CN114369165B - Bovine single-chain antibody of bovine-derived anti-staphylococcus aureus virulence factor GapC, preparation method and application thereof - Google Patents

Abstract

The invention discloses a bovine-derived single-chain antibody against Staphylococcus aureus virulence factor GapC, a preparation method and an application thereof. The sequential connection of VL-Linker-VH constitutes the bovine single-chain antibody fragment VL-Linker-VH, the VL amino acid sequence of the light chain variable region is shown in SEQ ID No.1, and the VH amino acid sequence of the heavy chain variable region As shown in SEQ ID No.2. After the single-chain antibody of the present invention is mixed with Staphylococcus aureus, it can specifically bind to Staphylococcus aureus glyceraldehyde-3-phosphate dehydrogenase GapC, and can be used for further research on the prevention and control of mastitis in dairy cows, and has good application prospect.

Description

A bovine-derived single-chain antibody against Staphylococcus aureus virulence factor GapC, its preparation method and its application

technical field

The invention relates to the field of genetic engineering prokaryotic expression single-chain antibody, in particular to a single-chain antibody for bovine anti-staphylococcus aureus virulence factor GapC, a preparation method and application thereof.

Background technique

Cow mastitis is a common frequently-occurring disease that affects the development of the dairy industry and causes huge losses to dairy production. There are many pathogenic bacteria that cause mastitis in dairy cows, among which Staphylococcus aureus is one of the most important pathogenic bacteria, and its prevalence rate reaches 50%, resulting in serious economic losses. Because Staphylococcus aureus is contagious and resistant to the antibiotics used for treatment, it is difficult to completely cure it. Staphylococcus aureus mainly causes disease by producing a variety of pathogenic factors. At present, vaccines against the whole strain of Staphylococcus aureus and various virulence factors are also used to prevent mastitis in cows, but the effects are not satisfactory.

Genetically engineered antibodies such as single-chain antibodies, with their unique antiviral and antibacterial effects and the advantages of large-scale engineering preparation, have shown great potential in the development of antibacterial drugs, and have been highly valued in this field.

The single-chain antibody is formed by linking the light chain variable region VL and the heavy chain variable region VH of the antibody through a short peptide linker head-to-tail through DNA recombination technology, and is the smallest functional fragment that retains the complete antigen-binding site. The expression forms of single-chain antibodies mainly include fusion expression, intracellular expression and secretory expression. Compared with intact antibodies, single-chain antibodies have the following advantages: 1) Small molecular weight, only one-sixth of the size of intact antibodies, and lower immunogenicity; 2) Strong tissue penetration, easy to enter microscopic cells around solid tumors circulation; 3) fast blood clearance, little accumulation in the kidney; 4) no Fc segment, low non-specific binding; 5) easy mass production through genetic engineering; 6) easy genetic manipulation, easier to construct recombinant immunotoxins.

Therefore, there is an urgent need in this field to develop single-chain antibodies with high specificity against whole bacteria of Staphylococcus aureus and various virulence factors.

Contents of the invention

In view of the above-mentioned defects of the prior art, the present invention provides a bovine-derived single-chain antibody against Staphylococcus aureus virulence factor GapC, a preparation method and use thereof.

The first aspect of the present invention is to provide a bovine single-chain antibody against Staphylococcus aureus virulence factor GapC, which is characterized in that it includes a light chain variable region VL, a heavy chain variable region VH, and a connecting peptide Linker, And connect according to the sequence of VL-Linker-VH to form the bovine single-chain antibody fragment VL-Linker-VH, the VL amino acid sequence of the light chain variable region is as shown in SEQ ID No.1, and the VH amino acid sequence of the heavy chain variable region The sequence is shown in SEQ ID No.2, and the amino acid sequence of the connecting peptide Linker is (GGGGGSGGGGS).

The amino acid sequence of the bovine single-chain antibody fragment VL-Linker-VH is shown in SEQ ID No.3.

The second aspect of the present invention is to provide a DNA molecule that can encode the bovine single-chain antibody against Staphylococcus aureus virulence factor GapC.

The third aspect of the present invention is to provide a medicament for inhibiting cow mastitis, the medicament comprises the bovine single-chain antibody against Staphylococcus aureus virulence factor GapC.

A fourth aspect of the present invention is to provide a test kit for Staphylococcus aureus, comprising the bovine-derived single-chain antibody against the virulence factor GapC of Staphylococcus aureus, or encoding the bovine-derived anti-Staphylococcus aureus The gene fragment of the single-chain antibody of the bacterial virulence factor GapC and the probe cross-linked thereto.

The fifth aspect of the present invention is to provide a bovine-derived single-chain antibody preparation method against Staphylococcus aureus virulence factor GapC, which is characterized in that it includes the following steps:

Step 1, PCR amplification of light chain variable region VL and heavy chain variable region gene VH

Collect the blood of dairy cows with mastitis, isolate peripheral blood leukocytes, extract total RNA, synthesize the first strand cDNA, design primers for amplifying the light and heavy chains of the antibody, and use RT-PCR to amplify the light chain variable region of the antibody coding gene VL gene and heavy chain variable region gene VH gene;

Step 2, Synthetic scFv gene

Using the SOE-PCR method to connect the VL gene of the light chain variable region and the heavy chain variable region gene to construct a bovine-derived single-chain antibody gene, that is, the scFv gene;

Step 3, construction of recombinant expression plasmid

The scFv gene obtained in step 2 and the pCANTAB5E vector were double digested respectively, and the scFv gene was inserted into the pCANTAB5E vector to construct a recombinant expression plasmid;

Preferably, the Dicer sites are Sfi I and Not I, wherein SfiI: GGCCCAGCCGGCC, NotI: GCGGCCGC;

Step 4. Establish primary single-chain antibody library

Transform the recombinant plasmid into Escherichia coli, cultivate and amplify with helper phage to establish a primary single-chain antibody library;

Step 5, using prokaryotically expressed Staphylococcus aureus virulence factor GapC as the coating antigen, enrichment and panning;

Step 6, using phage ELISA screening, using the prokaryotic expression of Staphylococcus aureus virulence factor GapC as the coating antigen, screening positive clones;

Step 7. Enzyme digest the positive clone obtained from the screening, recover the single-chain antibody encoding gene GapC-scFv, mix with the prokaryotic expression vector pGEX-4T-1 synchronously digested, ligate overnight at 14-16°C, and transform the ligated product into DH5α After competent cells, pick a single clone, colony PCR and plasmid double enzyme digestion to verify the correct clone for sequencing;

Step 8. Extract the plasmid from the clone with the correct sequencing, and then transform the recombinant plasmid into BL21 competent cells, pick a single clone, colony PCR and double enzyme digestion of the plasmid to verify the correct clone sequencing, and the sequenced one is the constructed one Single-chain antibody prokaryotic expression plasmid pGEX-4T-1-GapC-scFv.

Preferably, the restriction sites are BamH I and Xho I, wherein BamH I: GGATCC, Xho I: CTCGAG.

The primers for the light and heavy chains of the antibody are VL F, VL R, VH F and VH R respectively, and the nucleotide sequences are as SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 and SEQ ID No.7 As shown, VLF and VHR contain SfiI and NotI restriction sites respectively, VHF and VL R contain complementary Linker sequences, and the primers for colony PCR described in step 7 are VL-F and VH-R, and the nucleotide sequences are as follows: Shown in SEQ ID No.8 and SEQ ID No.9.

The PCR reaction system is 25 μL: 2×PCR mix 12.5 μL, template cDNA 2 μL, 25 μM upstream and downstream primers 1 μL, ddH ₂ O 8.5 μL; PCR amplification program: pre-denaturation at 95°C for 3 minutes; denaturation at 94°C for 40 seconds, 64°C Anneal for 40s, extend for 1min at 72°C, 30 cycles; finally extend for 10min at 72°C.

Preferably, 4 rounds of enrichment and panning are performed in step 5.

The sixth aspect of the present invention is to provide a prokaryotic expression plasmid pGEX-4T-1-GapC-scFv, including the single-chain antibody encoding gene of bovine anti-Staphylococcus aureus virulence factor GapC.

Beneficial technical effect of the present invention:

1. When constructing a recombinant bovine-derived single-chain antibody (scFv), according to the sequence of VL-Linker-VH, the antibody light chain variable region VL and the antibody heavy chain variable region VH are connected with the middle Linker to form a bovine-derived The single-chain antibody fragment VL-Linker-VH, the recombinant bovine scFv constructed by the present invention proved to be more effective than the general literature reports are connected in the order of VH-Linker-VL.

2. Cloning the single-chain antibody encoding gene (scFv) of the screened positive clone into the prokaryotic expression plasmid pET32a(+), constructing the single-chain antibody prokaryotic expression plasmid pET-32a-GapC-scFv, and combining the single-chain antibody with gold After Staphylococcus aureus is mixed and incubated in LB medium, it can specifically bind to Staphylococcus aureus glyceraldehyde-3-phosphate dehydrogenase GapC, which can be used for further research on the prevention and control of mastitis in dairy cows, and has good application prospect.

The idea, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings, so as to fully understand the purpose, features and effects of the present invention.

Description of drawings

Fig. 1 is the structural diagram of phagemid vector pCANTAB5E;

Figure 2 is the scFv positive clones screened by phage ELISA;

Fig. 3 is the electrophoresis diagram of the amplified fragment of the scFv positive clone gene screened by prokaryotic expression;

Fig. 4 is the protein SDS-PAGE detection chart of scFv gene expression;

Figure 5 is a protein Western Bloting detection diagram of scFv gene expression;

Fig. 6 is a test of GapC-scFv inhibiting the growth of Staphylococcus aureus.

Detailed ways

The following describes several preferred embodiments of the present invention with reference to the accompanying drawings, so as to make the technical content clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the protection scope of the present invention is not limited to the embodiments mentioned herein.

Example 1 Construction of bovine phage single-chain antibody library

1. Collect the blood of dairy cows suffering from mastitis. When the serum antibody titer is greater than 1:20000 by ELISA, continue the follow-up experiment. Bovine peripheral blood leukocytes were extracted with anticoagulated blood, and total RNA was extracted by Trizol method (TRIZOL Reagent was purchased from TaKaRa Company). Using the extracted total RNA as a template, Oligo primer was used to synthesize the first-strand cDNA according to the product instructions of the reverse transcription kit (cDNA first-strand synthesis kit was purchased from TaKaRa Company).

2. Analyze the variable region sequence of the bovine antibody coding gene in the published literature, and design primers for amplifying the light and heavy chains of the antibody according to the FR region (Table 1), wherein VHF and VHR are used to amplify the VH region; VL F and VL R are used to amplify the VL region. Wherein, VLF and VHR contain SfiI and NotI restriction sites respectively; VHF and VL R contain complementary Linker sequences (restriction sites and Linker sequences are underlined in Table 1). Primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.

Table 1 Primers for amplifying antibody variable regions and the size of their amplified fragments

3. Amplification of VH and VL genes. Using cDNA as a template, VH F and VH R were used as primers to amplify the VH gene; VL F and VL R were used as primers to amplify the VL gene. The PCR reaction system was 25 μL: 12.5 μL of 2×PCR mix, 2 μL of template cDNA, 1 μL of upstream and downstream primers (25 μM), and 8.5 μL of ddH ₂ O. The amplification program was as follows: pre-denaturation at 95°C for 3 min; denaturation at 94°C for 40 s, annealing at 64°C for 40 s, extension at 72°C for 1 min, 30 cycles; final extension at 72°C for 10 min. The product was identified by 1.5% agarose gel electrophoresis and the target gene was recovered (operated according to the gel recovery instructions provided by AxyGEN).

4. Acquisition of scFv gene. The VL and VH genes containing the Linker sequence were connected into a scFv gene (VL-linker-VH) by recombination chain extension reaction (SOE-PCR), and SfiI and NotI restriction sites were added.

5. Construction of primary library. As shown in the structural diagram of the phagemid vector pCANTAB5E in accompanying drawing 1, according to the conventional molecular cloning method (referring to the "Molecular Cloning Experiment Guide" edited by J. Sambrook et al.), the scFv gene and the pCANTAB5E vector were double-digested with SfiINotI respectively , Insert the scFv gene into the pCANTAB5E vector to construct a recombinant expression plasmid, and electrotransform it into TG1 competent cells for 50 times, combine all the electrotransformation culture fluids, take a small part of serial dilution and spread it on a 2YT-AG solid culture plate , overnight culture at 30°C to calculate the library capacity (pick clones for colony PCR and plasmid double-enzyme digestion verification, and sequencing to verify the diversity of the library); calculate the positive rate by colony PCR to obtain the actual library capacity. The primary library was established after the remaining bacterial culture was rescued by helper phage M13KO7.

Example 2 Screening of bovine-derived anti-Staphylococcus aureus glyceraldehyde-3-phosphate dehydrogenase GapC single-chain antibody

1. Enrichment and panning to prepare Staphylococcus aureus (ATCC25923) prokaryotic expression product of glyceraldehyde-3-phosphate dehydrogenase GapC of Staphylococcus aureus, which was used as antigen, and coated overnight at 4°C; Block the 96-well plate with PBST and incubate at 37°C for 2h; add the single-chain antibody phage antibody library prepared in the above steps to the 96-well plate, incubate at 37°C for 2h, wash with PBST and PBS 10 times each, and wash off unbound free Phage; Add 100ul 0.2mol/L Gly-Hcl buffer (PH=2.2) to each well to elute specifically bound phage, add 50ul 1mol/L Tris-Hcl (PH=9.1) to neutralize the eluate; After the eluate was infected with Escherichia coli TG1, the above steps were repeated. Repeat this for 3-5 rounds. After the first round, the stringency of washing should be increased: wash with PBS 20 times after elution with PBST for 20 times before elution.

2. phage ELISA screening 96 clones were randomly selected from the fourth round, rescued with M13K07 to prepare recombinant phage. The purified Staphylococcus aureus glyceraldehyde-3-phosphate dehydrogenase GapC prokaryotic expression protein was coated with 50mmol/L sodium bicarbonate salt solution (pH9.6) at 4°C overnight, and blocked with 4% skimmed milk powder solution for 1h. Wash 3 times with PBST (0.1% Tween20, the same below); add the phage single-chain antibody prepared above, react at 37°C for 2 h, wash 6 times with PBST and PBS; add 100 μL of HRP-antiM13 antibody (1:4000), 37 React at ℃ for 1 hour, wash with PBST and PBS for 6 times each; develop color with TMB, stop the reaction with 2mol/L sulfuric acid, read the OD450 value with a microplate reader, and set the helper phage M13K07 as a negative control. ELISA results are judged by P/N (P is the OD450 value of positive wells, N is the OD450 value of negative wells), P/N≥2.1 is positive; 1.5≤P/N＜2.1 is suspicious; P/N＜1.5 The results of scFv positive clones screened by negative phage ELISA are shown in Figure 2, where BlankControl is a blank control, Negative Control is a negative control, scFv is a positive clone, and the OD450 value of the positive clone is very high, close to 2.6; while the negative control The OD450 value is less than 0.4, and the ratio of the two is greater than 2.1.

Example 3 Prokaryotic expression and purification of single chain antibody pGEX-4T-1-GapC-scFv

1 Construction of the recombinant plasmid pGEX-4T-1-GapC-scFv Using the positive cloned strain as a template, use specific primers (as shown in Table 2, the underlined restriction site) to amplify the GapC-scFv target gene, select the restriction Endonucleases BamH I and XhoI double-digested the target gene and the prokaryotic expression vector pGEX-4T-1, ligated to obtain the recombinant plasmid after digestion, transformed it into DH5α competent, colony PCR and plasmid double-digestion verified correctness The clones were sent to Shanghai Boshang Biotechnology Co., Ltd. for sequencing;

Table 2 Primers for amplifying antibody variable regions and the size of their amplified fragments

The clones with correct sequencing were extracted from the plasmids, and then the recombinant plasmids were transformed into BL21 competent cells, and single clones were picked, colony PCR and plasmid double enzyme digestion verified that the correct clones were sent to Shanghai Boshang Biotechnology Co., Ltd. for sequencing, and the sequencing was correct The successful construction of the prokaryotic expression recombinant plasmid pGEX-4T-1-GapC-scFv is shown in Figure 3.

2 Purification of the single-chain antibody GapC-scFv protein The recombinantly expressed fusion protein contains GST-tag, and the protein is purified using a GST prepacked gravity column (purchased from Shanghai Sangon Bioengineering Co., Ltd.). The specific steps are as shown in the instructions. After purification, protein ultrafiltration was performed, and the collected eluate was analyzed by SDS-PAGE and Western Bloting. The protein size was 44kD. The results are shown in Figures 4 and 5. The protein concentration was determined by the Bradford method. According to the standard Based on the drawn standard curve and the measured OD values of the samples, the concentration of the single-chain antibody GapC-scfv protein was about 350 μg/mL.

Example 4 Recombinant scFv sequence analysis

The obtained single-chain antibody coding gene was sequenced to prove that it was inserted into the prokaryotic expression plasmid pGEX-4T-1 vector according to the correct reading frame sequence. The amino acid sequence is shown in SEQ ID No.3, and its sequence sequence is VL -Linker-VH, the amino acid sequence is as shown in SEQ ID No.3.

Example 5 Detection of single-chain antibody GapC-scFv inhibition of Staphylococcus aureus growth test

The test for detecting the inhibition of the growth of Staphylococcus aureus by the single-chain antibody GapC-scFv includes the following two steps, respectively:

1. Experimental methods and steps

The reagents used in this experiment were GapC-scFv recombinant protein, Staphylococcus aureus ATCC25923, and 4% bovine red blood cells.

Dilute GapC-scFv to 10, 20, 40, 50ug/mL with 0.9% normal saline, mix it with Staphylococcus aureus ATCC25923 with an OD600 of about 0.6 at a ratio of 1:1, add 200μL LB to culture and culture based on shaking at 37°C, respectively The OD600 of Staphylococcus aureus was measured at 6h and 12h, wherein 0.9% normal saline was used as a positive control, and the penicillin group was used as a negative control, and each group was set for three repetitions.

Add each solution sequentially according to the instructions, and pay attention to avoid generating air bubbles.

After the required solutions were added sequentially according to the instructions, the absorbance of each well was measured at 440nm with a microplate reader for a corresponding time.

2. Data statistics and experimental results

After co-incubating 10, 20, 40, 50, 100ug/mL of GapC-scFv with Staphylococcus aureus in the logarithmic growth phase, the results are shown in Figure 6, and different concentrations of GapC-scFv were obtained after 6 hours The inhibition of Staphylococcus aureus was significantly different from that of the normal saline group; after 12 hours, GapC-scFv with a concentration of 20ug/mL had a significant inhibitory effect on the growth of Staphylococcus aureus.

GapC has the activity of the key enzyme GAPDH in the glycolysis pathway of Staphylococcus aureus, and is also an important virulence factor for Staphylococcus aureus infected cow mastitis. The single-chain antibody inhibits the growth of Staphylococcus aureus, and the results show that the screened and prepared GapC-scFv has a good effect of inhibiting the growth of Staphylococcus aureus.

The preferred specific embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make many modifications and changes according to the concept of the present invention without creative efforts. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art shall be within the scope of protection defined by the claims.

sequence listing

<110> Shanghai Jiao Tong University

<120> A bovine single-chain antibody against Staphylococcus aureus virulence factor GapC, its preparation method and its application

<130> 01335-21298PIX

<160> 9

<170> SIPOSequenceListing 1.0

<210> 1

<211> 112

<212> PRT

<213> Artificial sequence

<400> 1

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

1 5 10 15

Gly Gln Arg Val Ser Ile Thr Cys Ser Gly Ser Ser Asn Asn Ile Gly

20 25 30

Arg Tyr Gly Val Gly Trp Tyr Gln Glu Val Pro Gly Ser Gly Leu Arg

35 40 45

Thr Ile Ile Tyr Ser Thr Thr Ser Arg Pro Ser Gly Val Pro Asp Arg

50 55 60

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

65 70 75 80

Leu Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ala Thr Ile Asp Gly

85 90 95

Ser Ser Gly Thr Ala Val Phe Gly Ser Gly Thr Thr Leu Thr Val Leu

100 105 110

<210> 2

<211> 121

<212> PRT

<213> Artificial sequence

<400> 2

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr

20 25 30

Gly Val Arg Trp Val Arg Gln Ala Pro Gly Lys Ala Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Arg Ala Gly Ser Thr Tyr Tyr Asn Pro Ala Leu Lys

50 55 60

Ser Arg Leu Ser Ile Thr Lys Asp Ser Ser Lys Ser Gln Val Ser Leu

65 70 75 80

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

85 90 95

Arg Val Val Ser Thr Thr His Val Ile Val Asp Ala Trp Gly Gln Gly

100 105 110

Leu Leu Val Thr Val Ser Ser Thr Ser

115 120

<210> 3

<211> 244

<212> PRT

<213> Artificial sequence

<400> 3

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

1 5 10 15

Gly Gln Arg Val Ser Ile Thr Cys Ser Gly Ser Ser Asn Asn Ile Gly

20 25 30

Arg Tyr Gly Val Gly Trp Tyr Gln Glu Val Pro Gly Ser Gly Leu Arg

35 40 45

Thr Ile Ile Tyr Ser Thr Thr Ser Arg Pro Ser Gly Val Pro Asp Arg

50 55 60

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

65 70 75 80

Leu Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ala Thr Ile Asp Gly

85 90 95

Ser Ser Gly Thr Ala Val Phe Gly Ser Gly Thr Thr Leu Thr Val Leu

100 105 110

Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Arg

115 120 125

Glu Ser Gly Pro Ser Leu Val Glu Pro Ser Gln Thr Leu Ser Leu Thr

130 135 140

Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr Gly Val Arg Trp Val

145 150 155 160

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

165 170 175

Ala Gly Ser Thr Tyr Tyr Asn Pro Ala Leu Lys Ser Arg Leu Ser Ile

180 185 190

Thr Lys Asp Ser Ser Lys Ser Gln Val Ser Leu Ser Val Ser Ser Val

195 200 205

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

210 215 220

Thr His Val Ile Val Asp Ala Trp Gly Gly Gln Gly Leu Leu Val Thr Val

225 230 235 240

Ser Ser Thr Ser

<210> 4

<211> 39

<212>DNA

<213> Artificial sequence

<400> 4

gtggcccagc cggccatggc ccaggctgtg ctgactcag 39

<210> 5

<211> 63

<212> DNA

<213> Artificial sequence

<400> 5

agatccgccg ccaccggatc caccaccgcc cgagccaccg ccacctagga cggtcagtgt 60

ggt 63

<210> 6

<211> 44

<212> DNA

<213> Artificial sequence

<400> 6

ggcggtggtg gatccggtgg cggcggatct caggtgcagc tgcg 44

<210> 7

<211> 34

<212>DNA

<213> Artificial sequence

<400> 7

ttgcggccgc actagtggag gagacggtga ccag 34

<210> 8

<211> 25

<212> DNA

<213> Artificial sequence

<400> 8

cgcggatcca tggcagtaaa agtag 25

<210> 9

<211> 28

<212>DNA

<213> Artificial sequence

<400> 9

ccgctcgagt ttagaaagtt cagctaag 28

Claims (6)

1. A bovine source anti-Staphylococcus aureus virulence factor GapC single-chain antibody is characterized in that, comprises light chain variable region VL, heavy chain variable region VH, connecting peptide Linker, and according to VL-Linker-VH The sequence connection of the bovine single-chain antibody fragment VL-Linker-VH, the amino acid sequence of the light chain variable region VL is shown in SEQ ID No.1, and the amino acid sequence of the heavy chain variable region VH is shown in SEQ ID No. 2. 2. the single-chain antibody of bovine source anti-staphylococcus aureus virulence factor GapC according to claim 1, is characterized in that, described bovine source single-chain antibody fragment VL-Linker-VH amino acid sequence is as SEQ ID No.3 shown. 3. A DNA molecule, characterized in that the single-chain antibody encoding the bovine-derived anti-Staphylococcus aureus virulence factor GapC according to claim 1 or 2. 4. A medicament for inhibiting cow mastitis, characterized in that the medicament comprises the bovine-derived single-chain antibody against Staphylococcus aureus virulence factor GapC according to claim 1 or 2. 5. A kit for Staphylococcus aureus, characterized by comprising the antibody of claim 1 or 2, or the DNA molecule of claim 3 and a probe cross-linked thereto. 6. a kind of prokaryotic expression plasmid pGEX-4T-1-GapC-scFv, it is characterized in that, described plasmid comprises the single-chain antibody coding gene of bovine source anti-staphylococcus aureus virulence factor GapC described in claim 1 or 2 .

Images