CN108659124B

CN108659124B - Single-chain antibody for resisting porcine epidemic diarrhea virus and application thereof

Info

Publication number: CN108659124B
Application number: CN201810510406.2A
Authority: CN
Inventors: 尹燕博; 孙举; 王冬冬; 刘宏
Original assignee: Qingdao Bolong Gene Engineering Co ltd
Current assignee: Qingdao Bolong Gene Engineering Co ltd
Priority date: 2018-05-24
Filing date: 2018-05-24
Publication date: 2020-08-25
Anticipated expiration: 2038-05-24
Also published as: CN108659124A

Abstract

The invention discloses a single-chain antibody for resisting porcine epidemic diarrhea virus and application thereof. The single-chain antibody provided by the invention is a polypeptide consisting of a light chain variable region, a connecting peptide and a heavy chain variable region; the linker peptide is between the light chain variable region and the heavy chain variable region; the light chain variable region is shown as amino acid residues from 1st to 107 th positions from the N terminal of a sequence 1 in a sequence table; the heavy chain variable region is shown as 123 th to 240 th amino acid residues from the N terminal of a sequence 1 in a sequence table. The single-chain antibody provided by the invention can neutralize porcine epidemic diarrhea virus, and has important application value in preventing and/or treating porcine epidemic diarrhea.

Description

Single-chain antibody for resisting porcine epidemic diarrhea virus and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a single-chain antibody for resisting porcine epidemic diarrhea virus and application thereof.

Background

Porcine Epidemic Diarrheic (PED) is an intestinal infectious disease caused by Porcine Epidemic Diarrheic Virus (PEDV), which is usually caused in cold winter and spring, and clinical symptoms mainly include Diarrhea, vomiting, emaciation, dehydration, and ultimately death. Although the application of the vaccine plays a certain role in controlling the disease at present, the epidemic disease still occurs and prevails to different degrees in partial areas.

The phage antibody display technology provides a more efficient way for efficient screening and directional modification of genetically engineered antibodies. With the development of animal immunization technology, the space will become narrower and narrower, and the in vitro method for screening antibodies will be more and more concerned. The phage display technology is different from the traditional monoclonal antibody in antibody screening, and the technology greatly shortens the screening time and steps by displaying on the surface of the phage, combining with a target antigen, and utilizing antigen-antibody combination through a simple 96-hole microplate method and through adsorption-elution-enrichment. The phage antibody display technology can realize the target animal source of the antibody and reduce the anti-antibody phenomenon of the antibody in the using process.

At present, a few porcine phage antibody display platforms are provided, the molecular weight of the antibody is large, the binding region with the antigen is easily sealed, the folding capacity of the single-chain antibody is limited, and the traditional monoclonal antibody is basically a murine antibody, so that the anti-antibody is easily generated in the using process, and the treatment effect of the monoclonal antibody is influenced. The existing porcine epidemic diarrhea vaccines can not completely prevent and control the outbreak of the disease, and bring huge economic loss to the pig industry.

Disclosure of Invention

The invention aims to provide a single-chain antibody for resisting porcine epidemic diarrhea virus.

The single-chain antibody provided by the invention is polypeptide consisting of a light chain variable region, a connecting peptide and a heavy chain variable region; the linker peptide is between the light chain variable region and the heavy chain variable region; the light chain variable region can be represented by amino acid residues from 1st to 107 th positions from the N terminal of a sequence 1 in a sequence table; the heavy chain variable region can be shown as 123 th to 240 th amino acid residues from the N terminal of a sequence 1 in a sequence table.

In the single-chain antibody, the connecting peptide can be shown as amino acid residues from 108 th to 122 th positions of the sequence 1 from the N terminal in a sequence table.

The single-chain antibody can be specifically a polypeptide shown as a sequence 1 in a sequence table.

The invention also provides a nucleic acid molecule encoding any one of the single-chain antibodies described above. The nucleic acid molecule of any of the above single-chain antibodies may be composed of a gene encoding the light chain variable region, a gene encoding the linker peptide, and a gene encoding the heavy chain variable region.

The encoding gene of the light chain variable region can be shown as nucleotides 1 to 321 from the 5' end of a sequence 2 in a sequence table.

The encoding gene of the heavy chain variable region can be shown as nucleotides 367 to 720 from the 5' end of the sequence 2 in the sequence table.

The coding gene of the connecting peptide can be shown as nucleotides 322 to 366 from the 5' end of a sequence 2 in a sequence table.

The nucleic acid molecule of any one of the above-mentioned single-chain antibodies may be specifically a DNA molecule represented by A1) or A2) or A3) or A4):

A1) the coding region is a DNA molecule shown as a sequence 2 in a sequence table;

A2) the nucleotide sequence is a DNA molecule shown in a sequence 2 in a sequence table;

A3) a DNA molecule having 75% or more 75% identity to the nucleotide sequence defined in A1) or A2) and encoding any of the above single-chain antibodies;

A4) a DNA molecule which hybridizes with the nucleotide sequence limited by A1) or A2) under strict conditions and codes for any one of the single-chain antibodies.

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

Wherein, the sequence 1 in the sequence table is composed of 720 nucleotides, and the nucleotide of the sequence 1 in the sequence table encodes an amino acid sequence shown as a sequence 2 in the sequence table.

The nucleotide sequence encoding the single-chain antibody of the present invention can be easily mutated by a person of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which are artificially modified to have 75% or more identity to the nucleotide sequence of the single-chain antibody of the present invention, as long as they encode the single-chain antibody and are resistant to porcine epidemic diarrhea virus, are derived from and identical to the nucleotide sequence of the present invention.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes a nucleotide sequence having 75% or more, or 80% or more, or 85% or more, or 90% or more, or 95% or more identity with the nucleotide sequence of the protein consisting of the amino acid sequence shown in sequence 2 of the sequence listing of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

The single-chain antibody or the nucleic acid molecule is also protected by the invention. The single-chain antibody or the nucleic acid molecule may be B1), B2), B3) or B4):

B1) preparing a product for neutralizing porcine epidemic diarrhea virus;

B2) neutralizing porcine epidemic diarrhea virus;

B3) preparing a product for preventing and/or treating diseases caused by porcine epidemic diarrhea viruses;

B4) preventing and/or treating diseases caused by porcine epidemic diarrhea virus.

In the above application, the product may be specifically a medicament.

The invention also provides a product containing the single-chain antibody; the function of the product may be C1) or C2):

C1) neutralizing porcine epidemic diarrhea virus;

C2) preventing and/or treating diseases caused by porcine epidemic diarrhea virus.

The product may in particular be a medicament.

Any of the porcine epidemic diarrhea viruses described above may specifically be porcine epidemic diarrhea virus S protein.

The disease caused by any of the porcine epidemic diarrhea viruses can be porcine epidemic diarrhea.

Experiments prove that the single-chain antibody provided by the invention can neutralize porcine epidemic diarrhea virus and has important application value in preventing and/or treating porcine epidemic diarrhea.

Drawings

FIG. 1 shows the results of 1.0% agarose gel electrophoresis of three PCR amplification products.

FIG. 2 shows the results of 1.0% agarose gel electrophoresis of scFv fragment A and scFv fragment B.

FIG. 3 is the identification of primary libraries of phage single chain antibodies.

FIG. 4 shows the phage antibody binding activity.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The experimental materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. In the quantitative experiments in the following examples, three replicates were set up and the results averaged.

XLI-Blue competent cells are well products of century Biotechnology Limited. The restriction enzyme SfiI is a product of NEB corporation in USA. Trizol reagent, PrimeScript^TMII 1st Strand cDNA Synthesis Kit and DNA maker DL2000 are products of Takara corporation. M13K07 is a product of Invitrogen. The kit for separating the peripheral blood lymphocytes of the pigs is a product of Beijing Solaibao science and technology Limited. The pComb3XSS vector is a product of Qingdao bonong laboratory animals Co. The porcine epidemic diarrhea virus S protein is a product of Qingdao Borong genetic engineering Co.

Example 1 preparation of phage antibody display library

First, design and synthesis of primer

Primers were designed based on the sequences of the porcine IgG heavy chain (GenBank accession No.: AM177137, light chain Kappa chain (GenBank accession No.: AY518084) and light chain lambda chain (GenBank accession No.: AF345512), respectively, the nucleotide sequences and the amplified fragment lengths of the respective primers are shown in Table 1.

TABLE 1

Note: the italicized part indicates the linker and the underline indicates the restriction recognition site of the restriction enzyme SfiI. The name contains "for" to indicate upstream, the name contains "back" to indicate downstream, VH is heavy chain, VL (Kappa) is light chain Kappa chain, VL (lambda) is light chain lambda chain, S is C or G, Y is C or Y, K is G or T, R is A or G, W is A or T.

Each of the primers shown in Table 1 was synthesized by Biotechnology engineering (Shanghai) Ltd.

Second, extraction of peripheral blood lymphocytes

1. The sick pig which is resistant to the porcine epidemic diarrhea is immunized 3 times by subcutaneous injection of porcine epidemic diarrhea vaccine (a product of Harbin veterinary institute of Chinese academy of agricultural sciences). The dose for each injection was 1 mL. The days between each immunization were 14 days.

2. And (4) 14d after the step 1 is completed, collecting peripheral blood of the pig.

3. Mixing 1 volume part of 10mL of porcine peripheral blood, 1-2 volume parts of whole blood and tissue diluent uniformly, and then extracting peripheral blood lymphocytes by adopting a porcine peripheral blood lymphocyte separation kit.

Preparation of primary library of phage single-chain antibody

1. And (3) extracting the total RNA of the peripheral blood lymphocytes prepared in the second step by using Trizol reagent.

2. And (3) carrying out reverse transcription to synthesize cDNA by using the total RNA extracted in the step 1 as a template according to the operation steps of the PrimeScriptTMII 1st Strand cDNAsSynthesis Kit.

3. And (3) performing PCR amplification by using the cDNA synthesized in the step (2) as a template and adopting a primer pair 1 (consisting of VH for and VH back), a primer pair 2 (consisting of VL (kappa) for and VL (kappa) back) and a primer pair 3 (consisting of VL (lambda) for and VL (lambda) back) respectively to obtain a PCR amplification product 1, a PCR amplification product 2 and a PCR amplification product 3 in sequence.

Reaction conditions are as follows: 5min at 95 ℃; 35 cycles of 95 ℃ for 30s, 58 ℃ for 30s, 72 ℃ for 40 s; extension at 72 ℃ for 10 min.

4. The PCR amplification product 1 was subjected to 1.0% agarose gel electrophoresis to recover a DNA fragment 1 of about 400 bp. The PCR amplification product 2 was subjected to 1.0% agarose gel electrophoresis to recover a DNA fragment 2 of about 350 bp. The PCR amplification product 3 was subjected to 1.0% agarose gel electrophoresis to recover a DNA fragment 3 of about 350 bp.

The results of 1.0% agarose gel electrophoresis of the three PCR amplification products are shown in FIG. 1(M is DNA maker DL2000, lane 1 is PCR amplification product 1, lane 2 is PCR amplification product 2, and lane 3 is PCR amplification product 3).

5. An EP tube was charged with 1 part by mass of the

DNA fragment

1 and 1 part by mass of the DNA fragment 2, and mixed to obtain a mixed DNA A. An EP tube was taken, and 1 part by mass of the

DNA fragment

1 and 1 part by mass of the DNA fragment 3 were added and mixed to obtain a mixed DNA B.

Taking the mixed DNA A as a template, and carrying out PCR amplification by adopting a primer pair consisting of VL (kappa) for and VH back to obtain a PCR amplification product, namely the spliced scFv fragment A. The structural formula of the scFv fragment A is as follows: VL (kappa) -linker-VH.

And (3) performing PCR amplification by using the mixed DNA B as a template and adopting a primer pair consisting of VL (lambda) for and VH back to obtain a PCR amplification product, namely the spliced scFv fragment B. The structural formula of the scFv fragment B is as follows: VL (Lambda) -linker-VH.

Reaction conditions are as follows: 5min at 95 ℃; 35 cycles of 95 ℃ for 30s, 58 ℃ for 30s and 72 ℃ for 40 s; extension at 72 ℃ for 10 min.

6. Taking the scFv fragment A, carrying out 1.0% agarose gel electrophoresis, and recovering; then, the DNA fragment A was obtained by restriction with the restriction enzyme SfiI, 1.0% agarose gel electrophoresis and recovery.

Replacing the scFv fragment A with the scFv fragment B according to the steps, and obtaining the DNA fragment B without changing other steps.

The results of 1.0% agarose gel electrophoresis of scFv fragment A and scFv fragment B are shown in FIG. 2(M is DNA makerDL2000, lane 1 is scFv fragment A, and lane 2 is scFv fragment B).

7. The pComb3XSS vector was digested with the restriction enzyme SfiI, and about 3800bp of the vector backbone was recovered.

8. And connecting the DNA fragment A with a carrier skeleton to obtain a connection product A.

And connecting the DNA fragment B with the vector skeleton to obtain a connection product B.

9. And transforming the 200 mu L of the ligation product A and the 200 mu L of the ligation product B into XLI-Blue competent cells by 10 times of electric shock, and mixing the bacterial liquid obtained 10 times to obtain a mixed bacterial liquid.

The steps of each electric shock conversion are as follows: and (3) adding 20 mu L of the ligation product A and 20 mu L of the ligation product B into 80 mu L of XLI-Blue competent cells, performing electric shock transformation (the electric shock parameters are 2.5KV and 800 omega), then re-suspending with 1mL of SOC culture medium, transferring to a centrifuge tube (the specification is 50mL), and performing shaking culture at 37 ℃ and 220r/min for 1h to obtain a bacterial solution.

And uniformly coating 100 mu L of the mixed bacterial liquid on an LB solid plate containing 100 mu g/mL of ampicillin, and detecting the recombination rate.

The detection result shows that the recombination rate of the mixed bacterial liquid is 90%.

10. Adding ampicillin and glucose into the residual mixed bacterial liquid obtained in the step 9 to obtain a culture system; the concentration of ampicillin in the culture system was 100. mu.g/mL, and the concentration of glucose was 0.02M. Culturing the culture system at 37 ℃ to OD_600nmWhen the concentration is 0.5, adding 20 mu L M13K07, and infecting for 30min at 37 ℃; then, the cells were cultured at 37 ℃ for 1 hour under shaking at 220r/min, the medium was discarded, an equal volume of SOC medium containing 100. mu.g/mL ampicillin, 50. mu.g/mL kanamycin and 0.1mM/mL IPTG was added, and the cells were cultured at 37 ℃ for 6 hours under shaking at 220 r/min.

11. After the step 10 is completed, centrifuging and collecting supernatant; then 1 part by volume of the supernatant and 5 parts by volume of PEG8000 were mixed and left overnight at 4 ℃; the precipitate was collected by centrifugation and resuspended in 1.3mL of 10mM PBS buffer, pH7.4, to obtain a primary pool of phage single-chain antibodies.

Fourth, identification of primary library of phage single-chain antibody

Taking 1 mu L of phage single-chain antibody primary library, respectively carrying out PCR amplification by using a primer pair 1 (consisting of VH for and VH back), a primer pair 2 (consisting of VL (kappa) for and VL (kappa) back), a primer pair 3 (consisting of VL (lambda) for and VL (lambda) back), a universal primer pair 1 (consisting of VL (kappa) for and VH back) and a universal primer pair 2 (consisting of VL (lambda) for and VH back) to obtain PCR amplification products.

Each PCR amplification product was subjected to 1.0% agarose gel electrophoresis. The results are shown in FIG. 3(M is DNA makerDL2000, lane 1 is primer pair 1, lane 2 is primer pair 2, lane 3 is primer pair 3, lane 4 is primer pair 4, and lane 5 is primer pair 5).

Fifth, determination of bacteriophage single-chain antibody primary library titer

mu.L of the primary phage single-chain antibody library was added to 999. mu.L of PBS buffer (pH 7.4, 10 mM) and mixed to give dilution 1. Adding 990 μ L of PBS buffer solution with pH7.4 and 10mM into 10 μ L of dilution 1 to obtain dilution 2; then diluting by 10 times, and diluting by 4-5 gradients.

From each dilution of the solution 20. mu.L, added 180. mu.L of OD_600nmThe bacterial suspension with a value of 0.5 was infected for 15min, and 20. mu.L of the suspension was taken out and spread on LB solid plates containing 100. mu.g/mL of ampicillin, and the titer of the primary pool of phage single-chain antibodies was calculated.

The results showed that the titer of the primary pool of phage single chain antibodies was 9.5 × 10⁹pfu/mL。

Sixth, preparation of phage antibody display library

Porcine epidemic diarrhea virus S protein (namely antigen) is taken and diluted by 0.1M sodium carbonate-sodium bicarbonate buffer solution with pH9.6 to obtain antigen dilution with the concentration of 100 mug/mu L.

Antibody libraries were screened using antigen-antibody binding. The primary phage single chain antibody library was subjected to 3 rounds of "adsorption-elution-enrichment" panning and titer determinations were performed for each round of input and output phage. Through panning, the titer of an antibody library (namely a phage antibody display library) is 80 times higher than that of a phage single-chain antibody primary library (see table 2), and phages with better specificity are effectively enriched. The method comprises the following specific steps:

1. round 1 of selection and panning

(1) A96-well plate was prepared, and 100. mu.L of the antigen dilution was added to each well, and the mixture was allowed to stand overnight at 4 ℃.

(2) After completion of step (1), the 96-well plate was taken, 100. mu.L of a blocking solution (obtained by dissolving 0.05g of BSA in 100mL of 10mM PBS buffer solution having pH 7.4) was added to each well, and the plate was allowed to stand at 37 ℃ for 2 hours.

(3) And (3) after the step (2) is completed, taking the 96-well plate, adding 100 mu L of phage single-chain antibody primary library into each well, and standing for 1h at 37 ℃.

(4) After completion of step (3), the 96-well plate was removed and washed with PBST buffer (8.0 g NaCl, 0.2g KCl, 1.15g Na₂HPO₄And 0.2g KH₂PO₄Dissolving in deionized water, and then using the deionized water to fix the volume to 1000 mL; finally, 100. mu.L of Tween 20 was added) and washed 3-4 times, and finally with Elution Buffer (0.5 g BSA and 0.75g glycine were dissolved in 100mL ddH)₂O, then pH adjusted to 2.2 with 1M hydrochloric acid) to give eluent 1.

The titer was determined by taking 20. mu.L of eluate 1.

2. Round 2 and round 3 panning

(1) And taking the residual eluent 1, and performing electric shock transformation on the residual eluent to XLI-Blue competent cells to obtain a bacterial liquid.

(2) Replacing the phage single-chain antibody primary library with the bacterial liquid obtained in the step (1) according to the steps (1) to (4) in the step 1, and obtaining an eluent 2 without changing other steps.

(3) The titer was determined by taking 20. mu.L of eluate 2.

(4) And taking the residual eluent 2, and performing electric shock transformation on the residual eluent to XLI-Blue competent cells to obtain bacterial liquid.

(5) Replacing the phage single-chain antibody primary library with the bacterial liquid obtained in the step (4) according to the steps (1) to (4) in the step 1, and obtaining an eluent 3 without changing other steps. The titer was determined by taking 20. mu.L of eluate 3.

Eluent 3 is the prepared phage antibody display library.

TABLE 2

Number of elutriations	Input phage (pfu)	Output phage (pfu)	Input/output
				1	1.0×10¹¹	3.0×10⁴	3.0×10^-7
2	5.0×10¹⁰	3.7×10⁴	7.4×10^-7
				3	5.0×10¹⁰	1.2×10⁶	2.4×10^-5

Example 2 detection of binding Activity of phage antibody display library

The binding activity of the phage antibody display library was detected by ELISA.

1. A96-well plate was prepared, and 100. mu.L of the antigen dilution was added to each well, and the mixture was allowed to stand overnight at 4 ℃.

2. After completion of step 1, the 96-well plate was taken, 100. mu.L of a blocking solution (obtained by dissolving 0.05g of BSA in 100ml of PBS buffer solution (pH 7.4) and 10 mM) was added to each well, and the plate was allowed to stand at 37 ℃ for 2 hours.

3. After completion of step 2, the 96-well plate was taken, 100. mu.L of phage antibody display library was added to each well, and allowed to stand at 37 ℃ for 2 hours.

4. After the step 3 is completed, taking the 96-well plate, adding 100 mu L of HRP-labeled anti-M13 antibody (serving as enzyme-labeled secondary antibody) into each well, standing for 1h at 37 ℃, and finally detecting OD (optical density) of each well by using an enzyme-labeling instrument_450nm。

The "phage antibody display library" in step 3 was replaced with M13K07, and all other steps were unchanged as negative controls.

The "phage antibody display library" in step 3 was replaced with 10mM PBS buffer, pH7.4, and the other steps were not changed, and used as a blank control.

Experimental results show that one strain has affinity with porcine epidemic diarrhea virus S proteinSingle-chain antibody with better activity against porcine epidemic diarrhea virus, and OD thereof_450nm0.762 (see FIG. 4, Control M13K07, CloneA is a single chain antibody with better affinity activity to porcine epidemic diarrhea virus). Through sequencing, the single-chain antibody for resisting the porcine epidemic diarrhea virus is shown as a sequence 1 in a sequence table. In sequence 1 in the sequence table, amino acid residues 1 to 107 from the N terminal are light chain variable regions, amino acid residues 108 to 122 are connecting peptides, and amino acid residues 123 to 240 are heavy chain variable regions.

<110> Qingdao Bolong Gene engineering Co., Ltd

<120> single-chain antibody for resisting porcine epidemic diarrhea virus and application thereof

<160>2

<170>PatentInversion3.5

<210>1

<211>720

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>1

gccatccaga tgacccagtc tccagcctcc ctggctgcat ctctcggaga cacggtctcc 60

atcacttgcc gggccagtca gagcattagc agttatttag cctggtatca acaacaacca 120

gggacggctc ctaaacgctt gatctatgct gcatccagtt tgcaaagtgg ggtcccatcc 180

cggttcaagg gcagtggatc tggcaccgat ttcaccctca ccatcagtgg cctgcaggct 240

gaagatgttg caacttatta ctgtttgcag aataataatg tacctccgac gttcggccaa 300

ggaaccaagc tggaactcaa acgggctgat gccaagccat ccgtcggtgg ttcctctaga 360

tcttcccagg agaagctggt ggagtctgga ggaggcctgg tgcagcctgg ggggtctctc 420

agactctcct gtgtcggctc tggattcacc ttcagtagta cctggattaa ctgggtccgc 480

caggctccag ggaaggggct ggagtggctg gcaggcattt atagtagtgc aggtagcacc 540

gtccactcag actctgtgaa gggccgattc accgtctcag gagacaactc ccagaacacg 600

gcctatctgc aaatgaacag cctgagaacc gaagacacgg cccgctatta ctgtacaaaa 660

tccaactggt atacgttgga tgtctggggc ccaggcgttg aggtcgtcgt gtcctcaggc 720

<210>2

<211>240

<212>PRT

<213> Artificial sequence

<220>

<223>

<400>2

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

1 5 10 15

Asp Thr Val Ser Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Gln Pro Gly Thr Ala Pro Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Lys Gly

50 55 60

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

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Leu Gln Asn Asn Asn Val Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Leu Lys Arg Ala Asp Ala Lys

100 105 110

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

115 120 125

Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys

130 135 140

Val Gly Ser Gly Phe Thr Phe Ser Ser Thr Trp Ile Asn Trp Val Arg

145 150 155 160

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

165 170 175

Ala Gly Ser Thr Val His Ser Asp Ser Val Lys Gly Arg Phe Thr Val

180 185 190

Ser Gly Asp Asn Ser Gln Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu

195 200 205

Arg Thr Glu Asp Thr Ala Arg Tyr Tyr Cys Thr Lys Ser Asn Trp Tyr

210 215 220

Thr Leu Asp Val Trp Gly Pro Gly Val Glu Val Val Val Ser Ser Gly

225 230 235 240

Claims

1. A single chain antibody is polypeptide composed of light chain variable region, connecting peptide and heavy chain variable region; the linker peptide is between the light chain variable region and the heavy chain variable region; the light chain variable region is shown as amino acid residues from 1st to 107 th positions from the N terminal of a sequence 2 in a sequence table; the heavy chain variable region is shown as 123 th to 240 th amino acid residues from the N terminal of a sequence 2 in a sequence table.

2. The single chain antibody of claim 1, wherein: the connecting peptide is shown as amino acid residues from 108 th to 122 th positions of the N terminal of a sequence 2 in a sequence table.

3. The single chain antibody of claim 1 or 2, wherein: the single-chain antibody is a polypeptide shown in a sequence 2 in a sequence table.

4. A nucleic acid molecule encoding the single-chain antibody of any one of claims 1 to 3, which comprises a gene encoding the light chain variable region, a gene encoding the linker peptide, and a gene encoding the heavy chain variable region.

5. The nucleic acid molecule of claim 4, wherein: the encoding gene of the light chain variable region is shown as nucleotides from 1st to 321 th site from the 5' end of a sequence 1 in a sequence table; the encoding gene of the heavy chain variable region is shown as 367 th to 720 th nucleotides from the 5' end of a sequence 1 in a sequence table.

6. The nucleic acid molecule of claim 4, wherein: the coding gene of the connecting peptide is shown as nucleotides 322 to 366 from the 5' end of a sequence 1 in a sequence table.

7. The nucleic acid molecule of any one of claims 4 to 6, wherein: the nucleic acid molecule is a DNA molecule shown in a sequence 1 in a sequence table.

8. Use of a single chain antibody according to any one of claims 1 to 3 or a nucleic acid molecule according to any one of claims 4 to 7, which is B1) or B2):

B1) preparing a product for neutralizing porcine epidemic diarrhea virus;

B2) the product for preventing and/or treating diseases caused by porcine epidemic diarrhea virus is prepared.

9. The use of claim 8, wherein: the product is a medicament.

10. A product containing a single chain antibody according to any one of claims 1 to 3; the function of the product is C1) or C2):

C1) neutralizing porcine epidemic diarrhea virus;

11. The product of claim 10, wherein: the product is a medicament.