CN114276447B - Bovine-derived single-chain antibody for inhibiting growth of staphylococcus aureus and preparation method and application thereof - Google Patents

Abstract

The invention discloses a bovine-derived single-chain antibody for inhibiting the growth of Staphylococcus aureus and its preparation method and application. RT-PCR is used to directly amplify the heavy chain variable region VH gene and the light chain variable region of the bovine antibody coding gene. VL gene, using the SOE-PCR method, link the linker with the VH gene and VL gene to construct a bovine scFv gene, and clone the scFv gene into the phagemid vector pCANTAB5E to construct a primary single-chain antibody library, and assist phage M13KO7 to rescue the primary library ; Prokaryotic expression of Staphylococcus aureus growth-promoting virulence factor GapC protein was used as a coating antigen after four rounds of enrichment and panning, and the positive clones were screened by phage ELISA method, which proved that the single-chain antibody had the ability to inhibit Staphylococcus aureus The role of growth.

Description

A bovine-derived single-chain antibody for inhibiting the growth of Staphylococcus aureus and its preparation method and application

technical field

The invention relates to the field of genetic engineering, in particular to a bovine single-chain antibody for inhibiting the growth of Staphylococcus aureus, a preparation method and application thereof.

Background technique

Single-chain antibody is a genetically engineered antibody, which 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 end-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.

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.

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.

Contents of the invention

The purpose of the present invention is to provide a bovine-derived single-chain antibody for inhibiting the growth of Staphylococcus aureus and its preparation method and application. The single-chain antibody can specifically bind to the growth-promoting virulence factor GapC protein of Staphylococcus aureus, and It has a certain effect of inhibiting the growth of Staphylococcus aureus, and can be used in the research of Staphylococcus aureus mastitis in cows.

To achieve the above object, the present invention adopts the following technical solutions:

The first aspect of the present invention is to provide a bovine single-chain antibody that inhibits the growth of Staphylococcus aureus, including the light chain variable region VL, the heavy chain variable region VH, the 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 of the body 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 shown.

Further, the amino acid sequence of the 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 drug for inhibiting mastitis in cows, which includes the single-chain antibody.

The third aspect of the present invention is to provide a dairy cow mastitis diagnostic kit, which is characterized by comprising the single-chain antibody, or the gene fragment encoding the single-chain antibody and a cross-linked probe.

The fourth aspect of the present invention is to provide a method for preparing a bovine-derived single-chain antibody that inhibits the growth of Staphylococcus aureus, comprising the following steps:

Step 1, amplifying the light chain variable region VL gene and the heavy chain variable region VH gene

Using RT-PCR to amplify the VL gene of the light chain variable region and the VH gene of the heavy chain variable region of the antibody coding gene from the RNA of peripheral blood mononuclear cells suffering from cow mastitis;

Step 2, Synthetic scFv gene

Using the SOE-PCR method to connect the intermediate connecting peptide linker, VH gene, and VL gene to construct a bovine-derived single-chain antibody gene, that is, the scFv gene;

Step 3, construction of recombinant expression plasmid

After digesting scFv gene and phage vector, construct recombinant expression plasmid;

Step 4. Establish primary single-chain antibody library

Transform the recombinant expression plasmid into host cells, culture and amplify with helper phage to establish a primary single-chain antibody library;

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

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

Step 7. Construction of single-chain antibody prokaryotic expression plasmid

Digest the positive clone obtained from the screening, recover the gene encoding the single-chain antibody GapC-scFv-1, mix it with the prokaryotic expression vector pET32a(+) that has been digested synchronously, and connect overnight at 14-16°C, and transform the ligated product into DH5α competent After cells, pick a single clone, colony PCR and plasmid double enzyme digestion to verify the correct clone for sequencing;

Step 8, constructing the bacterial strain of single-chain antibody prokaryotic expression plasmid

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 correct is the constructed single-chain antibody Prokaryotic expression plasmid pET32a-GapC-scFv-1;

Step 9. Expression and purification of single-chain antibody protein

Cultivate the strain pET32a-GapC-scFv-1-BL21 constructed in step 8 as 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 induce at 28°C After 16-20 hours of expression, the single-chain antibody protein was purified.

Preferably, the scFv genes described in step 2 are connected in the order of VL-Linker-VH.

Preferably, the antibody variable region VL and heavy chain variable region VH amino acid sequences are shown in SEQ ID No.1 and SEQ ID No.2, the scFv amino acid sequence is shown in SEQ ID No.3, and the light and heavy chains The forward and reverse primers are VL F, VL R, VH F and VH R respectively, and the nucleotide sequences are shown in SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 and SEQ ID No.7. VLF , VH R contain SfiI and NotI restriction sites respectively, VHF, VL R contain complementary Linker sequences, the colony PCR primers described in step 7 are respectively VL-F, VH-R, and the nucleotide sequence is as SEQ ID No .8 and shown in SEQ ID No.9.

Preferably, in step 7, when connecting with the pET32a (+) carrier, the preferred enzyme cutting sites are EcoRI and XhoI, wherein EcoRI: GAATTC, Xho I: CTCGAG.

Preferably, in step 4, the host cell is TG1 cell.

The fifth aspect of the present invention is to provide a single-chain antibody prokaryotic expression plasmid pET32a-GapC-scFv-1 and bacterial strain pET32a-GapC-scFv-1 obtained by using the method for preparing the bovine-derived anti-GapC protein single-chain antibody -BL21.

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;

Fig. 2 is the electrophoresis diagram of the amplified fragment of the scFv positive clone gene screened by prokaryotic expression;

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

Figure 4 is a protein Western Bloting detection diagram of scFv gene expression;

Figure 5 shows that GapC-scFv-1 inhibits the growth of Staphylococcus aureus in vitro at different concentrations.

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.

Among them, VLF and VHR contain Sfi I and Not I 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 growth-promoting virulence factor GapC single-chain antibody

1. Enrichment panning

Prepare the prokaryotic expression product of Staphylococcus aureus (ATCC25923) GapC virulence factor, use it as an antigen, and coat overnight at 4°C; seal the 96-well plate with PBST containing 4% skim milk powder, and incubate at 37°C for 2h; Add the single-chain antibody phage antibody library prepared in the above steps, 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-Hcl buffer to each well (PH=2.2) to elute the specifically bound phage, add 50ul 1mol/L Tris-Hcl (PH=9.1) to neutralize the eluate; infect the remaining part of the eluate to Escherichia coli TG1, repeat the above steps. 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 picked from the fourth round, and recombinant phages were prepared after rescue with M13K07. The purified Staphylococcus aureus growth-promoting virulence factor GapC virulence factor prokaryotic expression protein was coated with 50mmol/L sodium bicarbonate salt solution (pH9.6) at 4°C overnight, and 4% skimmed milk powder solution was blocked 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 Blank Control 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 of pET32a-GapC-scFv-1

1. The construction of the recombinant plasmid pET32a-GapC-scFv-1 uses the positive cloned strain as a template, and uses specific primers (as shown in Table 2, the underline is the enzyme cutting site) to amplify the GapC-scFv-1 target gene, and the selection is limited The target gene and the prokaryotic expression vector pET32a(+) were double-digested with EcoRI and Xho I, and then ligated to obtain a recombinant plasmid, which was transformed into DH5α competent, and the correctness was verified by colony PCR and double-digestion of the plasmid. 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 pET32a-GapC-scFv-1 is shown in Figure 3.

2. Purification of single-chain antibody GapC-scFv-1 protein

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

1) Fix the purification column, keep the temperature low with an ice pack around it, 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 low-temperature 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-30mM imidazole, Ni-native-50mM imidazole, Ni-native100mM imidazole, Ni-native-150mM imidazole, Ni-native-200mM imidazole, Ni-native-250mM imidazole to compete To elute the target protein, control the 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 50mM imidazole, 100mM imidazole, 150mM imidazole, and 200mM imidazole protein eluent respectively, add protein electrophoresis loading buffer and cook in 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 the effect of the single-chain antibody GapC-scFv-1 on inhibiting the growth of Staphylococcus aureus

The experiment for detecting the growth inhibition of Staphylococcus aureus by the single-chain antibody GapC-scFv-1 includes:

1. Experimental materials and methods

1) Experimental materials

Staphylococcus aureus ATCC25923 (preserved in our laboratory), agar powder, agarose, yeast extract, tryptone (purchased from Sigma Company), ampicillin (purchased from OXOID Company), and 96-well enzyme plate purchased from Corning, USA The company and other kinds of reagents are domestic analytical pure or chemical pure reagents.

2) Experimental method

After the purified scFv-GapC protein is detected with a protein concentration detection kit, it is diluted with normal saline to a concentration of 10, 20, 40, 50, 100 μg/ml, and 200 μl 106 cfu/ml of Staphylococcus aureus (standard strain ATCC25923 ) mixed, co-incubated in 400 μl LB medium, cultured with shaking at 37°C, and physiological saline and penicillin were used as negative and positive controls. Observe the changes in the concentration of the bacterial solution in each group, measure the OD600 every 6 hours until the concentration of the bacterial solution in the negative control does not change, and repeat the experiment three times.

2. Data statistics and experimental results

The experimental results are shown in Figure 5. Compared with the blank control group, the difference between the scFv treatment group and the blank control group is significant (P<0.01). In 0-12h, with the increase of time, the scFv's effect on the growth of Staphylococcus aureus Inhibition was dose-dependent.

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 inhibiting the growth of Staphylococcus aureus, characterized in that it comprises light chain variable region VL, heavy chain variable region VH, linker peptide Linker, and is connected in the order of VL-Linker-VH Constitute a bovine single-chain antibody fragment VL-Linker-VH, the amino acid sequence of the light chain variable region VL of the body 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 Show. 2. the bovine source according to claim 1 suppresses the single-chain antibody of Staphylococcus aureus growth, is characterized in that, described single-chain antibody fragment VL-Linker-VH aminoacid sequence is as shown in SEQ ID No.3. 3. A medicament for inhibiting cow mastitis, characterized in that the medicament comprises the single-chain antibody according to claim 1 or 2. 4. A dairy cow mastitis diagnostic kit, characterized in that it comprises the single-chain antibody described in claim 1 or 2, or the gene fragment encoding the single-chain antibody described in claim 1 or 2 and a probe cross-linked therewith. Needle. 5. A prokaryotic expression plasmid pET32a-GapC-scFv-1, characterized in that the plasmid comprises the gene encoding the bovine-derived single-chain antibody that inhibits the growth of Staphylococcus aureus according to claim 1 or 2. 6. A kind of prokaryotic expression bacterial strain pET32a-GapC-scFv-1-BL21, it is characterized in that, described bacterial strain is transfected with the coding gene of the single-chain antibody that the bovine source described in claim 1 or 2 suppresses the growth of Staphylococcus aureus .

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