CN114369165B - Bovine single-chain antibody of bovine-derived anti-staphylococcus aureus virulence factor GapC, preparation method and application thereof - Google Patents
Bovine single-chain antibody of bovine-derived anti-staphylococcus aureus virulence factor GapC, preparation method and application thereof Download PDFInfo
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- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
Abstract
The invention discloses a bovine-derived single-chain antibody against staphylococcus aureus virulence factor GapC, a preparation method and application, wherein the antibody comprises a light chain variable region VL, a heavy chain variable region VH and a connecting peptide Linker, and is connected according to the sequence of VL-Linker-VH to form a bovine-derived single-chain antibody fragment VL-Linker-VH, the light chain variable region VL amino acid sequence is shown as SEQ ID No.1, and the heavy chain variable region VH amino acid sequence is shown as SEQ ID No. 2. After being mixed with staphylococcus aureus, the single-chain antibody can be specifically combined with staphylococcus aureus glyceraldehyde-3-phosphate dehydrogenase GapC, can be used for further research on prevention and control of cow mastitis, and has good application prospect.
Description
Technical Field
The invention relates to the field of prokaryotic expression single-chain antibodies in genetic engineering, in particular to a single-chain antibody for bovine-derived anti-staphylococcus aureus virulence factor GapC, a preparation method and application thereof.
Background
Cow mastitis is a common frequently-occurring disease which affects the development of the dairy industry and causes great loss to dairy production. The pathogenic bacteria causing mastitis of the dairy cows are many, wherein staphylococcus aureus is one of the most important pathogenic bacteria, the prevalence rate reaches 50%, and serious economic loss is caused. Staphylococcus aureus is infectious and resistant to therapeutic antibiotics, making it difficult to cure it completely. Staphylococcus aureus can cause diseases mainly by producing various pathogenic factors, and at present, vaccines aiming at whole staphylococcus aureus and various virulence factors are also used for preventing mastitis of dairy cows, but the effect is not ideal.
The genetic engineering antibodies such as single-chain antibody and the like show great potential for developing antibacterial drugs by virtue of unique antiviral and antibacterial effects and the advantage of large-scale engineering preparation, and are highly regarded by the field.
The single-chain antibody is formed by connecting the light chain variable region VL and the heavy chain variable region VH of the antibody end to end through a section of connecting short peptide linker by a DNA recombination technology, and is a minimum functional fragment for reserving a complete antigen binding part. The expression form of the single-chain antibody mainly comprises three forms of fusion expression, intracellular expression and secretion expression. Compared with the intact antibody, the single-chain antibody has the following advantages: 1) The molecular weight is small, the size is only one sixth of that of a complete antibody, and the immunogenicity is low; 2) The tissue penetration is strong, and the tissue easily enters microcirculation around solid tumors; 3) Blood clearance is fast, and kidney accumulation is little; 4) No Fc segment and low non-specific binding; 5) Easy mass production by genetic engineering; 6) Easy gene operation and easy construction of recombinant immunotoxin.
Therefore, there is an urgent need in the art to develop highly specific single chain antibodies against staphylococcus aureus whole cells and various virulence factors.
Disclosure of Invention
In view of the defects in the prior art, the invention provides a bovine single-chain antibody of a virulence factor GapC of staphylococcus aureus, a preparation method and application.
The invention provides a bovine-derived single-chain antibody against staphylococcus aureus virulence factor GapC, which is characterized by comprising a light chain variable region VL, a heavy chain variable region VH and a connecting peptide Linker, wherein the light chain variable region VL, the heavy chain variable region VH and the connecting peptide Linker are connected in the sequence of VL-Linker-VH to form a bovine-derived single-chain antibody fragment VL-Linker-VH, the light chain variable region VL amino acid sequence is shown as SEQ ID No.1, the heavy chain variable region VH amino acid sequence is shown as SEQ ID No.2, and the connecting peptide Linker amino acid sequence is (GGGGGSGGS).
The bovine single-chain antibody fragment VL-Linker-VH amino acid sequence is shown in SEQ ID No. 3.
In a second aspect, the invention provides a DNA molecule encoding a single chain antibody against s.
The third aspect of the invention provides a medicament for inhibiting mastitis of dairy cows, which comprises the bovine-derived single-chain antibody against staphylococcus aureus virulence factor GapC.
The fourth aspect of the invention provides a kit for staphylococcus aureus, which comprises the bovine anti-staphylococcus aureus virulence factor GapC single-chain antibody, or a gene fragment encoding the bovine anti-staphylococcus aureus virulence factor GapC single-chain antibody and a probe crosslinked with the bovine anti-staphylococcus aureus virulence factor GapC single-chain antibody.
The fifth aspect of the invention provides a method for preparing a bovine-derived single-chain antibody against staphylococcus aureus virulence factor GapC, which is characterized by comprising the following steps:
Collecting cow blood with mastitis, separating peripheral blood leukocyte, extracting total RNA, synthesizing 1 st chain cDNA, designing primers for amplifying light and heavy chains of the antibody, and amplifying light chain variable region VL gene and heavy chain variable region gene VH gene of the antibody encoding gene by RT-PCR;
Connecting a light chain variable region VL gene and a heavy chain variable region gene by using an SOE-PCR method to construct a bovine-derived single-chain antibody gene, namely an scFv gene;
After the scFv gene and the pCANTAB5E vector obtained in the step 2 are subjected to double enzyme digestion respectively, the scFv gene is inserted into the pCANTAB5E vector to construct a recombinant expression plasmid;
preferably, the dicer sites are Sfi I and Not I, wherein Sfi: GGCCCAGCCGGCC, notI: GCGGCCGC;
step 4, establishing a primary single-chain antibody library
Transforming the recombinant plasmid into escherichia coli, culturing and amplifying by using helper phage to establish a primary single-chain antibody library;
and 8, extracting plasmids from the clones with correct sequencing, transforming the recombinant plasmids into BL21 competent cells, selecting single clones, verifying correct clone sequencing by colony PCR and plasmid double enzyme digestion, and obtaining the constructed single-chain antibody prokaryotic expression plasmid pGEX-4T-1-GapC-scFv with correct sequencing.
Preferably, the cleavage sites are BamH I and Xho I, wherein BamH I: GGATCC, xho I: CTCGAG.
The primers of the light chain and the heavy chain of the antibody are VL F, VL R, VH F and VH R respectively, the nucleotide sequences are shown as SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 and SEQ ID No.7, the VLF and the VH R respectively contain SfiI and NotI enzyme cutting sites, the VH F and the VL R contain complementary Linker sequences, the colony PCR primers in the step 7 are VL-F and VH-R, and the nucleotide sequences are shown as SEQ ID No.8 and SEQ ID No. 9.
The PCR reaction system is 25 mu L:2 XPCR mix 12.5. Mu.L, template cDNA 2. Mu.L, 25. Mu.M upstream and downstream primers 1. Mu.L each, ddH 2 O8.5 mu L; PCR amplification procedure: pre-denaturation at 95 ℃ for 3min; denaturation at 94 ℃ for 40s, annealing at 64 ℃ for 40s, extension at 72 ℃ for 1min, and 30 cycles; finally, extension is carried out for 10min at 72 ℃.
Preferably, there are 4 rounds of enrichment panning in step 5.
The sixth aspect of the invention provides a prokaryotic expression plasmid pGEX-4T-1-GapC-scFv which comprises a single-chain antibody coding gene of bovine anti-staphylococcus aureus virulence factor GapC.
The invention has the beneficial technical effects that:
1. when the recombinant bovine-derived single-chain antibody (scFv) is constructed, according to the sequence of VL-Linker-VH, an antibody light chain variable region VL and an antibody heavy chain variable region VH are connected by using a middle Linker to form a bovine-derived single-chain antibody fragment VL-Linker-VH, and thus the connection is proved to be more effective by the invention compared with the common literature reports that the constructed recombinant bovine-derived scFv is connected according to the sequence of VH-Linker-VL.
2. The screened positively cloned single-chain antibody coding gene (scFv) is cloned to a prokaryotic expression plasmid pET32a (+), so as to construct a single-chain antibody prokaryotic expression plasmid pET-32a-GapC-scFv, the single-chain antibody is mixed with staphylococcus aureus and then incubated in an LB culture medium, can be specifically combined with the staphylococcus aureus glyceraldehyde-3-phosphate dehydrogenase GapC, can be used for further research on prevention and control of cow mastitis, and has good application prospect.
The conception, the specific structure and the technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, the features and the effects of the present invention.
Drawings
FIG. 1 is a structural diagram of a phagemid vector pCANTAB 5E;
FIG. 2 shows scFv positive clones screened by phase ELISA;
FIG. 3 is an electrophoretogram of amplified fragments of scFv positive cloned genes selected by prokaryotic expression;
FIG. 4 is a SDS-PAGE map of scFv gene expression proteins;
FIG. 5 is a Western blotting detection chart of scFv gene expressed protein;
FIG. 6 is a GapC-scFv assay for inhibiting the growth of Staphylococcus aureus.
Detailed Description
The technical contents of the preferred embodiments of the present invention will be more clearly and easily understood by referring to the drawings attached to the specification. The present invention may be embodied in many different forms of embodiments and the scope of the invention is not limited to the embodiments set forth herein.
Example 1 construction of bovine-derived phage Single chain antibody library
1. Collecting the blood of the cow suffering from mastitis, and continuing the subsequent experiment when the ELISA method detects that the serum antibody titer is greater than 1. Bovine peripheral blood leukocytes were extracted with anticoagulated blood, and total RNA was extracted by Trizol method (available from TaKaRa). Using the extracted total RNA as a template, 1 st strand cDNA was synthesized using Oligo primer according to the protocol of the reverse transcription kit (cDNA 1 st strand synthesis kit available from TaKaRa).
2. The sequences of the variable regions of the genes encoding bovine antibodies in the published literature were analyzed, and primers for amplifying the light and heavy chains of the antibodies were designed based on the FR regions thereof (Table 1), wherein VH F and VH R were used for amplifying the VH regions; VL F and VL R were used to amplify the VL region. Wherein VLF and VH R respectively contain SfiI restriction site and NotI restriction site; VH F, VL R contain complementary Linker sequences (the cleavage sites and Linker sequences are underlined in Table 1). The primers were synthesized by Shanghai Bioengineering services, inc.
TABLE 1 primers for amplifying antibody variable regions and amplified fragment sizes thereof
3. Amplification of VH and VL genes. Amplifying VH genes by taking cDNA as a template and VH F and VH R as primers; VL F and VL R are primers for amplifying VL genes. The PCR reaction system was 25. Mu.L: 2 XPCR mix 12.5. Mu.L, template cDNA 2. Mu.L, upstream and downstream primers (25. Mu.L)M) 1. Mu.L each, ddH 2 O8.5. Mu.L. The amplification procedure was as follows: pre-denaturation at 95 ℃ for 3min; denaturation at 94 ℃ 40s, annealing at 64 ℃ 40s, extension at 72 ℃ for 1min,30 cycles; finally, extension is carried out for 10min at 72 ℃. The product was identified by 1.5% agarose gel electrophoresis and the gene of interest was recovered (according to the gel recovery instructions provided by AxyGEN).
4. Obtaining scFv gene. VL and VH genes containing Linker sequences were ligated into scFv genes (VL-Linker-VH) by recombinant chain extension reaction (SOE-PCR) and SfiI and NotI cleavage sites were added.
5. And (5) constructing a primary library. As shown in the structure diagram of the phagemid vector pCANTAB5E in the attached FIG. 1, according to the conventional molecular cloning method (refer to molecular cloning experimental guidance, mainly compiled by J. SammBruke, etc.), after the scFv gene and pCANTAB5E vector are subjected to SfiINOTI double digestion, the scFv gene is inserted into the pCANTAB5E vector to construct a recombinant expression plasmid, the recombinant expression plasmid is electrically transformed into TG1 competent cells for 50 times, all the electric transformation culture solutions are combined, a small part of the diluted electric transformation culture solutions are coated on a 2YT-AG solid culture plate, and the library capacity is calculated by overnight culture at 30 ℃ (the clone is selected for colony PCR and plasmid double digestion verification, and the diversity of a sequencing verification library is obtained); the positive rate was calculated by colony PCR to obtain the actual pool volume. The remaining bacterial culture was rescued by helper phage M13KO7 to create a primary library.
Example 2 screening of bovine-derived anti-Staphylococcus aureus glyceraldehyde-3-phosphate dehydrogenase GapC Single chain antibody
1. Enrichment panning is carried out to prepare staphylococcus aureus (ATCC 25923) glyceraldehyde-3-phosphate dehydrogenase GapC prokaryotic expression product of staphylococcus aureus, the prokaryotic expression product is used as antigen, and the prokaryotic expression product is coated overnight at 4 ℃; sealing the 96-well plate by PBST containing 4% skimmed milk powder, and incubating for 2h at 37 ℃; adding the single-chain antibody phage antibody library prepared in the step into a 96-well plate, incubating for 2h at 37 ℃, washing for 10 times by using PBST and PBS respectively, and washing away unbound free phage; specifically bound phage were eluted by adding 100ul 0.2mol/L Gly-Hcl buffer (pH = 2.2) per well, and the eluate was neutralized by adding 50ul 1mol/L Tris-Hcl (pH = 9.1); after the remaining fraction of the eluate was infected with E.coli TG1, the above procedure was repeated. This was repeated for 3-5 rounds, and after the first round the stringency of the washes was increased: elution was preceded by 20 PBST eluations and followed by 20 PBS washes.
2. phase ELISA screening 96 clones were randomly picked from the fourth round and rescued with M13K07 to make recombinant phage. Coating the purified staphylococcus aureus glyceraldehyde-3-phosphate dehydrogenase GapC prokaryotic expression protein with 50mmol/L sodium bicarbonate solution (pH 9.6) at 4 ℃ overnight, sealing with 4% skimmed milk powder solution for 1h, and washing with PBST (0.1% Tween20, the same below) 3 times; adding the prepared phage single-chain antibody, reacting at 37 ℃ for 2h, and washing by PBST and PBS for 6 times respectively; 100 μ L of HRP-anti M13 antibody (1; TMB color development, 2mol/L sulfuric acid termination reaction, enzyme labeling instrument read OD450 value, meanwhile, set the auxiliary phage M13K07 as negative control. The determination of the ELISA result is expressed by P/N (P is the OD450 value of a positive hole, N is the OD450 value of a negative hole), and P/N is more than or equal to 2.1 and is positive; P/N is more than or equal to 1.5 and less than 2.1, which is suspicious; the result of scFv positive clone screened by Negative phase ELISA with P/N < 1.5 is shown in figure 2, wherein Blank Control is Blank Control, negative Control is Negative Control, scFv is positive clone, and OD450 value of positive clone is very high and is close to 2.6; while the negative control had an OD450 value of less than 0.4, which was greater than 2.1.
EXAMPLE 3 prokaryotic expression and purification of Single-chain antibody pGEX-4T-1-GapC-scFv
1, constructing a recombinant plasmid pGEX-4T-1-GapC-scFv by taking a positive clone strain as a template, amplifying a GapC-scFv target gene by using specific primers (shown in a table 2, underlined is a restriction enzyme digestion site), selecting restriction enzymes BamH I and Xho I to carry out double enzyme digestion on the target gene and a prokaryotic expression vector pGEX-4T-1, connecting after enzyme digestion to obtain a recombinant plasmid, transforming the recombinant plasmid to DH5 alpha competence, and sending a clone with correct colony PCR and plasmid double enzyme digestion verification to Shanghai platinum biotechnology Limited for sequencing;
TABLE 2 primers for amplifying antibody variable regions and amplified fragment sizes thereof
Extracting plasmids from clones with correct sequencing, transforming the recombinant plasmids into BL21 competent cells, selecting single clones, carrying out colony PCR and plasmid double enzyme digestion verification to verify the correct clones, and sending the clones to Shanghai platinum biotechnology company Limited for sequencing, wherein the clones with correct sequencing are prokaryotic expression recombinant plasmids pGEX-4T-1-GapC-scFv successfully constructed, as shown in figure 3.
2 purification and recombinant expression of the single-chain antibody GapC-scFv protein comprises GST-tag, the protein is purified by a GST pre-loaded gravity column (purchased from Shanghai Biotechnology, ltd.), the specific steps are shown in the specification, protein ultrafiltration is carried out after purification, SDS-PAGE and Western blotting analysis are carried out on collected eluent, the protein size is 44kD, the result is shown in figures 4 and 5, the protein concentration is determined by a Bradford method, and the concentration of the single-chain antibody GapC-scFv protein is about 350 mu g/mL according to a standard curve drawn by a standard product and the OD value measured by a sample.
Example 4 sequence analysis of recombinant scFv
Sequencing the obtained single-chain antibody coding gene, and proving that the single-chain antibody coding gene is inserted into a prokaryotic expression plasmid pGEX-4T-1 vector according to a correct reading frame sequence, wherein the amino acid sequence is shown as SEQ ID No.3, the sequence is VL-Linker-VH, and the amino acid sequence is shown as SEQ ID No. 3.
EXAMPLE 5 assay for detecting Single chain antibody GapC-scFv inhibiting Staphylococcus aureus growth
The test for detecting the single-chain antibody GapC-scFv to inhibit the growth of staphylococcus aureus comprises the following two steps:
1. experimental methods and procedures
The reagents used in the experiment are GapC-scFv recombinant protein, staphylococcus aureus ATCC25923 and 4% bovine red blood cells.
GapC-scFv was diluted to 10, 20, 40, 50ug/mL using 0.9% physiological saline at 1:1 ratio and about 0.6 OD600 of Staphylococcus aureus ATCC25923, adding 200 u LLB culture medium, performing vibration culture at 37 ℃, and measuring the OD600 of the Staphylococcus aureus of 6h and 12h respectively, wherein 0.9% physiological saline is used as a positive control, penicillin is used as a negative control, and each group is provided with three replicates.
The solutions were added sequentially according to the order of the specification, taking care to avoid the formation of bubbles.
After the required solutions are added in sequence according to the specification, the action lasts for the corresponding time, and the absorbance of each hole is measured at the position of 440nm of an enzyme-labeling instrument.
2. Data statistics and experimental results
After 10, 20, 40, 50 and 100ug/mL of GapC-scFv and staphylococcus aureus in logarithmic phase are incubated together, the results are shown in figure 6, and after 6 hours, the inhibition of the obtained GapC-scFv with different concentrations on the staphylococcus aureus is obviously different from that of a normal saline group; after 12 hours, the growth of staphylococcus aureus is obviously inhibited by GapC-scFv with the concentration of 20 ug/mL.
GapC has the activity of a key enzyme GAPDH in a glycolysis pathway of staphylococcus aureus and is an important virulence factor of staphylococcus aureus infecting cow mastitis, so that the growth of the staphylococcus aureus is inhibited by preparing a single-chain antibody of the GapC virulence factor and utilizing the single-chain antibody of the GapC, and the result shows that the screened and prepared GapC-scFv has a good effect of inhibiting the growth of the staphylococcus aureus.
The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.
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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 Gln Gly Leu Leu Val Thr Val
225 230 235 240
Ser Ser Thr Ser
<210> 4
<211> 39
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 4
gtggcccagc cggccatggc ccaggctgtg ctgactcag 39
<210> 5
<211> 63
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 5
agatccgccg ccaccggatc caccaccgcc cgagccaccg ccacctagga cggtcagtgt 60
ggt 63
<210> 6
<211> 44
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 6
ggcggtggtg gatccggtgg cggcggatct caggtgcagc tgcg 44
<210> 7
<211> 34
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 7
ttgcggccgc actagtggag gagacggtga ccag 34
<210> 8
<211> 25
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 8
cgcggatcca tggcagtaaa agtag 25
<210> 9
<211> 28
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 9
ccgctcgagt ttagaaagtt cagctaag 28
Claims (6)
1. A bovine-derived single-chain antibody against staphylococcus aureus virulence factor GapC is characterized by comprising a light chain variable region VL, a heavy chain variable region VH and a connecting peptide Linker, wherein the light chain variable region VL, the heavy chain variable region VH and the connecting peptide Linker are connected in the sequence of VL-Linker-VH to form a bovine-derived single-chain antibody fragment VL-Linker-VH, the amino acid sequence of the light chain variable region VL is shown as SEQ ID No.1, and the amino acid sequence of the heavy chain variable region VH is shown as SEQ ID No. 2.
2. The bovine-derived single-chain antibody against staphylococcus aureus virulence factor GapC according to claim 1, wherein the bovine-derived single-chain antibody fragment VL-Linker-VH amino acid sequence is shown in SEQ ID No. 3.
3. A DNA molecule encoding a single chain antibody of bovine origin against s.
4. A medicament for inhibiting mastitis in a bovine animal, which comprises the bovine-derived single-chain antibody against staphylococcus aureus virulence factor GapC of claim 1 or 2.
5. A kit for staphylococcus aureus comprising an antibody according to claim 1 or 2, or a DNA molecule according to claim 3, and a probe cross-linked thereto.
6. A prokaryotic expression plasmid pGEX-4T-1-GapC-scFv, comprising the gene encoding a single chain antibody of bovine anti-s.
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| CN111848790A (en) * | 2020-08-07 | 2020-10-30 | 上海交通大学 | Bovine-derived single-chain antibody for resisting staphylococcus aureus and preparation and application thereof |
| CN113493510A (en) * | 2021-07-07 | 2021-10-12 | 上海交通大学 | Bovine-derived single-chain antibody for resisting staphylococcus aureus LukD virulence factor and preparation and application thereof |
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| US20160244511A1 (en) * | 2013-10-17 | 2016-08-25 | Arsanis Biosciences Gmbh | Cross-reactive staphylococcus aureus antibody sequences |
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| CN111848790A (en) * | 2020-08-07 | 2020-10-30 | 上海交通大学 | Bovine-derived single-chain antibody for resisting staphylococcus aureus and preparation and application thereof |
| CN113493510A (en) * | 2021-07-07 | 2021-10-12 | 上海交通大学 | Bovine-derived single-chain antibody for resisting staphylococcus aureus LukD virulence factor and preparation and application thereof |
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