcgttt catagggagt ggttatggga cagatttcac tctcaccatt 600

agcaatgtgc agtctgaaga cttggcagag tatttctgtc agcaatataa caactatccg 660

tacacgttcg gaggggggac caagctggaa ataaaacggg ctggtggtgg tggttctggc 720

ggcggcggct ccggtggtgg tggatccgag gtccagctgc aacagtctgg acctgagctg 780

gtgaagcctg gggcttcagt gaagatttcc tgcaggactt ctggatacac attcactgaa 840

tacaccattc actgggtgaa gcggagccat ggaaagagcc ttgagtggat tggacatatt 900

tatcctaaca atggtggtgc taactacaac cagaagttca agggcaaggc cactttgact 960

gtagacaggt cctccagcac agcctacatg gaactccgca gcctgacatc tgaggattct 1020

gcggtctatt actgtgctac ttctgtttac gacgggggct ggtctgttta ctggggccaa 1080

gggactctgg tcactgtctc tgcatcgggc ggtggcggtt cacaggatat tttgatgacc 1140

caaactccac tctccctgcc tgtcagtctt ggagatcaag cctccatctc ttgcagatct 1200

agtcagagca ttgtacatag taatggaaac acctatttag aatggtacct gcagaaacca 1260

ggccagtctc caaagctcct gatctacaaa gtttccaacc gattttctgg ggtcccagac 1320

aggttcagtg tcagtggatc agggacagat ttcacactca agatcagcag agtggaggct 1380

gaggatctgg gagtttatta ctgctttcaa ggttcacatg ttcctccgac gttcggtgga 1440

ggcaccaagc tggaaatcaa acgggct 1467

<210> 4

<211> 489

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Pro Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Ser Gly Gly Ser Thr Tyr Tyr Leu Asp Asn Val Glu

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Ser Ala Arg Asn Ile Leu Tyr Leu

65 70 75 80

Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala

85 90 95

Arg Gly Arg Gly Gly Tyr Asp Trp Phe Ala Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ala Ser Gly Gly Gly Gly Ser Gln Asp Ile Val

115 120 125

Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly Asp Arg Val

130 135 140

Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn Val Ala Trp

145 150 155 160

Ser Gln Gln Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile Tyr Ser Ala

165 170 175

Ser His Arg Tyr Ser Gly Val Pro Asp Arg Phe Ile Gly Ser Gly Tyr

180 185 190

Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser Glu Asp Leu

195 200 205

Ala Glu Tyr Phe Cys Gln Gln Tyr Asn Asn Tyr Pro Tyr Thr Phe Gly

210 215 220

Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Gly Gly Gly Gly Ser Gly

225 230 235 240

Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Gln Gln Ser

245 250 255

Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Arg

260 265 270

Thr Ser Gly Tyr Thr Phe Thr Glu Tyr Thr Ile His Trp Val Lys Arg

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Ser His Gly Lys Ser Leu Glu Trp Ile Gly His Ile Tyr Pro Asn Asn

290 295 300

Gly Gly Ala Asn Tyr Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr

305 310 315 320

Val Asp Arg Ser Ser Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu Thr

325 330 335

Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Thr Ser Val Tyr Asp Gly

340 345 350

Gly Trp Ser Val Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala

355 360 365

Ser Gly Gly Gly Gly Ser Gln Asp Ile Leu Met Thr Gln Thr Pro Leu

370 375 380

Ser Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser

385 390 395 400

Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr

405 410 415

Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser

420 425 430

Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Val Ser Gly Ser Gly

435 440 445

Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly

450 455 460

Val Tyr Tyr Cys Phe Gln Gly Ser His Val Pro Pro Thr Phe Gly Gly

465 470 475 480

Gly Thr Lys Leu Glu Ile Lys Arg Ala

485

<210> 5

<211> 247

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Asp Ile Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Val Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly

130 135 140

Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser

145 150 155 160

Tyr Pro Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp

165 170 175

Val Ala Ser Ile Ser Ser Gly Gly Ser Thr Tyr Tyr Leu Asp Asn Val

180 185 190

Glu Gly Arg Phe Thr Ile Ser Arg Asp Ser Ala Arg Asn Ile Leu Tyr

195 200 205

Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys

210 215 220

Ala Arg Gly Arg Gly Gly Tyr Asp Trp Phe Ala Tyr Trp Gly Gln Gly

225 230 235 240

Thr Leu Val Thr Val Ser Ala

245

<210> 6

<211> 243

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn

20 25 30

Val Ala Trp Ser Gln Gln Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile

35 40 45

Tyr Ser Ala Ser His Arg Tyr Ser Gly Val Pro Asp Arg Phe Ile Gly

50 55 60

Ser Gly Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser

65 70 75 80

Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Asn Asn Tyr Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Gly Gly Gly

100 105 110

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu

115 120 125

Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Val Lys Ile

130 135 140

Ser Cys Arg Thr Ser Gly Tyr Thr Phe Thr Glu Tyr Thr Ile His Trp

145 150 155 160

Val Lys Arg Ser His Gly Lys Ser Leu Glu Trp Ile Gly His Ile Tyr

165 170 175

Pro Asn Asn Gly Gly Ala Asn Tyr Asn Gln Lys Phe Lys Gly Lys Ala

180 185 190

Thr Leu Thr Val Asp Arg Ser Ser Ser Thr Ala Tyr Met Glu Leu Arg

195 200 205

Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Thr Ser Val

210 215 220

Tyr Asp Gly Gly Trp Ser Val Tyr Trp Gly Gln Gly Thr Leu Val Thr

225 230 235 240

Val Ser Ala