阅读说明：本技术 一种抗非洲猪瘟病毒的ScFv抗体VH-VLλ11及其制备方法 (ScFv antibody VH-VL lambda 11 for resisting African swine fever virus and preparation method thereof ) 是由 郑海学 王丽娟 �田宏 石正旺 罗俊聪 杨波 张克山 茹毅 李丹 杨帆 于 2020-12-12 设计创作，主要内容包括：本发明涉及一种抗非洲猪瘟病毒的ScFv抗体VH-VLλ11及其制备方法。ScFv抗体的重链可变区的氨基酸序列如SEQ ID NO:1所示,ScFv抗体的轻链可变区的氨基酸序列如SEQ ID NO:2所示。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(DE3)感受态细胞,筛选得到一个针对于非洲猪瘟病毒的ScFv抗体VH-VLλ11,对筛选得到的ScFv抗体VH-VLλ11进行初步活性鉴定发现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支持。(The invention relates to an ScFv antibody VH-VL lambda 11 for resisting African swine fever virus and a preparation method thereof. The amino acid sequence of the heavy chain variable region of the ScFv antibody is shown in SEQ ID NO. 1, and the amino acid sequence of the light chain variable region of the ScFv antibody is shown in SEQ ID NO. 2. The invention separates lymphocytes from peripheral blood of naturally infected African swine fever immune tolerant pigs, extracts and separates total mRNA of the lymphocytes, obtains total cDNA fragments by an RT-PCR method, obtains a swine ScFv antibody gene sequence by an SOE-PCR method under the action of corresponding primers with Linker joints by taking cDNA as a template, constructs the ScFv antibody gene sequence to a PET-30a vector, converts BL21(DE3) competent cells, screens to obtain an ScFv antibody VH-VL lambda 11 aiming at African swine fever viruses, and performs primary activity identification on the screened ScFv antibody VH-VL lambda 11 to find that 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 provides a new technical support for controlling epidemic spread of the African swine fever as soon as possible.)

1. An ScFv antibody VH-VL lambda 11 for resisting African swine fever virus is characterized in that the amino acid sequence of the heavy chain variable region of the ScFv antibody VH-VL lambda 11 is shown as SEQ ID NO. 1, and the amino acid sequence of the light chain variable region of the ScFv antibody VH-VL lambda 11 is shown as SEQ ID NO. 2.

2. A DNA fragment encoding the ScFv antibody against African swine fever virus, VH-VL lambda 11, according to claim 1.

3. The DNA fragment of claim 2, wherein the DNA fragment encoding the heavy chain variable region of ScFv antibody VH-VL λ 11 is represented by SEQ ID NO. 3 and the DNA fragment encoding the light chain variable region of ScFv antibody VH-VL λ 11 is represented by SEQ ID NO. 4.

4. A recombinant expression vector comprising the DNA fragment of claim 2 encoding the anti-African swine fever virus ScFv antibody VH-VL λ 11.

5. A host cell comprising the recombinant expression vector of claim 4.

6. Use of the ScFv antibody against african swine fever virus VH-VL λ 11 of claim 1, the DNA fragment encoding the ScFv antibody against african swine fever virus VH-VL λ 11 of claim 2, the recombinant expression vector of claim 4, or the host cell of claim 5 for the preparation of a diagnostic agent for african swine fever virus.

7. Use of the ScFv antibody against african swine fever virus VH-VL λ 11 of claim 1, the DNA fragment encoding the ScFv antibody against african swine fever virus VH-VL λ 11 of claim 2, the recombinant expression vector of claim 4, or the host cell of claim 5 for the preparation of an african swine fever virus therapeutic agent.

8. Use of the ScFv antibody against african swine fever virus VH-VL λ 11 of claim 1, the DNA fragment encoding the ScFv antibody against african swine fever virus VH-VL λ 11 of claim 2, the recombinant expression vector of claim 4 or the host cell of claim 5 for the preparation of an epitope-investigative product.

9. The method for preparing an ScFv antibody VH-VL lambda 11 against African swine fever virus according to claim 1, comprising the steps of:

(1) lymphocyte separation:

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

(2) extraction of total mRNA from lymphocytes:

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

(3) and (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; w1 and Q1 are primers to carry out the first round of amplification, and the amplification procedures are respectively as follows: 2min at 98 ℃, 10s at 98 ℃, 5s at 52 ℃, 30s at 72 ℃ and 30 cycles, 10min at 72 ℃; carrying out electrophoresis on the amplification product for 30 cycles of 98 ℃ for 2min, 98 ℃ for 10s, 53 ℃ for 5s, 72 ℃ for 30s and 72 ℃ for 10min, purifying and collecting a target product, and storing at-20 ℃ for later use;

(5) and (3) second round amplification:

taking the first round amplification product as a template, and taking P1 and R2 as templates; w2 and Q1 are primers to carry out the second round of amplification, and the amplification procedures are respectively as follows: 2min at 98 ℃, 10s at 98 ℃, 5s at 56 ℃, 30s at 72 ℃ and 35 cycles, 10min at 72 ℃; performing electrophoresis at 98 ℃ for 2min, 98 ℃ for 10s, 68 ℃ for 30s, 35 cycles, and 72 ℃ for 10min, purifying and collecting a target product from gel by using a gel recovery kit, and storing at-20 ℃ for later use;

(6) third round of amplification

And (3) performing third round amplification by using the second round amplification product as a template and using P1 and Q1 as primers, wherein the amplification procedure is as follows:no primer: 2min at 98 ℃, 10s at 98 ℃, 30s at 68 ℃, 10 cycles, 10min at 72 ℃;comprises the following primers: 2min at 98 ℃, 10s at 98 ℃, 5s at 55 ℃, 50s at 72 ℃ and 35 cycles, 10min at 72 ℃; performing electrophoresis on the amplification product, purifying and collecting a target product from the gel by using a gel recovery kit, and storing at-20 ℃ for later use;

(7) T-A clone identification

: treating the third round of amplification product by adding ' A ' to the 3 ' end, wherein the reaction program is 65 ℃ for 10 min;

: attaching A-plus product to PMD^TM20-T carrier, and the connection program is 16 ℃ for 30 min;

: conversion of the ligation product toETransforming 10 muL of connecting products by 100 muL of competent cells in the competence of coli JM109, and uniformly coating transformed bacteria liquid on LB-A⁺On a flat plate, carrying out inverted culture at 37 ℃ for 8-16h until a monoclonal antibody appears, and picking a 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 VH-VL lambda 11:

preparing BL21 competent cells, converting 1 muL of ScFv-PET-30a plasmid constructed by correctly sequenced amplification products every 50 muL of competent cells, and uniformly coating the converted bacterial liquid on LB-K⁺On a flat plate, carrying out inverted culture at 37 ℃ for 8-16h until a monoclonal antibody appears, and picking a monoclonal antibody colony on LB-K⁺Inducing and expressing the antibody protein in a liquid culture medium, and collecting, washing and purifying the expressed antibody protein to obtain the anti-African swine fever ScFv antibody VH-VL lambda 11.

10. The method for preparing ScFv antibody VH-VL lambda 11 against African swine fever virus according to claim 9,

the lymphocyte separation is carried out according to the following steps:

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

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

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

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an ScFv antibody VH-VL lambda 11 for resisting African swine fever virus and a preparation method thereof.

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 a key effector molecule in humoral immunity and is also a core component in vaccines and diagnostic reagents, and the development of the antibody plays a crucial 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 invention patent of China (publication number CN 110078819A) is an antibody against African swine fever and a preparation method thereof, the antibody is obtained by immunizing healthy female poultry with African swine fever virus, collecting egg yolk when the content of the African swine fever antibody in the egg yolk of the immunized poultry exceeds 40ng/mL, extracting water-soluble components in the egg yolk and purifying. The antibody obtained in the patent is an avian African swine fever antibody, has a heterologous reaction, and does not overcome the problem of large molecular weight of the conventional antibody.

Single-chain antibodies have attracted considerable attention as one of the third-generation antibodies (engineered antibodies). 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, the conventional antibody cannot meet the requirements of the current antibody market, and there is an urgent need for a flexible antibody which is easy and convenient to prepare, has strong specificity and high stability, and has a small molecular weight, and can be efficiently applied to a specific site. 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.

Chinese invention patent (publication No. CN 111040031A) and Chinese invention patent (publication No. CN 111560069A) both disclose an ScFv antibody against African swine fever virus, but the light chains of the two antibodies are kappa light chains. The prior art does not disclose ScFv antibodies against African swine fever virus, in which the light chain is other than kappa light chain.

Disclosure of Invention

The invention provides an African swine fever virus specific ScFv antibody VH-VL lambda 11 for resisting African swine fever virus and a preparation method thereof.

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

in a first aspect, the invention provides an ScFv antibody VH-VL lambda 11 for resisting African swine fever virus, wherein the amino acid sequence of the heavy chain variable region of the ScFv antibody VH-VL lambda 11 is shown as SEQ ID NO. 1, and the amino acid sequence of the light chain variable region of the ScFv antibody VH-VL lambda 11 is shown as SEQ ID NO. 2.

SEQ ID NO:1

MEVKLVESGGGLVQPGGSLRLSCVGSEYEFRGSLIHWVRQAPGKGLEWLATICTTDPITEYADSVKGRFTISRDSSQNTAYLQMDSLRGEDTARYYCGVAFSYGGGCHRMDLRGQGVEVVV；

SEQ ID NO:2：

MSQTVIQEPAMSVSPGGTVTLTCAFSSGSESTRKYPIRFQQTPGQPPRLLIYHTNSRPTGVPSRFSGAISGNKAALTITGAQAEDEADYFCSLYIRTYNVPFGGGTHLTVL。

In a second aspect, the present invention provides a DNA fragment encoding the heavy chain variable region of ScFv antibody VH-VL λ 11 and a DNA fragment encoding the light chain variable region of ScFv antibody VH-VL λ 11.

Preferably, the DNA fragment encoding the heavy chain variable region of ScFv antibody VH-VL λ 11 is shown in SEQ ID NO:3, and the DNA fragment encoding the light chain variable region of ScFv antibody VH-VL λ 11 is shown in SEQ ID NO: 4.

SEQ ID NO:3：

5’-ATGGAGGTGAAGCTGGTGGAGTCTGGAGGAGGCCTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGTCGGCTCTGAATACGAATTCCGTGGATCCTTGATTCACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGCTTGCAACTATCTGTACTACTGATCCTATAACGGAATACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAGCTCCCAGAACACGGCCTATCTGCAAATGGACAGCCTGAGAGGCGAAGACACGGCCCGCTATTACTGTGGAGTTGCCTTCAGCTATGGGGGGGGCTGTCATCGAATGGATCTCAGGGGCCAAGGCGTTGAAGTCGTCGTG-3’；

SEQ ID NO:4：

5’-ATGTCTCAGACTGTGATCCAGGAGCCGGCGATGTCAGTGTCTCCTGGAGGGACCGTCACACTCACCTGTGCCTTTAGCTCAGGGTCAGAATCTACTAGGAAGTACCCCATTAGGTTCCAGCAGACACCAGGCCAGCCTCCCCGACTGCTGATCTACCACACCAATAGCCGCCCGACTGGGGTCCCCAGTCGCTTCTCTGGAGCCATCTCTGGGAACAAAGCCGCCCTCACTATCACGGGGGCCCAGGCTGAGGACGAGGCCGACTATTTCTGTAGTCTCTATATAAGGACTTATAATGTTCCCTTCGGCGGTGGGACCCATCTGACCGTCCTCG-3’。

In a third aspect, the present invention provides a recombinant expression vector comprising a DNA fragment encoding the heavy chain variable region of ScFv antibody VH-VL λ 11 and a DNA fragment encoding the light chain variable region of ScFv antibody VH-VL λ 11.

In a fourth aspect, the present invention provides a host cell comprising the above recombinant expression vector.

In a fifth aspect of the invention, a method for preparing an ScFv antibody VH-VL lambda 11 against African swine fever virus is provided, which comprises the following steps:

(1) lymphocyte separation:

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

(2) extraction of total mRNA from lymphocytes:

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

(3) and (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; w1 and Q1 are primers to carry out the first round of amplification, and the amplification procedures are respectively as follows: 2min at 98 ℃, 10s at 98 ℃, 5s at 52 ℃, 30s at 72 ℃ and 30 cycles, and 10min at 72 ℃; 2min at 98 ℃, 10s at 98 ℃, 5s at 53 ℃, 30s 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’-CACGACGACTTCAACGCCTGGG-3’，

R1:5’-GAGGWGAAGCTGGTGGAGTCTG-3’，

W1:5’-CGAGGACGGTCAGATGGGTC-3’，

Q1:5’-TCTCAGACTGTGATCCAGGAGC-3’。

(5) and (3) second round amplification:

taking the first round amplification product as a template, and taking P1 and R2 as templates; w2 and Q1 are primers to carry out the second round of amplification, and the amplification procedures are respectively as follows: 2min at 98 ℃, 10s at 98 ℃, 5s at 56 ℃, 30s at 72 ℃ and 35 cycles, and 10min at 72 ℃; 2min at 98 ℃, 10s at 98 ℃, 30s at 68 ℃ and 35 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’-GCCGCCTGACCCTCCGCCACCGAGGWGAAGCTGGTGGAGTCTG-3’，W2:5’-GGTGGCGGAGGGTCAGGCGGCCGAGGACGGTCAGATGGGTC-3’。

(6) and a third round of amplification:

and (3) performing third round amplification by using the second round amplification product as a template and using P1 and Q1 as primers, wherein the amplification procedure is as follows: firstly, no primer: 2min at 98 ℃, 10s at 98 ℃, 30s at 68 ℃ and 10 cycles, and 10min at 72 ℃. ② there is primer: 2min at 98 ℃, 10s at 98 ℃, 5s at 55 ℃, 50s at 72 ℃ and 35 cycles, and 10min at 72 ℃. 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) And (3) identifying T-A clone:

i: treating the third round of amplification product by adding ' A ' to the 3 ' end, wherein the reaction program is 65 ℃ for 10 min;

II: attaching A-plus product to PMD^TM20-T carrier, and the connection procedure is 16 ℃ (metal bath) for 30 min;

III: coli JM109 was transformed with the ligation products, 10. mu.L of the ligation products were transformed with 100. mu.L of competent cells, and the transformed bacterial suspension was uniformly spread on LB-A⁺On a flat plate, carrying out inverted culture at 37 ℃ for 8-16h until a monoclonal antibody appears, and picking a 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 VH-VL lambda 11:

BL21(DE3) 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 flat plate, carrying out inverted culture at 37 ℃ for 8-16h until a monoclonal antibody appears, and picking a monoclonal antibody colony on LB-K⁺Inducing and expressing the antibody protein in a liquid culture medium, and collecting, washing and purifying the expressed antibody protein to obtain the anti-African swine fever ScFv antibody VH-VL lambda 11.

In the present invention, preferably, the lymphocyte separation is performed according to the following steps:

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

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

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

In the present invention, preferably, the extraction of total mRNA of lymphocytes is performed 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.

In the present invention, preferably, 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.

In the present invention, preferably, the first round of amplification is performed by sequentially adding the following components to a 0.2mL PCR amplification tube:

①:

reagent material	Volume of
		5 XPCR buffer	10μL
dNTP Mixture	4μl
		full-Length cDNA	2.5μl
P1 primer (10 pmol/. mu.L)	1.5μl
		R1 primer (10 pmol/. mu.L)	1.5μl
DNA polymerase mixture	0.5μL
		DEPC water	30μl
Total volume	50μl

②:

Reagent material	Volume of
		5 XPCR buffer	10μL
dNTP Mixture	4μl
		full-Length cDNA	2.5μl
W1 primer (10 pmol/. mu.L)	1.5μl
		Q1 primer (10 pmol/. mu.L)	1.5μl
DNA polymerase mixture	0.5μL
		DEPC water	30μl
Total volume	50μl

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

In the present invention, preferably, the second round of amplification is performed by sequentially adding the following components to a 0.2mL PCR amplification tube:

①:

②:

reagent material	Volume of
		5 XPCR buffer	10μL
dNTP Mixture	4μl
		W2 primer (10 pmol/. mu.L)	1.5μl
Q1 primer (10 pmol/. mu.L)	1.5μl
		First round amplification product②	0.5μl
DNA polymerase mixture	0.5μL
		DEPC water	32μl
Total volume	50μl

And (4) carrying out second round amplification under the amplification procedure in the step (5), carrying out electrophoresis on the amplification product, purifying and collecting the target product from the gel by using a gel recovery kit, and storing at-20 ℃ for later use.

In the present invention, preferably, the third round of amplification is performed by sequentially adding the following components to a 0.2mL PCR amplification tube:

①:

reagent material	Volume of
		5 XPCR buffer	10μL
dNTP Mixture	4μl
		Second round amplification products①	1μl
Second round amplification products②	1μl
		DNA polymerase mixture	0.5μL
DEPC water	31.5μl
		Total volume	48μl

② supplementary primers

Reagent material	Volume of
		P1 primer (10 pmol/. mu.L)	1μL
Q1 primer (10 pmol/. mu.L)	1μL

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

In the present invention, preferably, the T-A clone identification is performed by performing I, II 2 procedures in sequence in a 0.2mL PCR amplification tube, and performing procedure III in a 1.5mL centrifuge tube:

I

reagent material	Volume of
		Third round of amplification products	8μl
10×Buffer	1μL
		dATP Mixture	0.5μl
A-overhang enzyme	0.5μl
		Total volume	10μl

II

Reagent material	Volume of
		I addition of A product	2μl
PMDTM20-T vector	1μL
		DEPC water	2μl
Ligation Mighty Mix	5μl
		Total volume	10μl

III

Reagent material	Volume of
		II all ligation products	10μl
E.coli JM109	100μL

In the sixth aspect, the invention provides an application of ScFv antibody VH-VL lambda 11 resisting African swine fever virus, a DNA fragment encoding ScFv antibody VH-VL lambda 11, a recombinant expression vector or a host cell in preparation of an African swine fever virus diagnostic reagent, an African swine fever virus therapeutic reagent or an antigen epitope research product.

The invention has the following beneficial effects:

the invention adopts ELISA detection technology and indirect immunofluorescence assay (IFA) to perform reactivity identification on the ScFv antibody VH-VL lambda 11 of the anti-African swine fever virus, and the result shows that the ScFv antibody VH-VL lambda 11 of the anti-African swine fever virus has African swine fever reactivity.

The ScFv antibody VH-VL lambda 11 of the invention for resisting African swine fever virus has the African swine fever reaction activity which is obviously higher than that of VH-VL kappa, as shown in figure 6OD of reaction product of ScFv antibody VH-VL. lamda.11 and African swine fever holovirus_450nmThe value can reach 1.12, and the OD of the reactant of ScFv antibody VH-VL lambda 11 and African swine fever virus P30 protein_450nmThe value is about 2.95.

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 is a first round of amplification of VH agarose gel electrophoresis of porcine peripheral blood lymphocyte ds cDNA shown as 1 a; m: DL2000 relative molecular mass standard; 1-2: a first round of amplification of VH products;

1b is a first round of amplification VL lambda agarose gel electrophoresis of porcine peripheral blood lymphocyte ds cDNA; m: DL2000 relative molecular mass standard; 1-2: first round amplification of VL λ product.

FIG. 2a is a diagram of agarose gel electrophoresis of a second round of amplification of VH-Linker from porcine peripheral blood lymphocyte ds cDNA; m: DL2000 relative molecular mass standard; 1: a destination strip;

2b is the agarose gel electrophoresis image of a second round of amplification of VL lambda-Linker from porcine peripheral blood lymphocyte ds cDNA. M: DL2000 relative molecular mass standard; 1-2: the destination strip.

FIG. 3 is a third round of amplified VH-VL lambda agarose gel electrophoresis of porcine peripheral blood lymphocyte ds cDNA; m: DL2000 relative molecular mass standard; 1: the destination strip.

FIG. 4 shows the PCR amplification result of the plasmid for the cloned identification of porcine peripheral blood lymphocyte T-A; m: DL2000 relative molecular mass standard; 1-22: plasmid PCR results of monoclonal colonies.

FIG. 5 shows the specific protein bands expressed by VH-VL λ 11 gene. M: protein molecular mass standard, 1, 2: the anti-African swine fever ScFv antibody VH-VL lambda 11 expresses a purified product.

FIG. 6 is a diagram showing the result of detecting African swine fever virus complete virus by using the product expressed by VH-VL lambda 11 gene. Positive represents VH-VL lambda 11 coated plate + ASFV + Positive serum + secondary pig antibody, while Negative represents VH-VL lambda 11 coated plate + ASFV + Negative serum + secondary pig antibody; blank control represents VH-VL lambda 11 coated plates + ASFV + serum diluent + porcine secondary antibody, and Positive control represents VH-VL lambda 11 coated plates + ASFV + serum diluent + P30-HRP.

FIG. 7 is a diagram showing the results of detecting the expression product of VH-VL lambda 11 gene with ASFV inactivated antigen. ScFv represents ASFV-envelope plate + VH-VL. lamda.11 + HIS secondary antibody; negative control indicates ASFV coated plate + diluent + HIS secondary antibody.

FIG. 8 is a graph showing the results of detection of ASFV-p30 protein by the expression product of VH-VL lambda 11 gene. ScFv represents ASFV-p30 protein coated plate + VH-VL lambda 11+ HIS secondary antibody, Negative control represents ASFV-p30 protein coated plate + diluent + HIS secondary antibody.

FIG. 9 depicts IFA identification of ScFv antibody VH-VL. lamda.11 activity. ASFV + represents ASFV-vaccinated group, ASFV-represents control group; DAPI is a cell nucleus staining result graph, Alexa Fluor 555 is a commercial product, represents fluorescent dye and a wavelength channel thereof, and represents red fluorescence; merge represents a combined graph.

Detailed Description

The present invention is described in detail below with reference to specific examples, but the scope of the present invention is not limited to the following examples, and any technical solutions that can be conceived by those skilled in the art based on the present invention and the common general knowledge in the art are within the scope of the present invention.

Preparation of anti-African swine fever virus ScFv antibody VH-VL lambda 11

1. Materials and methods

1.1 materials

1.1.1 test animals

An African swine fever immune-tolerant pig (5 months, from a certain pig farm in New county in Henan).

1.1.2 strains and reagents

LTS1110 porcine peripheral blood lymphocyte isolate Kit was purchased from Tianjin tertiary-ocean biologicals, Inc.; PMD^TM20-T vector, E.coli JM109 competent,HS DNA Polymerase, RNase-free Water, DL2,000 DNA Marker, 6 × Loading Buffer were purchased from Takara Bio Inc.; unstanated protein MW marker (26610) was purchased from Thermo Fisher Scientific(ii) a BL21(DE3) was competently purchased from Beijing Quanyu gold; trizol, nickel affinity chromatography resin, DNA fragment recovery kit and plasmid extraction kit are OMEGA products; molecular biology reagents were from Sigma; other biochemical reagents are all made in China and analyzed to be pure.

1.2 methods

1.2.1 lymphocyte isolation

Collecting 100mL of anticoagulated blood of a pig by using a jugular vein, adding a whole blood diluent in equal amount, and uniformly mixing; adding lymphocyte separation liquid in a volume ratio of 5:9, slowly adding diluted anticoagulation blood, and performing horizontal centrifuge at 1800rpm for 20min at 18-22 ℃; carefully collecting lymphocyte layer in a new collecting tube, diluting the lymphocyte layer by 5 times of cell washing liquid, and horizontally centrifuging at 1500rpm at 18-22 ℃ for 10 min; discarding the supernatant, diluting the precipitate with a cell washing solution, and centrifuging by a horizontal centrifuge at 1500rpm, 18-22 ℃ for 10 min; discarding supernatant, precipitating to obtain separated lymphocyte, collecting supernatant of suspension cell, counting, suspending in RNA preservation solution (RNAlater), and storing at-70 deg.C.

1.2.2 extraction of Total mRNA from lymphocytes

Sucking 400ul of the above lymphocyte fluid, adding 1mLTRIzol, mixing, standing at 4 deg.C for 5 min; adding 250ul of chloroform, mixing, standing at 4 deg.C for 10min, 12000r/min, and centrifuging at 4 deg.C for 15 min; sucking 450ul of supernatant, adding equivalent isopropanol, standing at-20 deg.C for 30min, 12000r/min, and 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; 25ul of RNase-free water-soluble precipitate was the Total mRNA harvested, and the Total mRNA concentration was 356ng/ul measured by ND2000, with a purity OD260/OD280 of 1.87.

1.2.3 design of primers

First round amplification primers:

P1:5’-CACGACGACTTCAACGCCTGGG-3’，

R1:5’-GAGGWGAAGCTGGTGGAGTCTG-3’，

W1:5’-CGAGGACGGTCAGATGGGTC-3’，

Q1:5’-TCTCAGACTGTGATCCAGGAGC-3’。

wherein, the primers P1 and R1 are used for amplifying VH sequence, and the primers W1 and Q1 are used for amplifying VL lambda sequence.

Second round amplification primers:

P1:5’-CACGACGACTTCAACGCCTGGG-3’，

R2:5’-GCCGCCTGACCCTCCGCCACCGAGGWGAAGCTGGTGGAGTCTG-3’，

W2:5’-GGTGGCGGAGGGTCAGGCGGCCGAGGACGGTCAGATGGGTC-3’。

Q1:5’-TCTCAGACTGTGATCCAGGAGC-3’。

wherein, the primers P1 and R2 are used for amplifying VH-Linker sequences, and the primers W2 and Q1 are used for amplifying VL lambda-Linker sequences.

Third round of amplification primers:

P1:5’-CACGACGACTTCAACGCCTGGG-3’，

Q1:5’-TCTCAGACTGTGATCCAGGAGC-3’。

primers P1, Q1 were used to amplify VH-VL λ sequences.

1.2.4 cDNA Synthesis of double strands and purification

1.2.4.1 Total Gene cDNA Synthesis

The following components were added sequentially to a 0.2mL PCR amplification tube:

reagent material	Volume of
		DEPC water	4.5μL
5×First strand Buffer	4μL
		DTT(0.1M)	2μL
Oligo(dT)	1μL
		RRI	1μL
dATP	1μL
		Radom primers	0.5μL
M-MLV reverse transcription (200u/ul)	1μL
		mRNA	5μL
Total volume	20μL

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.

1.2.4.2 first round amplification

The following components are respectively and sequentially added into a 0.2mL PCR amplification tube:

amplification of VH sequences:

reagent material	Volume of
		5 XPCR buffer	10μL
dNTP Mixture	4μl
		full-Length cDNA	2.5μl
P1 primer (10 pmol/. mu.L)	1.5μl
		R1 primer (10 pmol/. mu.L)	1.5μl
DNA polymerase mixture	0.5μL
		DEPC water	30μl
Total volume	50μl

Amplification of VL Lambda sequences

Reagent material	Volume of
		5 XPCR buffer	10μL
dNTP Mixture	4μl
		full-Length cDNA	2.5μl
W1 primer (10 pmol/. mu.L)	1.5μl
		Q1 primer (10 pmol/. mu.L)	1.5μl
DNA polymerase mixture	0.5μL
		DEPC water	30μl
Total volume	50μl

The amplification procedures were: 2min at 98 ℃, 10s at 98 ℃, 5s at 52 ℃, 30s at 72 ℃ and 30 cycles, and 10min at 72 ℃; secondly, the step of: 2min at 98 ℃, 10s at 98 ℃, 5s at 53 ℃, 30s at 72 ℃ and 30 cycles, and 10min at 72 ℃. And (4) carrying out electrophoresis on the amplification product, purifying and collecting a target product, and storing at-20 ℃ for later use.

1.2.4.3 second round amplification

The following components are respectively and sequentially added into a 0.2mL PCR amplification tube:

amplification of VH-Linker sequence

Reagent material	Volume of
		5 XPCR buffer	10μL
dNTP Mixture	4μl
		P1 primer (10 pmol/. mu.L)	1.5μl
R2 primer (10 pmol/. mu.L)	1.5μl
		First round amplification product①	0.5μl
DNA polymerase mixture	0.5μL
		DEPC water	32μl
Total volume	50μl

② for amplifying VL lambda-Linker sequences

The amplification procedures were as follows: the method comprises the following steps: 2min at 98 ℃, 10s at 98 ℃, 5s at 56 ℃, 30s at 72 ℃ and 35 cycles, and 10min at 72 ℃; secondly, the step of: 2min at 98 ℃, 10s at 98 ℃, 30s at 68 ℃ and 35 cycles, and 10min at 72 ℃. Performing electrophoresis on the amplification product, purifying and collecting a target product from the gel by using a gel recovery kit, and storing at-20 ℃ for later use;

1.2.4.4 third round of amplification

The following components were added sequentially to a 0.2mL PCR amplification tube:

pre-splicing of VH-VL lambda without primers

Reagent material	Volume of
		5 XPCR buffer	10μL
dNTP Mixture	4μl
		Second round amplification products①	1μl
Second round amplification products②	1μl
		DNA polymerase mixture	0.5μL
DEPC water	31.5μl
		Total volume	48μl

Amplification of VH-VL. lamda.

Reagent material	Volume of
		P1 primer (10 pmol/. mu.L)	1μL
Q1 primer (10 pmol/. mu.L)	1μL

The amplification procedures were as follows: firstly, no primer: 2min at 98 ℃, 10s at 98 ℃, 30s at 68 ℃ and 10 cycles, and 10min at 72 ℃. ② there is primer: 2min at 98 ℃, 10s at 98 ℃, 5s at 55 ℃, 50s at 72 ℃ and 35 cycles, and 10min at 72 ℃. And (3) carrying out electrophoresis on the amplified product, purifying and collecting a target product from the gel by using a gel recovery kit, namely the amplified target product VH-Linker-VL lambda 6 (VH-VL lambda 6 for short), and storing at-20 ℃ for later use.

1.2.5T-A clone identification

The following 2 procedures were performed in 0.2mL PCR amplification tubes followed by procedure III in 1.5mL centrifuge tubes:

i third round of amplification product sequence followed by "A"

II I addition of A product to T vector

Reagent material	Volume of
		I addition of A product	2μl
PMDTM20-T vector	1μL
		DEPC water	2μl
Ligation Mighty Mix	5μl
		Total volume	10μl

III transformation

II all ligation products	10μl
		E.coli JM109	100μL

The above procedures are respectively I: 10min at 65 ℃; II: 16 deg.C (metal bath) for 30 min; III: uniformly coating the transformed bacterial liquid on LB-A⁺On a flat plate, carrying out inverted culture at 37 ℃ for 8-16h until a monoclonal antibody appears, and picking a monoclonal antibody colony on LB-A⁺Culturing in liquid culture medium, extracting plasmid, and sequencing.

1.2.6 alignment of Gene sequences

And (3) comparing the sequencing result with a heavy chain variable region sequence and a light chain lamda chain of the porcine antibody released on an NCBI website by using DNA star as comparison software, and after the comparison is finished, sending the correct gene sequence to ScFv-PET-30a plasmid construction by Wuhan Kingkurui company to obtain a VH-VL lambda 11-PET-30a plasmid, wherein the VH-VL lambda 11 is positioned between NdeI and XhoI enzyme cutting sites of the PET-30 a.

1.2.7 construction and preparation of antibodies

BL21(DE3) competent cells were prepared, 1. mu.L of VH-VL. lamda.11-PET-30 a plasmid was transformed into 50. mu.L of competent cells, and the transformed bacterial suspension was uniformly spread 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 (3) inducing and expressing the antibody protein in a liquid culture medium at 37 ℃, and collecting, washing and purifying the expressed antibody protein to obtain the anti-African swine fever ScFv antibody VH-VL lambda 11.

1.2.8 ELISA for identification of antibody Activity

1.2.8.1

Taking a purified ScFv antibody VH-VL lambda 11(0.18mg/mL), coating a detection plate according to the proportion of 1:4, adding 50 mu L of inactivated African swine fever virus antigen after coating, incubating in an incubator at 37 ℃ for 30min, washing with 250 mu L of PBST washing liquid for 4 times, patting dry, respectively adding 50 mu L of African swine fever positive serum 2 holes diluted by 1:5, healthy swine serum 2 holes diluted by 1:5 and serum diluent 4 holes, incubating in the incubator at 37 ℃ for 30min, washing with 250 mu L of PBST washing liquid for 4 times, patting dry, adding 50 mu L of HRP-labeled secondary pig antibody diluted by 1:15000 into the first 6 holes, adding 50 mu L of P30-HRP diluted by 1:15000 into the second 2 holes, incubating in the incubator at 37 ℃ for 30min, washing with 250 mu L of PBST washing liquid for 4 times, drying, adding 50 mu L of TMB substrate solution, incubating in the incubator at 37 ℃ for 15min, adding 50 mu L of stop solution (H2SO4), the OD450nm values of the reactions were read on a spectrophotometer.

1.2.8.2

Coating ASFV inactivated antigen 1:2 with a detection plate, respectively adding 50 μ L VH-VL λ 11 antibody protein (0.4mg/mL) diluted 1:8 and diluent, incubating at 37 deg.C for 30min, washing with 250 μ L PBST cleaning solution for 4 times, patting to dry, adding 50 μ L rabbit anti-pig HIS-HRP diluted 1:5000, incubating at 37 deg.C for 30min, washing with 250 μ L PBST cleaning solution for 4 times, patting to dry, adding 50 μ L TMB substrate solution, incubating at 37 deg.C for 15min, adding 50 μ L stop buffer (H)₂SO₄) Reading the OD of the reaction on a spectrophotometer_450nmThe value is obtained.

1.2.8.3

Taking maltose-tagged P30 protein 1: 400 coating detection plate, after coating, respectively adding 50 μ L of VH-VL λ 11 antibody protein (0.4mg/mL) diluted 1:16, diluent, incubating at 37 deg.C for 30min, washing with 250 μ L of PBST washing solution for 4 times, patting to dry, adding 50 μ L of rabbit anti-swine HIS-HRP diluted 1:5000, incubating at 37 deg.C for 30min, washing with 250 μ L of PBST washing solution for 4 times, patting to dry, adding 50 μ L of TMB substrate solution, incubating at 37 deg.C for 15min, and adding 50 μ L of stop solution (H)₂SO₄) Reading the OD of the reaction on a spectrophotometer_450nmThe value is obtained.

1.2.8.4

Taking maltose-tagged P30 protein 1: 40 coatingDetecting plate, after coating is finished, 1: gradient adding ASFV positive serum, ASFV negative serum and serum diluent into a column at 4, incubating at 37 deg.C for 30min, washing with 250 μ L PBST washing solution for 4 times, patting to dryness, adding 1:80 diluted VH-VL λ 11 protein (0.4mg/mL), incubating at 37 deg.C for 30min, washing with 250 μ L PBST washing solution for 4 times, patting to dryness, adding 50 μ L rabbit anti-swine HIS-HRP diluted at 1:5000, incubating at 37 deg.C for 30min, washing with 250 μ L PBST washing solution for 4 times, patting to dryness, adding 50 μ L TMB substrate solution, incubating at 37 deg.C for 15min, adding 50 μ L stop buffer (H)₂SO₄) Reading the OD of the reaction on a spectrophotometer_450nmThe value is obtained.

1.2.9 IFA identification of antibody Activity

Inoculating porcine PAMS cells with ASFV, setting up a negative control at the same time, fixing with 4% paraformaldehyde for 30min, washing with PBS for three times, penetrating the membrane for 15min at room temperature with 0.1% Triton-100, washing with PBS for three times, sealing with 5% Blocker BSA for 15min at room temperature, adding VH-VL lambda 11 Antibody (1:200), incubating overnight at 4 ℃, washing with PBS for three times, 6x-His Tag Monoclonal Antibody (HIS. H8), and Alexa Fluor 555(1:60), dyeing in the dark at room temperature for 1h, washing with PBS for three times, adding DAPI to stain nuclei for 10min, washing with PBS for three times, and observing by using a mirror.

2. Results

2.1 agarose gel electrophoresis of cDNA Synthesis duplexes

The ds cDNA product from the first round of amplification was electrophoresed to collect a 300bp band. And (4) taking the ds cDNA product amplified in the second round for electrophoresis, and collecting a band about 300 bp. The third round of amplified ds cDNA product was electrophoresed to collect a band of about 800 bp. As shown in fig. 1, 2 and 3.

2.2 plasmid PCR results after cloning of T-A

PMD to be connected^TM20-T-A is used for carrying out plasmid PCR identification, plasmids with the gene size of about 800bp are selected and sequenced, and the result of the plasmid PCR identification is shown in figure 4.

14 monoclonals are selected from VH-Linker-VL lambda, and after sequencing, the sequences of the monoclonals are basically consistent with the framework regions of the pig immunoglobulin variable region sequences, and the CDR regions are different and accord with the gene structure of the pig light and heavy chain variable regions, wherein the VH part is 342 bp-375 bp, the VL lambda part is 331 bp-346 bp, and the Linker base sequences between the heavy chain and the light chain are all correct.

Wherein, the size of VH-Linker-VL lambda 11 is 720bp, the nucleotide sequence is shown as follows, and the sequence shown by underlining is the Linker sequence:

5’-ATGGAGGTGAAGCTGGTGGAGTCTGGAGGAGGCCTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGTCGGCTCTGAATACGAATTCCGTGGATCCTTGATTCACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGCTTGCAACTATCTGTACTACTGATCCTATAACGGAATACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAGCTCCCAGAACACGGCCTATCTGCAAATGGACAGCCTGAGAGGCGAAGACACGGCCCGCTATTACTGTGGAGTTGCCTTCAGCTATGGGGGGGGCTGTCATCGAATGGATCTCAGGGGCCAAGGCGTTGAAGTCGTCGTGGGTGGC GGAGGGTCAGGCGGCATGTCTCAGACTGTGATCCAGGAGCCGGCGATGTCAGTGTCTCCTGGAGGGACCGTCACACTCACCTGTGCCTTTAGCTCAGGGTCAGAATCTACTAGGAAGTACCCCATTAGGTTCCAGCAGACACCAGGCCAGCCTCCCCGACTGCTGATCTACCACACCAATAGCCGCCCGACTGGGGTCCCCAGTCGCTTCTCTGGAGCCATCTCTGGGAACAAAGCCGCCCTCACTATCACGGGGGCCCAGGCTGAGGACGAGGCCGACTATTTCTGTAGTCTCTATATAAGGACTTATAATGTTCCCTTCGGCGGTGGGACCCATCTGACCGTCCTCG-3’。

2.3 ScFv antibody Gene sequence alignment

And (3) comparing the gene sequence suitable for sequencing with the swine antibody variable region sequence published on the NCBI website.

2.3.1 VH sequence alignment

VH sequence of porcine IGG:

EENLVESGGGLVQPGGSLRLSCVGSGFTSSSYGMWVRQAPGKGLEWLACIYSSGSRTYYADSVKGRCTISRDNSQNTAYLQMDSLRTEDTAHYYCVTGDASWCPFRKVHLWGPGVEVVVSS。

VH sequence of VH-VL λ 11:

MEVKLVESGGGLVQPGGSLRLSCVGSEYEFRGSLIHWVRQAPGKGLEWLATICTTDPITEYADSVKGRFTISRDSSQNTAYLQMDSLRGEDTARYYCGVAFSYGGGCHRMDLRGQGVEVVV。

2.3.2 VL Lambda sequence alignment

VL λ sequence of porcine IGG:

VIQEPAMSVSLGGTVTLTCAFSSGSVTSSDYPGWFQQTPGQPPRTVIYSTNSRPTGVPSRFSGAISGNKATLTITGAQAEDEADYFCALRKSGGTVTFGGGTHVTVL。

VL λ sequence of VH-VL λ 11:

MSQTVIQEPAMSVSPGGTVTLTCAFSSGSESTRKYPIRFQQTPGQPPRLLIYHTNSRPTGVPSRFSGAISGNKAALTITGAQAEDEADYFCSLYIRTYNVPFGGGTHLTVL。

2.4 expression of the ScFv antibody VH-VL. lamda.11 Gene

After induction at 37 ℃ 1 specific protein band of 27kDa appeared, which is in agreement with the expectation, as shown in FIG. 5. And when the concentration of the inducer is 1/1000 (the final concentration is 0.1mM) and the induction time is 6h, the expression level reaches a peak.

2.5 ELISA identification of ScFv antibody VH-VL. lamda.11 Activity

2.5.1

The VH-VL lambda 11 gene expression product is coated on a detection plate and detected according to the program of 1.2.8.1, and the detection result shows that the expressed product can be specifically combined with the African swine fever holovirus antigen, as shown in figure 6. And most likely to be directed to the P30 site.

2.5.2

The ASFV inactivated antigen is coated on a detection plate and detected according to the program 1.2.8.2, and the detection result shows that the ASFV and the expression product are specifically combined, as shown in figure 7.

2.5.3

ASFV-P30 protein is coated on a detection plate and detected according to the program 1.2.8.3, and the detection result shows that the ASFV-P30 is specifically combined with the expression product, as shown in figure 8.

2.5.4

The concentration of the P30 protein is fixed, and the detection is carried out according to the program of 1.2.8.4, and the detection result shows that the VH-VL lambda 11 antibody has a blocking effect.

TABLE 1 blocking ELISA identification of VH-VL λ 11 proteins

	VH-VLλ11	Negative control
			Blocking rate	72.66％	29.1％

2.6 IFA identification of ScFv antibody VH-VL. lamda.11 Activity

Fluorescent staining of VH-VL. lamda.11 Antibody with 6X-His Tag Monoclonal Antibody (HIS. H8), Alexa Fluor 555, showed that VH-VL. lamda.11 could react with ASFV in cytoplasm, as shown in FIG. 9.

The above embodiments are merely preferred embodiments of the present invention, and not intended to limit the scope of the invention, so that equivalent changes or modifications made based on the structure, characteristics and principles of the invention should be included in the claims of the present invention.

SEQUENCE LISTING

<110> Lanzhou veterinary research institute of Chinese academy of agricultural sciences

<120> ScFv antibody VH-VL lambda 11 for resisting African swine fever virus and preparation method thereof

<130> do not

<160> 10

<170> PatentIn version 3.5

<210> 1

<211> 121

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 1

Met Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Val Gly Ser Glu Tyr Glu Phe Arg Gly

20 25 30

Ser Leu Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

35 40 45

Leu Ala Thr Ile Cys Thr Thr Asp Pro Ile Thr Glu Tyr Ala Asp Ser

50 55 60

Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Ser Ser Gln Asn Thr Ala

65 70 75 80

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

85 90 95

Cys Gly Val Ala Phe Ser Tyr Gly Gly Gly Cys His Arg Met Asp Leu

100 105 110

Arg Gly Gln Gly Val Glu Val Val Val

115 120

<210> 2

<211> 111

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Ser Gln Thr Val Ile Gln Glu Pro Ala Met Ser Val Ser Pro Gly

1 5 10 15

Gly Thr Val Thr Leu Thr Cys Ala Phe Ser Ser Gly Ser Glu Ser Thr

20 25 30

Arg Lys Tyr Pro Ile Arg Phe Gln Gln Thr Pro Gly Gln Pro Pro Arg

35 40 45

Leu Leu Ile Tyr His Thr Asn Ser Arg Pro Thr Gly Val Pro Ser Arg

50 55 60

Phe Ser Gly Ala Ile Ser Gly Asn Lys Ala Ala Leu Thr Ile Thr Gly

65 70 75 80

Ala Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Leu Tyr Ile Arg

85 90 95

Thr Tyr Asn Val Pro Phe Gly Gly Gly Thr His Leu Thr Val Leu

100 105 110

<210> 3

<211> 363

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atggaggtga agctggtgga gtctggagga ggcctggtgc agcctggggg gtctctgaga 60

ctctcctgtg tcggctctga atacgaattc cgtggatcct tgattcactg ggtccgccag 120

gctccaggga aggggctgga gtggcttgca actatctgta ctactgatcc tataacggaa 180

tacgcagact ctgtgaaggg ccgattcacc atctccagag acagctccca gaacacggcc 240

tatctgcaaa tggacagcct gagaggcgaa gacacggccc gctattactg tggagttgcc 300

ttcagctatg gggggggctg tcatcgaatg gatctcaggg gccaaggcgt tgaagtcgtc 360

gtg 363

<210> 4

<211> 334

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atgtctcaga ctgtgatcca ggagccggcg atgtcagtgt ctcctggagg gaccgtcaca 60

ctcacctgtg cctttagctc agggtcagaa tctactagga agtaccccat taggttccag 120

cagacaccag gccagcctcc ccgactgctg atctaccaca ccaatagccg cccgactggg 180

gtccccagtc gcttctctgg agccatctct gggaacaaag ccgccctcac tatcacgggg 240

gcccaggctg aggacgaggc cgactatttc tgtagtctct atataaggac ttataatgtt 300

cccttcggcg gtgggaccca tctgaccgtc ctcg 334

<210> 5

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

cacgacgact tcaacgcctg gg 22

<210> 6

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gaggwgaagc tggtggagtc tg 22

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

cgaggacggt cagatgggtc 20

<210> 8

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

tctcagactg tgatccagga gc 22

<210> 9

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gccgcctgac cctccgccac cgaggwgaag ctggtggagt ctg 43

<210> 10

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

ggtggcggag ggtcaggcgg ccgaggacgg tcagatgggt c 41