阅读说明：本技术 一种抗非洲猪瘟病毒的单链抗体及其制备方法和用途 (Single-chain antibody for resisting African swine fever virus and preparation method and application thereof ) 是由 郑海学 王丽娟 �田宏 石正旺 罗俊聪 杨帆 于 2020-05-15 设计创作，主要内容包括：本发明公开了一种抗非洲猪瘟病毒的单链抗体及其制备方法和用途,本发明从自然感染非洲猪瘟免疫耐过猪的外周血中分离得到淋巴细胞,提取分离到的淋巴细胞的总mRNA,通过RT-PCR的方法得到总cDNA片段,以cDNA为模板,在相应的带有Linker接头的引物的作用下,通过SOE-PCR的方法得到猪源ScFv抗体基因序列,将ScFv抗体基因序列构建至pET-30a载体,转化BL21感受态细胞,从转化后的单个菌落中筛选得到一个针对于非洲猪瘟病毒的ScFv抗体(VH-VL<Sub>κ6</Sub>),采用ELISA试验对筛选得到的ScFv抗体(VH-VL<Sub>κ6</Sub>)进行初步活性鉴定,表明此抗体具有非洲猪瘟反应活性。本发明的提出为非洲猪瘟的早期诊断与防控提供了新的材料,为尽快控制非洲猪瘟的疫情传播提供了新的技术手段。(The invention discloses a single-chain antibody for resisting African swine fever virus, a preparation method and application thereof, the invention separates lymphocytes from peripheral blood of naturally infected African swine fever immune resistant pigs, extracts total mRNA of the separated lymphocytes, obtains total cDNA fragments by an RT-PCR method, takes cDNA as a template, obtains a swine ScFv antibody gene sequence by an SOE-PCR method under the action of a corresponding primer with a Linker joint, and obtains the ScFv antibody gene sequenceConstructing into pET-30a vector, transforming BL21 competent cells, screening out an ScFv antibody (VH-VL) against African swine fever virus from the transformed single colony κ6 ) The ScFv antibody (VH-VL) obtained by screening was subjected to ELISA assay κ6 ) The primary activity identification is carried out, and the antibody has African swine fever reaction activity. The invention provides a new material for early diagnosis and prevention and control of the African swine fever and a new technical means for controlling epidemic spread of the African swine fever as soon as possible.)

1. A single chain antibody (ScFv) against african swine fever virus, encoded by the sequence of SEQ id No. 1.

2. The single-chain antibody (ScFv) according to claim 1, wherein said single-chain antibody is obtained by expression and purification of the sequence of SEQ ID No.1 in a prokaryotic expression system.

3. A preparation method of a single-chain antibody (ScFv) for resisting African swine fever virus is characterized by comprising the following steps:

(1) lymphocyte isolation

Separating lymphocytes from peripheral blood of naturally infected African swine fever immune-tolerant pigs, and storing for later use;

(2) extraction of lymphocyte Total mRNA

Extracting total mRNA of the lymphocyte separated in the step (1);

(3) whole Gene cDNA Synthesis

Synthesizing a complete gene cDNA by taking the total mRNA of the lymphocytes extracted in the step (2) as a template;

(4) first round amplification

Taking the whole gene cDNA synthesized in the step (3) as a template, and taking P1 and R1; f1, B1₁、B1₂、B1₃The first round of amplification was performed for the primers, and the amplification procedures were: 2min at 98 ℃,10 s at 98 ℃, 5s at 52 ℃,30 s at 72 ℃ and 30 cycles, and 10min at 72 ℃; 2min at 98 ℃,10 s at 98 ℃, 5s at 54 ℃,30 s at 72 ℃ and 30 cycles, and 10min at 72 ℃; carrying out electrophoresis on the amplified product, purifying and collecting a 300bp product, and storing at-20 ℃ for later use;

P1:5-ACGACGACTTCAACGCCTGG-3

R1:5-GAGGWGAAGCTGGTGGAGTCYGG-3

F1:5-TTTGAKYTCCAGCTTGGTCCC-3

B1₁:5-GCCATYGTGCTGACCCAGASTCC-3

B1₂:5-GAGCTCGTSATGACCCAGTCTCC-3

B1₃:5-GAGCTGCGTGATACACAGTCTCC-3

(5) second round of amplification

Taking the first round amplification product as a template, and taking P1 and R2 as templates; f2, B1₁、B1₂、B1₃For the second round of amplification of the primers, the amplification procedures were: 2min at 98 ℃,10 s at 98 ℃, 5s at 56 ℃,30 s at 72 ℃ and 35 cycles, and 10min at 72 ℃; 2min at 98 ℃,10 s at 98 ℃,30 s at 68 ℃ and 30 cycles, and 10min at 72 ℃. Performing electrophoresis on the amplified product, purifying and collecting a 320bp product from the gel by using a gel recovery kit, and storing at-20 ℃ for later use;

R2:

5-GCCGCCTGACCCTCCGCCACCGAGGWGAAGCTGGTGGAGTCYGG-3F2:5-GGTGGCGGAGGGTCAGGCGGCTTTGAKYTCCAGCTTGGTCCC-3

(6) third round of amplification

Using the second round amplification product as a template, and using P1 and B1₁、B1₂、B1₃The third round of amplification was performed with ① primers at 98 ℃ for 2min, 98 ℃ for 10s, 68 ℃ for 30s, 10 cycles, 72 ℃ for 10min, ② primers at 98 ℃ for 2min, 98 ℃ for 10s, 55 ℃ for 5s, 72 ℃ for 50s, 35 cycles, 72 ℃ for 10min.And (4) performing electrophoresis on the amplified product, purifying and collecting the 800bp product from the gel by using a gel recovery kit, and storing at-20 ℃ for later use.

(7) T-A clone identification

I, treating the third round of amplification product by adding ' A ' to the 3 ' end, wherein the reaction procedure is 10min at 65 ℃;

II ligation of the A-addition product to PMD^TM20-T carrier, and the connection program is 16 ℃ for 30 min;

coli JM109 competent cells to 10. mu.L of the ligation products, and the transformed cells were spread on LB-A⁺On a plate, inversely culturing for 8-16h at 37 ℃ until a monoclonal antibody appears, and picking the monoclonal antibody colony on LB-A⁺Culturing in liquid culture medium, extracting plasmid, sequencing, and screening out plasmid with correct gene sequence;

(8) construction and preparation of ScFv antibody

BL21 competent cells were prepared, 1. mu.L of ScFv-PET-30a plasmid constructed from the correctly sequenced amplification product was transformed into 50. mu.L of competent cells, and the transformed suspension was spread uniformly on LB-K⁺On a plate, inversely culturing for 8-16h at 37 ℃ until a monoclonal antibody appears, and picking a monoclonal antibody colony on LB-K⁺And inducing to express the antibody protein in a liquid culture medium, and collecting, washing and purifying the expressed antibody protein to obtain the single-chain antibody for resisting the African swine fever virus.

4. A method according to claim 3, wherein said lymphocyte separation is performed according to the following steps:

(1) adding the same amount of whole blood diluent into peripheral blood of a naturally infected African swine fever immune resistant pig, and uniformly mixing to obtain the whole blood diluent;

(2) measuring the lymphocyte separating medium according to the volume ratio of the whole blood diluent to the lymphocyte separating medium of 5:9, slowly and quickly adding the anticoagulant diluent obtained in the step (1), and performing horizontal centrifuge at 1800rpm, 18-22 ℃ for 30 min;

(3) carefully collecting lymphocyte layer in a new collection tube, diluting with 5 times of cell washing solution, horizontally centrifuging at 1500rpm at 18-22 deg.C for 10 min; discarding the supernatant, diluting the precipitate with cell washing solution, centrifuging with horizontal centrifuge at 1500rpm, 18-22 deg.C for 10 min; and (3) resuspending the precipitate by using 5mL of RNase-free water, adding 45mL of RPIM-1640 culture solution, uniformly mixing, centrifuging at the temperature of 18-22 ℃ for 10min at 1000rpm to obtain the precipitate, namely the lymphocyte, resuspending the lymphocyte in RNAlater preservation solution, and preserving at the temperature of-70 ℃ for later use.

5. A method according to claim 3, wherein the extraction of total mRNA from lymphocytes is carried out by: sucking 400 μ L of the above lymphocyte fluid, adding 1mL of TRizol, mixing, and standing at 4 deg.C for 5 min; adding 250 μ L of chloroform, mixing, standing at 4 deg.C for 10min, 12000r/min, and centrifuging at 4 deg.C for 15 min; sucking 450 μ L of supernatant, adding equal amount of isopropanol, standing at-20 deg.C for 30min, 12000r/min, centrifuging at 4 deg.C for 15 min; slightly discarding the supernatant, adding 1mL of 75% ethanol into the precipitate, 12000r/min, centrifuging at 4 ℃ for 5 min; drying the precipitate in a fume hood for 10 min; 25 μ L of RNase free water-solubilized pellet was the total mRNA harvested.

6. The method of claim 3, wherein the whole gene cDNA synthesis is performed by sequentially adding the following components to a 0.2ml PCR amplification tube:

after uniformly mixing, placing the PCR amplification tube in a PCR instrument, wherein the reaction procedure is as follows: 10min at 25 ℃, 60min at 37 ℃ and 15min at 72 ℃. The obtained product is the whole genome cDNA, and is stored at-20 ℃ for later use.

7. The method of claim 3, wherein the first round of amplification comprises sequentially adding the following components to a 0.2ml PCR amplification tube:

①:

②:

performing a first round of amplification under the amplification procedure in the step (4), performing electrophoresis on the amplification product, purifying and collecting a 300bp product, and storing at-20 ℃ for later use;

the second round of amplification is to add the following components in a 0.2ml PCR amplification tube in turn:

①:

②:

performing second amplification under the amplification program in the step (5), performing electrophoresis on the amplification product, purifying and collecting a 320bp product from the gel by using a gel recovery kit, and storing at-20 ℃ for later use;

the third round of amplification is to add the following components in a 0.2ml PCR amplification tube in sequence:

①:

② supplementary primers

And (4) carrying out third amplification under the amplification program in the step (6), carrying out electrophoresis on the amplification product, purifying and collecting the 800bp product from the gel by using a gel recovery kit, and storing at-20 ℃ for later use.

8. The method of claim 1, wherein the T-A clone identification is performed by performing the following procedures I and II in sequence in a 0.2mL PCR amplification tube, and performing procedure III in a 1.5mL centrifuge tube:

I

II

III

II Total ligation product 10. mu.l

E.coli JM109 100μL

T-A cloning identification is carried out under the program in the step (7), and plasmids with correct gene sequences are screened to construct ScFv-PET-30a plasmids.

9. Use of the single-chain antibody against African swine fever virus according to claim 1 or 2 in the preparation of a reagent for detecting African swine fever virus.

10. Nucleotide sequence encoding a single chain antibody against African swine fever virus according to claim 1, preferably, said nucleotide sequence is shown in SEQ ID No. 1.

Technical Field

The invention relates to a single-chain antibody for resisting African swine fever virus. In addition, the invention also relates to a preparation method and application of the antibody. The invention belongs to the field of biotechnology.

Background

African Swine Fever (ASF) is a highly lethal hemorrhagic disease of pigs caused by African Swine Fever Virus (ASFV), and has the characteristic of high contact infection. ASFV is a regular icosahedral double-stranded DNA virus with a diameter of 200nm, is the only member of African swine fever virus family, and is the only known virus transmitted by vector worms. The African swine fever firstly breaks out in African kenia in 1921, is introduced into China in 2018 in 8 months, and has 24 provinces in China as long as 2019 in 2 months, so that the African swine fever epidemic situation occurs, and huge economic loss is caused to the pig industry. At present, no effective vaccine for the virus exists worldwide, so the early diagnosis of the African swine fever plays a crucial role in the prevention and control of the disease. The antibody is an important immune molecule in the immune system of the body and is also a core component in vaccines and diagnostic reagents, and the development of the antibody plays an important promoting role in the prevention and control of African swine fever. However, the genome of ASFV is complex, and at present, the mechanism of action of each functional protein is poorly understood, so the development of the traditional antibody is very difficult, and at the moment, the development and preparation of a novel antibody may become a breakthrough for preventing and controlling the disease.

The single chain antibody (scFv) is an antibody formed by connecting an antibody heavy chain variable region and an antibody light chain variable region through a short peptide (linker) of 15-20 amino acids. Single-chain antibodies have attracted considerable attention as one of the third-generation antibodies (engineered antibodies). In 1988, Bird, Huston and the like developed single-chain antibodies for the first time, and researchers have not been interrupted to research the single-chain antibodies in the three decades since then, so that not only are various antibody forms of single-chain antibody multimers, bispecific single-chain antibodies and the like developed, but also various display systems of the single-chain antibodies are established. The single-chain antibody is an antibody with a molecular weight smaller than that of a conventional antibody, and only contains a heavy chain variable region and a light chain variable region, so that the single-chain antibody has good penetrability, is easy to contact with target cells through a blood vessel wall, and is suitable for diagnosis and treatment of tumors; in addition, the single-chain antibody still has accurate specificity and affinity to the original antigen because the variable region is well reserved; the single-chain antibody is used as an artificially synthesized antibody, is easy to modify molecules and can directly kill target cells; the single-chain antibody has the most prominent characteristics of overcoming the characteristic of strong mouse source property of the traditional monoclonal antibody, eliminating the heterologous reaction in the application of the antibody and widening the application range of the antibody. By virtue of the characteristics, the single-chain antibody has great advantages compared with the common antibody. Therefore, single chain antibodies are of great value in the treatment and diagnosis of disease.

In view of the development of the antibody field in recent years, we can see that the traditional antibody can not meet the requirements of the current antibody market, and people need a flexible antibody which is simple and convenient to prepare, strong in specificity, high in stability and small in molecular weight, so that the antibody can efficiently act on a specific part. Therefore, the realization of the miniaturization of antibodies and the targeting of effector molecules or other target cells in cancer cells is always an important target and research hotspot of antibody engineering and anticancer research.

In conclusion, the preparation of the antibody with ASFV specificity, good biological activity, strong antigen binding capacity and potential neutralization function, especially the novel antibody such as single-chain antibody, has important significance for establishing a sensitive ASFV clinical diagnosis method and controlling the spread of the epidemic situation of the ASF.

Disclosure of Invention

The invention aims to solve the technical problem of providing a single-chain antibody (ScFv) for resisting African swine fever virus and a preparation method thereof.

In order to achieve the purpose, the invention adopts the following technical means:

the invention relates to a single-chain antibody (ScFv) for resisting African swine fever virus, which is coded by a sequence shown in SEQ ID NO. 1.

Wherein, preferably, the single-chain antibody is obtained by expressing and purifying the sequence of SEQ ID NO.1 through a prokaryotic expression system.

Furthermore, the invention also provides a preparation method of the single-chain antibody (ScFv) for resisting African swine fever virus, which comprises the following steps:

(1) lymphocyte isolation

Separating lymphocytes from peripheral blood of naturally infected African swine fever immune-tolerant pigs, and storing for later use;

(2) extraction of lymphocyte Total mRNA

Extracting total mRNA of the lymphocyte separated in the step (1);

(3) whole Gene cDNA Synthesis

Synthesizing a complete gene cDNA by taking the total mRNA of the lymphocytes extracted in the step (2) as a template;

(4) first round amplification

Taking the whole gene cDNA synthesized in the step (3) as a template, and taking P1 and R1; f1, B1₁、B1₂、B1₃The first round of amplification was performed for the primers, and the amplification procedures were: 2min at 98 ℃,10 s at 98 ℃, 5s at 52 ℃,30 s at 72 ℃ and 30 cycles, and 10min at 72 ℃; 2min at 98 ℃,10 s at 98 ℃, 5s at 54 ℃,30 s at 72 ℃ and 30 cycles, and 10min at 72 ℃; carrying out electrophoresis on the amplified product, purifying and collecting a 300bp product, and storing at-20 ℃ for later use;

P1:5-ACGACGACTTCAACGCCTGG-3

R1:5-GAGGWGAAGCTGGTGGAGTCYGG-3

F1:5-TTTGAKYTCCAGCTTGGTCCC-3

B1₁:5-GCCATYGTGCTGACCCAGASTCC-3

B1₂:5-GAGCTCGTSATGACCCAGTCTCC-3

B1₃:5-GAGCTGCGTGATACACAGTCTCC-3

(5) second round of amplification

Using the first round amplification product as a templateP1, R2; f2, B1₁、B1₂、B1₃For the second round of amplification of the primers, the amplification procedures were: 2min at 98 ℃,10 s at 98 ℃, 5s at 56 ℃,30 s at 72 ℃ and 35 cycles, and 10min at 72 ℃; 2min at 98 ℃,10 s at 98 ℃,30 s at 68 ℃ and 30 cycles, and 10min at 72 ℃. Performing electrophoresis on the amplified product, purifying and collecting a 320bp product from the gel by using a gel recovery kit, and storing at-20 ℃ for later use;

R2:

5-GCCGCCTGACCCTCCGCCACCGAGGWGAAGCTGGTGGAGTCYGG-3

F2:

5-GGTGGCGGAGGGTCAGGCGGCTTTGAKYTCCAGCTTGGTCCC-3

(6) third round of amplification

Using the second round amplification product as a template, and using P1 and B1₁、B1₂、B1₃The third round of amplification is carried out on the primers, wherein the amplification procedure comprises ① no primer, 98 ℃ for 2min, 98 ℃ for 10s, 68 ℃ for 30s, 10 cycles, 72 ℃ for 10min, ② with primers, 98 ℃ for 2min, 98 ℃ for 10s, 55 ℃ for 5s, 72 ℃ for 50s, 35 cycles, 72 ℃ for 10min, electrophoresis of the amplification product, purification and collection of 800bp product from gel by a gel recovery kit, and storage at-20 ℃ for later use.

(7) T-A clone identification

I, treating the third round of amplification product by adding ' A ' to the 3 ' end, wherein the reaction procedure is 10min at 65 ℃;

II ligation of the A-addition product to PMD^TM20-T carrier, and the connection program is 16 ℃ for 30 min;

coli JM109 competent cells to 10. mu.L of the ligation products, and the transformed cells were spread on LB-A⁺On a plate, inversely culturing for 8-16h at 37 ℃ until a monoclonal antibody appears, and picking the monoclonal antibody colony on LB-A⁺Culturing in liquid culture medium, extracting plasmid, sequencing, and screening out plasmid with correct gene sequence;

(8) construction and preparation of ScFv antibody

BL21 competent cells were prepared, 1. mu.L of ScFv-PET-30a plasmid constructed from the correctly sequenced amplification product was transformed into 50. mu.L of competent cells, and the transformed suspension was spread uniformly on LB-K⁺On a plate, inversely culturing for 8-16h at 37 ℃ until a monoclonal antibody appears, and picking a monoclonal antibody colony on LB-K⁺And inducing to express the antibody protein in a liquid culture medium, and collecting, washing and purifying the expressed antibody protein to obtain the single-chain antibody for resisting the African swine fever virus.

Wherein, preferably, the lymphocyte separation is carried out according to the following steps:

(1) adding the same amount of whole blood diluent into peripheral blood of a naturally infected African swine fever immune resistant pig, and uniformly mixing to obtain the whole blood diluent;

(2) measuring the lymphocyte separating medium according to the volume ratio of the whole blood diluent to the lymphocyte separating medium of 5:9, slowly and quickly adding the anticoagulant diluent obtained in the step (1), and performing horizontal centrifuge at 1800rpm, 18-22 ℃ for 30 min;

(3) carefully collecting lymphocyte layer in a new collection tube, diluting with 5 times of cell washing solution, horizontally centrifuging at 1500rpm at 18-22 deg.C for 10 min; discarding the supernatant, diluting the precipitate with cell washing solution, centrifuging with horizontal centrifuge at 1500rpm, 18-22 deg.C for 10 min; and (3) resuspending the precipitate by using 5mL of RNase-free water, adding 45mL of RPIM-1640 culture solution, uniformly mixing, centrifuging at the temperature of 18-22 ℃ for 10min at 1000rpm to obtain the precipitate, namely the lymphocyte, resuspending the lymphocyte in RNAlater preservation solution, and preserving at the temperature of-70 ℃ for later use.

Preferably, the extraction of the total mRNA of the lymphocytes is carried out according to the following method: sucking 400 μ L of the above lymphocyte fluid, adding 1mL of TRizol, mixing, and standing at 4 deg.C for 5 min; adding 250 μ L of chloroform, mixing, standing at 4 deg.C for 10min, 12000r/min, and centrifuging at 4 deg.C for 15 min; sucking 450 μ L of supernatant, adding equal amount of isopropanol, standing at-20 deg.C for 30min, 12000r/min, centrifuging at 4 deg.C for 15 min; slightly discarding the supernatant, adding 1mL of 75% ethanol into the precipitate, 12000r/min, centrifuging at 4 ℃ for 5 min; drying the precipitate in a fume hood for 10 min; 25 μ L of RNase free water-solubilized pellet was the total mRNA harvested.

Wherein, preferably, the whole gene cDNA synthesis is to add the following components in sequence in a 0.2ml PCR amplification tube:

after uniformly mixing, placing the PCR amplification tube in a PCR instrument, wherein the reaction procedure is as follows: 10min at 25 ℃, 60min at 37 ℃ and 15min at 72 ℃. The obtained product is the whole genome cDNA, and is stored at-20 ℃ for later use.

Preferably, the first round of amplification is to add the following components in a 0.2ml PCR amplification tube in sequence:

①:

②:

performing a first round of amplification under the amplification procedure in the step (4), performing electrophoresis on the amplification product, purifying and collecting a 300bp product, and storing at-20 ℃ for later use;

the second round of amplification is to add the following components in a 0.2ml PCR amplification tube in turn:

①:

②:

performing second amplification under the amplification program in the step (5), performing electrophoresis on the amplification product, purifying and collecting a 320bp product from the gel by using a gel recovery kit, and storing at-20 ℃ for later use;

the third round of amplification is to add the following components in a 0.2ml PCR amplification tube in sequence:

①:

② supplementary primers

And (4) carrying out third amplification under the amplification program in the step (6), carrying out electrophoresis on the amplification product, purifying and collecting the 800bp product from the gel by using a gel recovery kit, and storing at-20 ℃ for later use.

Preferably, the T-A clone identification is performed by performing the following two procedures I and II in sequence in a 0.2mL PCR amplification tube, and performing procedure III in a 1.5mL centrifuge tube:

I

II

III

II Total ligation product 10. mu.l

E.coli JM109 100μL

T-A cloning identification was carried out under the procedure described in step (7), and plasmids having the correct gene sequence were selected for construction of ScFv-pET-30a plasmid.

Furthermore, the invention also provides application of the single-chain antibody for resisting the African swine fever virus in preparation of a reagent for detecting the African swine fever virus.

The nucleotide sequence of the single-chain antibody for resisting the African swine fever virus is also within the protection scope of the invention, and preferably, the nucleotide sequence is shown as SEQ ID NO. 1.

Compared with the prior art, the invention has the beneficial effects that:

the invention provides a single-chain antibody (ScFv) for resisting African swine fever virus, which is coded by a sequence shown in SEQ ID NO. 1. The inventors of the present invention prepared ScFv antibody (VH-VL) against African swine fever virus_κ6) The gene sequence of the antibody is compared and analyzed, and the result shows that the gene sequence of the antibody is basically consistent with the gene sequence of the swine antibody, thereby proving that the constructed gene sequence of the antibody is correct. The prepared ScFv antibody (VH-VL) for resisting African swine fever virus is detected by ELISA technology_κ6) The reaction activity identification is carried out, and the result shows that the prepared ScFv antibody (VH-VL) for resisting African swine fever virus_κ6) Has African swine fever reaction activity. The invention provides a new material for early diagnosis and prevention and control of the African swine fever and provides a new technical support for controlling epidemic spread of the African swine fever as soon as possible.

Drawings

FIG. 1a shows VH gene amplification products;

wherein, M: DL2000DNA marker; 1-2: VH;

FIG. 1b is VL_κA gene amplification product;

wherein, M: DL2000DNA marker; 1: VL_κ；

FIG. 2a shows the amplification product of VH-Linker gene;

wherein, M: DL2000DNA marker; 1: VH-Linker;

FIG. 2b shows the VL kappa-Linker gene amplification product;

wherein, M: DL2000DNA marker; 1-2: VL kappa-Linker;

FIG. 3 shows ScFv (VH-VL)_κ) PCR amplification products;

wherein, M: DL2000DNA marker; 1: VH-VL_κ；

FIG. 4 shows the PCR amplification result of the plasmid for the cloned identification of porcine peripheral blood lymphocyte T-A;

wherein, M: DL2000 relative molecular mass standard; 1-22 plasmid PCR results of monoclonal colonies;

FIG. 5 shows ScFv antibody (VH-VL)_κ6)The heavy chain variable region of the protein is compared with the sequence of the swine source antibody variable region published on the NCBI website;

FIG. 6 shows ScFv antibody (VH-VL)_κ6) The result of sequence comparison between the light chain variable region of the (D) and the pig source antibody variable region sequence published on NCBI website;

FIG. 7 shows ScFv antibody (VH-VL)_κ6) Induced expression of the gene.

Wherein, M: protein molecular mass standard; 1: anti-African swine fever ScFv antibody (VH-VL)_κ6) Expressing the purified product.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.