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

Abstract

The invention discloses a bovine-derived single-chain antibody against Staphylococcus aureus virulence factor Coa coagulase and its preparation and application. The single-chain antibody comprises a light chain variable region VL, a heavy chain variable region VH, a connecting Peptide Linker, and connected in the order of VL-Linker-VH to form a bovine single-chain antibody fragment VL-Linker-VH. The invention can obviously inhibit the coagulation effect of Staphylococcus aureus Coa coagulase, thereby weakening the pathogenicity of Staphylococcus aureus to bovine mammary glands.

Description

A bovine-derived single-chain antibody against 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 against Coa coagulase and its preparation method and application.

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. At present, vaccines against the whole strain of Staphylococcus aureus and various virulence factors are also used to prevent mastitis in dairy cows, but the effects are not satisfactory. Coagulase (Coa) is a coagulation-promoting protein secreted by Staphylococcus aureus, which can non-enzymatically activate prothrombin and play a key role in the host infection process.

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 low 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 in the prior art, the technical problem to be solved by the present invention is to provide a bovine antibody against Staphylococcus aureus and its preparation method and application.

The first aspect of the present invention provides a bovine-derived single-chain antibody against Staphylococcus aureus coagulase Coa, comprising a light chain variable region VL, a heavy chain variable region VH, a connecting peptide Linker, and according to VL-Linker -The sequential connection of VH constitutes the bovine single-chain antibody fragment VL-Linker-VH, the amino acid sequence of the VL variable region of the light chain is shown in SEQ ID No.1, and the VH amino acid sequence of the variable region of the heavy chain is shown in SEQ ID Shown in No.2.

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 provides a DNA molecule encoding the bovine single-chain antibody against Staphylococcus aureus coagulase Coa.

The third aspect of the present invention provides a medicament for inhibiting cow mastitis, which comprises the bovine single-chain antibody against Staphylococcus aureus coagulase Coa.

The fourth aspect of the present invention provides a detection kit for Staphylococcus aureus, characterized in that it includes the bovine-derived single-chain antibody against Staphylococcus aureus coagulase Coa, or encodes the bovine-derived anti-Staphylococcus aureus A single-chain antibody gene fragment of coccal coagulase Coa and a probe cross-linked 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, which is characterized in that it comprises the following steps:

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

The blood of dairy cows suffering from mastitis was collected, peripheral blood leukocytes were isolated, total RNA was extracted, the first strand cDNA was synthesized, primers were designed to amplify the VL gene of the light chain variable region and the VH gene of the heavy chain variable region gene, and amplified by RT-PCR. Increase the light chain variable region VL gene and the heavy chain variable region gene VH gene of the antibody coding gene;

Step 2, Synthetic scFv gene

Using the SOE-PCR method to connect the VL gene of the light chain variable region and the VH gene of the heavy chain variable region 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;

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 Coa coagulase protein as the coating antigen, enrichment and panning;

Step 6, using phage ELISA screening, using the prokaryotic expression of Staphylococcus aureus Coa coagulase protein as the coating antigen, screening positive clones;

Step 7. Enzyme digest the positive clone obtained in step 6, recover the single-chain antibody coding gene Coa-scFv, mix it with the prokaryotic expression vector pET32a(+) that was digested synchronously, and ligate overnight at 14-16°C; transform the ligation product After DH5α competent cells, pick the first monoclonal, amplify the colony of the first monoclonal by PCR and extract the first plasmid; Perform sequencing on the first single clone that has been verified to be connected correctly, and obtain the first single clone with correct sequencing;

Step 8. Extract the recombinant plasmid of the first monoclonal sequence obtained in step 7 to obtain the first recombinant plasmid. After the first recombinant plasmid is transformed into BL21 competent cells, the second monoclonal is picked, and the second recombinant plasmid is selected. Colony PCR amplification of the single clone and extraction of the second plasmid; the colony PCR amplification product of the second single clone and the second plasmid were respectively verified by double enzyme digestion, and the verified correct second single clone was sequenced to obtain the sequenced correct The second monoclonal; the plasmid extracted from the second monoclonal with correct sequencing is the constructed single-chain antibody prokaryotic expression plasmid pET32a-Coa-scFv, and the second monoclonal colony pET32a-Coa-scFv-BL21 is subcultured and purified, and stored for future use ;

Step 9: Culture the pET32a-Coa-scFv-BL21 strain pET32a-Coa-scFv-BL21 constructed in step 8 into a prokaryotic expression plasmid for single-chain antibody at 37°C. When the bacterial OD value is 0.4-0.6, add 0.6mM protein inducer IPTG and proceed at 28°C The expression was induced for 16-20 hours, and the single-chain antibody protein was obtained and purified.

Further, 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 such as SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, and SEQ ID No.6. As shown in ID No.7, VLF and VHR contain SfiI and NotI restriction sites respectively, VHF and VL R contain complementary Linker sequences, and the colony PCR primers described in step 7 are VL-F and VH-R respectively, The nucleotide sequence is shown in SEQ ID No.8 and SEQ ID No.9.

Further, the PCR reaction system is 25 μL: 12.5 μL of 2×PCR mix, 2 μL of template cDNA, 1 μL of 25 μM upstream and downstream primers, and 8.5 μL of ddH2O.

PCR amplification program: 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.

Preferably, the preferred cutting enzyme sites in step 3 are Sfi I and Not I, wherein SfiI: GGCCCAGCCGGCC, NotI: GCGGCCGC; when connected with the pET32a (+) vector, the preferred enzyme cutting sites are EcoRI and XhoI, wherein EcoR I: GAATTC, Xho I: CTCGAG.

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

The sixth aspect of the present invention provides a prokaryotic expression plasmid strain, which is characterized in that it includes the single-chain antibody prokaryotic expression plasmid pET32a-Coa-scFv, which can express bovine-derived single-chain antibody against Staphylococcus aureus coagulase Coa protein.

Beneficial technical effect of the present invention:

1. When constructing a recombinant bovine single-chain antibody (scFv), according to the sequence of VL-Linker-VH, the bovine antibody light chain variable region VL and bovine antibody heavy chain variable region VH are connected with the middle Linker to form a bovine antibody The source single-chain antibody fragment VL-Linker-VH, and the recombinant bovine scFv constructed by the present invention proved to be more effective than the general literature reports that 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 pET32a-Coa-scFv, and combining the single-chain antibody with Grape aureus After the cocci are mixed, they are incubated in LB medium, which can inhibit the coagulation of Staphylococcus aureus Coa coagulase and thus weaken the pathogenicity of Staphylococcus aureus to bovine mammary glands. The single-chain antibody is used for Staphylococcus aureus cow mastitis The relevant research has a good application prospect.

Description of drawings

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

Fig. 2 is the PCR amplified fragment electrophoresis figure of Coa-scFv positive clone gene;

Fig. 3 is the protein SDS-PAGE detection chart of Coa-scFv recombinant gene expression;

Fig. 4 is the protein Western Bloting detection chart of Coa-scFv gene expression;

Fig. 5 is a comparison chart of coagulation inhibition experiment of Coa-scFv Staphylococcus aureus Coa coagulase.

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, VHF and VHR 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 is 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 ddH2O. 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 conventional molecular cloning methods (referring to "Molecular Cloning Experiment Guide" edited by J. After cleavage, insert the scFv gene into the pCANTAB5E vector to construct a recombinant expression plasmid, and electrotransform TG1 competent cells with it for 50 times. Combine all electrotransformation culture fluids, take a small part of serial dilution and spread on 2YT-AG solid The culture plate was cultivated overnight 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); the positive rate was calculated 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 Coa coagulase single-chain antibody

1. Prepare the prokaryotic expression product of Coa coagulase of Staphylococcus aureus (ATCC25923) by enrichment and panning, use it as an antigen, and coat at 4°C overnight; seal the 96-well plate with PBST containing 4% skimmed milk powder, 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 2 hours, wash with PBST and PBS 10 times each, and wash off unbound free phage; add 100ul 0.2mol/L Gly to each well -Hcl buffer (PH=2.2) to elute the specifically bound phage, add 50ul 1mol/L Tris-Hcl (PH=9.1) to neutralize the eluate; after infecting the rest of the eluate to Escherichia coli TG1, repeat the above step. 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 Coa coagulase prokaryotic expression protein was coated with 50mmol/L sodium bicarbonate salt solution (pH9.6) at 4°C overnight, blocked with 4% skim milk powder solution for 1h, and PBST (0.1% Tween20, The same below) and wash 3 times; add the phage single-chain antibody prepared above, react at 37°C for 2 hours, wash with PBST and PBS 6 times each; add 100 μL of HRP-antiM13 antibody (1:4000), react at 37°C for 1 hour, PBST and PBS Each was washed 6 times; TMB was used for color development, 2mol/L sulfuric acid was used to terminate the reaction, and the OD450 value was read by a microplate reader. At the same time, the helper phage M13K07 was set 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 pET32a-Coa-scFv

1. Construction of the recombinant plasmid pET32a-Coa-scFv Use the positive cloned strain as a template, use specific primers (as shown in Table 2, the underline is the enzyme cutting site) to amplify the Coa-scFv target gene, and select the restriction endonuclease EcoRI and Xho I performed double enzyme digestion on the target gene and the prokaryotic expression vector pET32a(+), ligated to obtain the recombinant plasmid after enzyme digestion, transformed it into DH5α competent, colony PCR and double enzyme digestion of the plasmid verified that the correct clone was sent to Shanghai Sequencing by Bosun Biotechnology Co., Ltd.;

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 pET32a-Coa-scFv is shown in Figure 3.

2. Purification of single-chain antibody Coa-scFv protein

The fusion protein expressed by the pET32a(+) vector carries a His-tag, so the Coa coagulase recombinant protein can be affinity-purified using a Ni-NTA prepacked gravity column for His affinity purification. The specific experimental method is as follows:

(1) Fix the purification column, keep the temperature low with ice packs around, and let the preservation solution flow out;

(2) Add 5-10 times the column volume of Ni-native-0buffer to balance the purification column, and control the flow rate to about 1mL/min;

(3) Add the supernatant obtained after sonication and cryogenic centrifugation in 2.1.2, and control the flow rate to about 0.5mL/min;

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

(5) Add 5-10 times column volume Ni-native-20mM imidazole, Ni-native-50mM imidazole, Ni-native100mM imidazole, Ni-native-200mM imidazole, Ni-native-500mM imidazole to competitively elute the target protein, Control flow rate 0.5-1mL/min;

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

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

(8) Fill up with 20% ethanol and store the purification column at 4°C.

Take 5 protein eluates with different imidazole concentrations, add protein electrophoresis Loading Buffer to boiling water bath for 10min,

Solubility identification was performed using SDS-PAGE. SDS-PAGE electrophoresis conditions: adjust the voltage to 80V in constant voltage mode, increase the voltage to 120V after 30 minutes of electrophoresis, and continue electrophoresis for about 1 hour until the moving position of the Loading Buffer is close to the bottom. After electrophoresis, stain with Coomassie brilliant blue for 45 minutes and decolorize for 12 hours, and observe the electrophoresis in a gel imaging system. The obtained protein concentration was measured with BCA protein concentration assay kit.

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 pET32a(+) vector according to the correct reading frame order. The amino acid sequence is shown in SEQ ID No.3, and its sequence order is VL- Linker-VH.

Example 5 Detection of single-chain antibody Coa-scFv inhibiting coagulation of Staphylococcus aureus Coa coagulase

The coagulation assay for detecting the inhibition of Coa coagulase by the single-chain antibody Coa-scFv of Staphylococcus aureus includes:

1. Experimental materials and methods

1) Experimental materials

Coa coagulase prokaryotic expression antigen protein and scFv prokaryotic expression protein, SPF grade adult New Zealand white rabbit (Shanghai Jiesijie Experimental Animal Co., Ltd.), PEG8000, agarose, bovine fibrinogen (Beijing Suo Lai Bao Technology Co., Ltd.), physiological Saline, sodium heparin vacuum blood collection tube, blood collection needle, glass test tube, 60mm petri dish.

2) Experimental method

Validation of coagulation function of scFv inhibiting Coa coagulase by using agarose blood plate

Take a prepared agarose blood plate, add 20 μL of normal saline to one small hole; add 20 μL of Coa coagulase antigen protein of a determined concentration to the other small hole as a control, and add Coa coagulation at a final concentration of 1x μg/mL to the remaining small holes. 20 μL of the mixture of enzyme antigen protein and scFv protein diluted with normal saline to a final concentration of 1x, 0.5x, 0.25x μg/mL, labeled and incubated at 37°C for 12 hours, then measure and record the diameter of the coagulation ring, and each group of experiments was set up three times repeat.

2. Data statistics and experimental results

Prepare an agarose blood plate, punch 5 small holes evenly, add a mixture of a certain concentration of Coa coagulase and different concentrations of scFv, physiological saline as a control, and observe after 12 hours. The results are shown in Fig. 5, the coagulation ring is no longer visible to the naked eye around the hole after adding the scFv, and the coagulation effect of the Coa antigen is inhibited.

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.

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Claims (6)

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.

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