CN114292333B

CN114292333B - Bovine-derived single-chain antibody for resisting staphylococcus aureus coagulase Coa, preparation method and application

Info

Publication number: CN114292333B
Application number: CN202210093028.9A
Authority: CN
Inventors: 朱建国; 程曼玲
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2021-12-31
Filing date: 2022-01-26
Publication date: 2023-04-18
Anticipated expiration: 2042-01-26
Also published as: CN114292333A

Abstract

The invention discloses a bovine-derived single-chain antibody against staphylococcus aureus virulence factor Coa coagulase, and a preparation method and application thereof. The invention has the function of obviously inhibiting the coagulation of the Coa coagulase of staphylococcus aureus, thereby weakening the pathogenicity of the staphylococcus aureus to bovine mammary gland.

Description

Bovine-derived single-chain antibody for resisting staphylococcus aureus coagulase Coa, preparation method and application

Technical Field

The invention relates to the field of genetic engineering antibodies, in particular to a bovine antibody aiming at Coa coagulase and 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. The existing vaccine aiming at staphylococcus aureus whole bacteria and multiple virulence factors is also used for preventing the mastitis of the dairy cattle, but the effect is not ideal. Coagulase (Coa) is a coagulation-promoting protein secreted by staphylococcus aureus, can activate prothrombin non-enzymatically, and plays a key role in the host infection process.

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 valued in 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 complete 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 above-mentioned defects of the prior art, the technical problem to be solved by the present invention is to provide a bovine-derived antibody against staphylococcus aureus, and a preparation method and a use thereof.

The invention provides a bovine-derived single-chain antibody against staphylococcus aureus coagulase Coa, which comprises a light chain variable region VL, a heavy chain variable region VH and a connecting peptide Linker, wherein the light chain variable region VL is connected with the connecting peptide Linker 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 has an amino acid sequence shown as SEQ ID No.1, and the heavy chain variable region VH has an amino acid sequence shown as SEQ ID No. 2.

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 the bovine-derived single-chain antibody against Staphylococcus aureus coagulase Coa.

The third aspect of the invention provides a medicament for inhibiting the mastitis of dairy cows, which comprises the single-chain antibody of bovine origin against staphylococcus aureus coagulase Coa.

The invention provides a kit for detecting staphylococcus aureus, which is characterized by comprising the bovine single-chain antibody against staphylococcus aureus coagulase Coa, or a gene fragment of the bovine single-chain antibody against staphylococcus aureus coagulase Coa and a probe crosslinked with the gene fragment.

The fifth aspect of the present invention provides a method for preparing a bovine-derived single-chain antibody against staphylococcus aureus coagulase Coa, comprising the steps of:

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

Collecting cow blood with mastitis, separating peripheral blood leukocyte, extracting total RNA, synthesizing 1 st chain cDNA, designing light chain variable region VL gene and heavy chain variable region gene VH gene amplification primers, and amplifying light chain variable region VL gene and heavy chain variable region gene VH gene of antibody encoding gene by RT-PCR;

step 2, synthesis of scFv Gene

Connecting a light chain variable region VL gene and a heavy chain variable region VH gene by utilizing an SOE-PCR method to construct a bovine-derived single-chain antibody gene, namely an scFv gene;

step 3, constructing recombinant expression plasmid

After the ScFv gene and the pCANTAB5E vector obtained in the step 2 are subjected to double enzyme digestion respectively, inserting the ScFv gene into the pCANTAB5E vector to construct a recombinant expression plasmid;

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;

step 5, using the prokaryotic expression staphylococcus aureus Coa coagulase protein as a coating antigen, and carrying out enrichment panning;

step 6, screening by adopting a phase ELISA, and screening positive clones by using a prokaryotic expression staphylococcus aureus Coa coagulase protein as a coating antigen;

step 7, performing enzyme digestion on the positive clone obtained by screening in the step 6, recovering a single-chain antibody coding gene Coa-scFv, uniformly mixing the single-chain antibody coding gene Coa-scFv with a prokaryotic expression vector pET32a (+) subjected to synchronous enzyme digestion, and connecting the mixture at 14-16 ℃ overnight; after the ligation product is transformed into DH5 alpha competent cells, selecting a first monoclonal, carrying out PCR amplification on a colony of the first monoclonal, and extracting a first plasmid; performing double enzyme digestion verification on a colony PCR amplification product of the first monoclonal and the first plasmid respectively, and sequencing the first monoclonal which is correctly connected by the verification to obtain a first monoclonal which is correctly sequenced;

step 8, extracting the correctly sequenced recombinant plasmid of the first monoclonal obtained in the step 7 to obtain a first recombinant plasmid, transforming the first recombinant plasmid into BL21 competent cells, selecting a second monoclonal, performing PCR amplification on a colony of the second monoclonal, and extracting the second plasmid; the colony PCR amplification product of the second monoclonal and the second plasmid are verified by double digestion respectively, and the second monoclonal which is verified to be correct is sequenced to obtain the second monoclonal which is correctly sequenced; the plasmid extracted from the second monoclonal with correct sequencing is constructed single-chain antibody prokaryotic expression plasmid pET32a-Coa-scFv, and the second monoclonal bacterial colony pET32a-Coa-scFv-BL21 is subjected to passage purification and is stored for later use;

and 9, culturing the bacterial strain pET32a-Coa-scFv-BL21 of the single-chain antibody prokaryotic expression plasmid constructed in the step 8 at 37 ℃, adding 0.6mM protein inducer IPTG when the OD value of the bacteria is 0.4-0.6, and carrying out induced expression for 16-20 h at 28 ℃ to obtain the single-chain antibody protein and purifying the single-chain antibody protein.

Furthermore, primers of the light chain and the heavy chain of the antibody are VL F, VL R, VH F and VH R respectively, nucleotide sequences are shown as SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 and SEQ ID No.7, VLF and VH R respectively contain SfiI and NotI enzyme cutting sites, VH F and VL R contain complementary Linker sequences, the colony PCR primers in the step 7 are VL-F and VH-R respectively, and the nucleotide sequences are shown as SEQ ID No.8 and SEQ ID No. 9.

Further, the PCR reaction system is 25 μ L:2 XPCR mix 12.5. Mu.L, template cDNA 2. Mu.L, 25. Mu.M upstream and downstream primers 1. Mu.L each, ddH2O 8.5. 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, preferred nicking enzyme sites in step 3 are Sfi I and Not I, wherein SfiI: GGCCCAGCCGGCC, notI: GCGGCCGC; when ligated to the pET32a (+) vector, the preferred cleavage sites are EcoRI and XhoI, where EcoR I: GAATTC, xho I: CTCGAG.

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

The sixth aspect of the invention provides a bacterial strain of prokaryotic expression plasmid, which is characterized by comprising the single-chain antibody prokaryotic expression plasmid pET32a-Coa-scFv and being capable of expressing bovine-derived single-chain antibody protein of anti-staphylococcus aureus coagulase Coa.

The invention has the beneficial technical effects that:

1. when the recombinant bovine-derived single-chain antibody (scFv) is constructed, a bovine-derived single-chain antibody fragment VL-Linker-VH is formed by connecting a bovine-derived antibody light chain variable region VL and a bovine-derived antibody heavy chain variable region VH by using a middle Linker according to the sequence of 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 pET32a-Coa-scFv, the single-chain antibody is mixed with staphylococcus aureus and then incubated in an LB culture medium, so that the coagulation effect of staphylococcus aureus Coa coagulase can be inhibited, the pathogenicity of the staphylococcus aureus to cow mammary gland can be weakened, and the single-chain antibody is used for related research on bovine mastitis of staphylococcus aureus, and has good application prospect.

Drawings

FIG. 1 is a structural diagram of a phagemid vector pCANTAB 5E;

FIG. 2 is an electrophoretogram of a PCR-amplified fragment of a Coa-scFv positive cloned gene;

FIG. 3 is an SDS-PAGE profile of the protein expressed by the Coa-scFv recombinant gene;

FIG. 4 is a Western blotting assay of the protein expressed by the Coa-scFv gene;

FIG. 5 is a graph comparing the experimental coagulation inhibition of Coa-scFv Staphylococcus aureus Coa coagulase.

Detailed Description

The technical contents of the preferred embodiments of the present invention will be made clear 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 anticoagulation, 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 region; VL F and VL R were used to amplify the VL region. Wherein, VLF and VH R respectively contain SfiI and NotI enzyme cutting sites; 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 Biotechnology engineering 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.M) each 1. Mu.L, ddH2O 8.5. Mu.L. The amplification procedure was as follows: 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 ℃. The product was identified by 1.5% agarose gel electrophoresis and the gene of interest was recovered (following 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. Construction of the 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. SammBruk et al), after the scFv gene and the pCANTAB5E vector are subjected to SfiI and NotI double enzyme digestion respectively, 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 enzyme digestion verification and sequencing verification library diversity); 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 Coa coagulase Single-chain antibody

1. Enrichment panning is carried out to prepare a prokaryotic expression product of Coa coagulase of staphylococcus aureus (ATCC 25923), the prokaryotic expression product is used as an 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); the above procedure was repeated after infecting the remaining fraction of the eluate with E.coli TG 1. This was repeated for 3-5 cycles, after the first cycle, to increase the stringency of the washes: 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 produce recombinant phage. Coating the purified staphylococcus aureus Coa coagulase 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) for 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-antiM13 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 the 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 the 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 the Single-chain antibody pET32a-Coa-scFv

1. Constructing a recombinant plasmid pET32a-Coa-scFv by taking a positive clone strain as a template, amplifying a Coa-scFv target gene by using a specific primer (shown in table 2, underlined is a restriction enzyme cutting site), selecting restriction enzymes EcoRI and Xho I to carry out double enzyme cutting on the target gene and a prokaryotic expression vector pET32a (+), connecting after enzyme cutting to obtain a recombinant plasmid, converting the recombinant plasmid into DH5 alpha competence, and sending the clone with correct verification of colony PCR and plasmid double enzyme cutting 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 to verify that the correct clones are sent to Shanghai platinum biotechnology company Limited for sequencing, and obtaining the successfully constructed prokaryotic expression recombinant plasmid pET32a-Coa-scFv with correct sequencing, as shown in figure 3.

2. Purification of Single chain antibody Coa-scFv proteins

The fusion protein expressed by the pET32a (+) vector carries His-tag, so that the Coa coagulase recombinant protein can be subjected to His affinity purification by using a Ni-NTA pre-loaded gravity column, and the specific experimental method is as follows:

(1) Fixing the purification column, and keeping the periphery at low temperature by using an ice bag to allow the preservation solution to flow out;

(2) Adding Ni-native-0buffer balance purification column with 5-10 times column volume, and controlling flow rate to be about 1mL/min;

(3) Adding supernatant obtained by ultrasonic crushing and low-temperature centrifugation in 2.1.2, and controlling the flow rate to be about 0.5 mL/min;

(4) Adding Ni-native-0buffer with 5-10 times of column volume to clean the purification column, and controlling the flow rate to be about 1mL/min;

(5) Sequentially adding Ni-native-20mM imidazole, ni-native-50mM imidazole, ni-native100mM imidazole, ni-native-200mM imidazole and Ni-native-500mM imidazole in column volumes of 5-10 times, and competitively eluting the target protein, and controlling the flow rate to be 0.5-1mL/min;

(6) Adding Ni-native-0buffer with 5-10 times of column volume to clean the purification column, and controlling the flow rate to be about 1mL/min;

(7) Adding deionized water with 5-10 times of column volume to clean the purification column, and controlling the flow rate to be about 1mL/min;

(8) Adding 20% ethanol, and storing the column at 4 deg.C.

Respectively taking 5 protein eluents with different imidazole concentrations, adding protein electrophoresis Loading Buffer, boiling water bath, boiling for 10min,

solubility characterization was performed using SDS-PAGE. SDS-PAGE conditions: and adjusting the voltage to be 80V in the constant voltage mode, increasing the voltage to be 120V after electrophoresis is carried out for 30min, and continuing electrophoresis for about 1h until the Loading Buffer moving position is close to the bottom. After electrophoresis is finished, dyeing is carried out for 45min by Coomassie brilliant blue, then decoloring is carried out for 12h, and the electrophoresis condition is observed in a gel imaging system. The protein concentration obtained was determined with the BCA protein concentration assay kit.

Example 4 recombinant scFv sequence analysis

Sequencing the obtained single-chain antibody coding gene, and proving that the single-chain antibody coding gene is inserted into a prokaryotic expression plasmid pET32a (+) vector according to a correct reading frame sequence, wherein the amino acid sequence is shown as SEQ ID No.3, and the sequence is VL-Linker-VH.

Example 5 detection of the coagulation Effect of the Single chain antibody Coa-scFv on the inhibition of Staphylococcus aureus Coa coagulase

The experiment for detecting the coagulation effect of the single-chain antibody Coa-scFv on inhibiting staphylococcus aureus Coa coagulase comprises the following steps:

1. experimental materials and methods

1) Experimental Material

Coa coagulase prokaryotic expression antigen protein and scFv prokaryotic expression protein, SPF-grade adult New Zealand white rabbit (Shanghai Jiesi laboratory animals Co., ltd.), PEG8000, agarose, bovine fibrinogen (Beijing Solibao science and technology Co., ltd.), normal saline, heparin sodium vacuum blood collection tube, blood collection needle, glass test tube, and 60 mm-sized culture dish.

2) Experimental methods

Verification of coagulation function of scFv (single chain variable fragment) inhibiting Coa coagulase by adopting agarose blood plate

Taking a prepared agarose blood plate, and adding 20 mu L of normal saline into a small hole; another well was added 20. Mu.L of the Coa coagulase antigen protein at a defined concentration as a control, and the remaining wells were added 20. Mu.L of a mixture of Coa coagulase antigen protein at a final concentration of 1 Xg/mL and scFv protein diluted with physiological saline at final concentrations of 1X, 0.5X, 0.25 Xg/mL, respectively, and after labeling, the loop diameters were measured and recorded after incubation at 37 ℃ for 12h, with three replicates per set of experiments.

2. Data statistics and experimental results

Agarose blood plates were prepared, 5 wells were punched evenly, a mixture of Coa coagulase of defined concentration and scFv of different concentration was added, and a physiological saline solution was used as a control, and observed after 12 hours. As a result, as shown in FIG. 5, the coagulation loop is not visible around the well after the addition of scFv, and the coagulation effect of the Coa antigen is inhibited.

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 concept. 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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Claims

1. A bovine-derived single-chain antibody against Staphylococcus aureus coagulase Coa 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 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.

2. The bovine-derived single-chain antibody against staphylococcus aureus coagulase Coa of claim 1, wherein the amino acid sequence of the bovine-derived single-chain antibody fragment VL-Linker-VH is shown as SEQ ID No. 3.

3. A DNA molecule encoding the bovine-derived single-chain antibody against staphylococcus aureus coagulase Coa of claim 1 or 2.

4. A medicament for inhibiting mastitis in a milk cow, which comprises the single-chain antibody against staphylococcus aureus coagulase Coa of the bovine origin as claimed in claim 1 or 2.

5. A kit for the detection of 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 bacterial strain of prokaryotic expression plasmid, comprising a single-chain antibody prokaryotic expression plasmid pET32a-Coa-scFv capable of expressing the bovine-derived single-chain antibody protein against staphylococcus aureus coagulase Coa of claim 1 or 2.