CN114276447B - Bovine-derived single-chain antibody for inhibiting growth of staphylococcus aureus and preparation method and application thereof - Google Patents
Bovine-derived single-chain antibody for inhibiting growth of staphylococcus aureus and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a bovine-derived single-chain antibody for inhibiting the growth of staphylococcus aureus and a preparation method and application thereof, wherein a VH gene of a heavy chain variable region and a VL gene of a light chain variable region of a bovine antibody coding gene are directly amplified by RT-PCR (reverse transcription-polymerase chain reaction), a linker is connected with the VH gene and the VL gene by utilizing an SOE-PCR (sequence of amplification-polymerase chain reaction) method to construct a bovine-derived scFv gene, the scFv gene is cloned into a phagemid vector pCANTAB5E to construct a single-chain antibody primary library, and an auxiliary phage M13KO7 rescues the primary library; after four rounds of enrichment and panning by using prokaryotic expression staphylococcus aureus growth-promoting virulence factor GapC protein as a coating antigen, positive clones are screened by adopting a phase ELISA method, and the single-chain antibody is proved to have the effect of inhibiting the growth of the staphylococcus aureus.
Description
Technical Field
The invention relates to the field of genetic engineering, in particular to a bovine-derived single-chain antibody for inhibiting the growth of staphylococcus aureus, and a preparation method and application thereof.
Background
The single-chain antibody is a genetic engineering antibody, and is formed by connecting the light chain variable region VL and the heavy chain variable region VH of an antibody end to end through a section of connecting short peptide linker by a DNA recombination technology, and is a minimum functional fragment for retaining the whole antigen binding part. The expression forms of the single-chain antibody mainly comprise three forms of fusion expression, intracellular expression and secretory 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.
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.
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.
Disclosure of Invention
The invention aims to provide a bovine-derived single-chain antibody for inhibiting the growth of staphylococcus aureus, a preparation method and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a bovine single-chain antibody for inhibiting the growth of staphylococcus aureus, 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, the heavy chain variable region VH and the connecting peptide Linker are connected in the sequence of VL-Linker-VH to form a bovine 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.
Furthermore, the amino acid sequence of the single-chain antibody fragment VL-Linker-VH is shown in SEQ ID No. 3.
In a second aspect, the invention provides a medicament for inhibiting mastitis in a cow, which comprises the single-chain antibody.
The third aspect of the invention provides a diagnostic kit for mastitis in dairy cows, which is characterized by comprising the single-chain antibody, or a gene fragment encoding the single-chain antibody and a probe crosslinked with the single-chain antibody.
The fourth aspect of the invention provides a preparation method of a bovine-derived single-chain antibody for inhibiting the growth of staphylococcus aureus, which comprises the following steps:
Amplifying a light chain variable region VL gene and a heavy chain variable region VH gene of an antibody encoding gene from peripheral blood mononuclear cell RNA of the mastitis of the dairy cow by adopting RT-PCR;
Connecting the intermediate connecting peptide linker, the VH gene and the VL gene by using an SOE-PCR method to construct a bovine-derived single-chain antibody gene, namely an scFv gene;
Carrying out enzyme digestion on the scFv gene and a phage vector, and constructing a recombinant expression plasmid;
step 4, establishing a primary single-chain antibody library
Transforming the recombinant expression plasmid into host cells, culturing and amplifying by using helper phage to establish a primary single-chain antibody library;
step 6, screening by adopting phase ELISA, and screening positive clones by using prokaryotic expression staphylococcus aureus GapC protein as a coating antigen;
Carrying out enzyme digestion on the screened positive clone, recovering a single-chain antibody GapC-scFv-1 coding gene, uniformly mixing the coding gene with a prokaryotic expression vector pET32a (+) subjected to synchronous enzyme digestion, connecting the coding gene and the prokaryotic expression vector pET32a (+) at 14-16 ℃ overnight, transforming a DH5 alpha competent cell by a connecting product, then picking the single clone, and sequencing the clone which is verified to be correct by colony PCR and plasmid double enzyme digestion;
Extracting plasmids from clones with correct sequencing, transforming recombinant plasmids into BL21 competent cells, picking single clones, verifying correct clone sequencing by colony PCR and plasmid double enzyme digestion, and obtaining the constructed single-chain antibody prokaryotic expression plasmid pET32a-GapC-scFv-1 with correct sequencing;
And (3) culturing the bacterial strain pET32a-GapC-scFv-1-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, carrying out induced expression at 28 ℃ for 16-20h, and purifying the single-chain antibody protein.
Preferably, the scFv genes in step 2 are linked in the order VL-Linker-VH.
Preferably, the amino acid sequences of the variable region VL and the variable region VH of the heavy chain of the antibody are shown in SEQ ID No.1 and SEQ ID No.2, the amino acid sequence of the scFv is shown in SEQ ID No.3, the forward primers and the reverse primers of the light chain and the heavy chain are respectively VL F, VL R, VH F and VH R, the nucleotide sequences are shown in 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 primer in the step 7 is VL-F, VH-R, and the nucleotide sequences are shown in SEQ ID No.8 and SEQ ID No. 9.
Preferably, in step 7, when ligated to the pET32a (+) vector, the preferred cleavage sites are EcoRI and XhoI, where EcoR I: GAATTC, xho I: CTCGAG.
Preferably, in step 4, the host cell is a TG1 cell.
The fifth aspect of the invention provides a single-chain antibody prokaryotic expression plasmid pET32a-GapC-scFv-1 and a strain pET32a-GapC-scFv-1-BL21 which are obtained by the preparation method of the bovine-derived anti-GapC protein single-chain antibody.
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 is an electrophoretogram of amplified fragments of scFv positive cloned genes selected by prokaryotic expression;
FIG. 3 is a SDS-PAGE map of scFv gene expression proteins;
FIG. 4 is a Western blotting detection chart of the protein expressed by scFv gene;
FIG. 5 shows the inhibition of Staphylococcus aureus growth in vitro at various concentration levels of GapC-scFv-1.
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. Niu Waizhou white blood cells were extracted by anticoagulation, and total RNA was extracted by Trizol method (available from TaKaRa, TRIZOL Reagent). 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.
The VLF and the VH R respectively contain Sfi I restriction enzyme cutting sites and Not I restriction 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 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.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 ℃ 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
The 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 growth-promoting virulence factor GapC Single-chain antibody against Staphylococcus aureus
1. Enrichment panning
Preparing prokaryotic expression product of staphylococcus aureus (ATCC 25923) GapC virulence factor, using the prokaryotic expression product as antigen, and coating 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
From the fourth round, 96 clones were randomly picked and rescued with M13K07 to prepare recombinant phages. The purified staphylococcus aureus growth-promoting virulence factor GapC virulence factor prokaryotic expression protein was coated with 50mmol/L sodium bicarbonate solution (pH 9.6) overnight at 4 ℃, blocked with 4% skim milk powder solution for 1h, washed 3 times with PBST (0.1% Tween20, the same below); 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 of pET32a-GapC-scFv-1
1. Constructing a recombinant plasmid pET32a-GapC-scFv-1 by taking a positive clone strain as a template, amplifying a GapC-scFv-1 target gene by using a specific primer (shown in a table 2, wherein underlining 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, transforming the recombinant plasmid to DH5 alpha competence, and sending a clone with correct colony PCR and plasmid double enzyme cutting 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 to verify that the correct clones are sent to Shanghai platinum biotechnology company Limited for sequencing, wherein the clones with correct sequencing are the successfully constructed prokaryotic expression recombinant plasmids pET32a-GapC-scFv-1, as shown in figure 3.
2. Purification of Single chain antibody GapC-scFv-1 protein
The fusion protein expressed by the pET32a (+) vector carries His-tag, so that the GapC 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 a Ni-native-0buffer balance purification column with the column volume of 5-10 times, and controlling the 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-30mM imidazole, ni-native-50mM imidazole, ni-native100mM imidazole, ni-native-150mM imidazole, ni-native-200mM imidazole and Ni-native-250mM imidazole in column volumes of 5-10 times, 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 50mM imidazole, 100mM imidazole, 150mM imidazole and 200mM imidazole protein eluent, adding protein electrophoresis Loading Buffer, boiling in water bath, boiling for 10min,
solubility identification 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 the electrophoresis is finished, dyeing is carried out for 45min by using 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 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 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 testing of the Effect of the Single chain antibody GapC-scFv-1 on the inhibition of Staphylococcus aureus growth
The experiment for detecting the effect of the single-chain antibody GapC-scFv-1 on inhibiting the growth of staphylococcus aureus comprises the following steps:
1. experimental materials and methods
1) Experimental materials
Staphylococcus aureus ATCC25923 (stored in the laboratory), agar powder, agarose, yeast extract, tryptone (purchased from Sigma), ampicillin (purchased from OXOID), 96-well enzyme-linked plate (purchased from American Corning company), and other various reagents are domestic analytical pure or chemical pure reagents.
2) Experimental methods
The purified scFv-GapC protein is diluted to the concentration of 10, 20, 40, 50 and 100 mu g/ml by using a protein concentration detection kit, mixed with 200 mu l of 106cfu/ml staphylococcus aureus (standard strain ATCC 25923), incubated in 400 mu l of LB culture medium together, cultured by shaking at 37 ℃, and the physiological saline and penicillin are used as negative and positive controls. And observing the concentration change of the bacterial liquid of each group, measuring the OD600 every 6h until the concentration of the bacterial liquid of the negative control is not changed, and repeating the experiment for three times.
2. Data statistics and experimental results
The experimental results are shown in figure 5, the difference between the scFv treated group and the blank control group is significant (P < 0.01), and the inhibition of the growth of Staphylococcus aureus by scFv shows a dose dependency with the time increase in 0-12 h.
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 (6)
1. A bovine-derived single-chain antibody for inhibiting the growth of staphylococcus aureus 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 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 for inhibiting the growth of staphylococcus aureus according to claim 1, wherein the amino acid sequence of the single-chain antibody fragment VL-Linker-VH is shown as SEQ ID No. 3.
3. A medicament for inhibiting mastitis in a cow which comprises the single-chain antibody according to claim 1 or 2.
4. A diagnostic kit for mastitis in a cow, comprising the single-chain antibody according to claim 1 or 2, or a gene fragment encoding the single-chain antibody according to claim 1 or 2, and a probe crosslinked thereto.
5. A prokaryotic expression plasmid pET32a-GapC-scFv-1, wherein the plasmid comprises the gene encoding the bovine-derived single-chain antibody that inhibits the growth of staphylococcus aureus of claim 1 or 2.
6. A prokaryotic expression strain pET32a-GapC-scFv-1-BL21, which is transfected with the gene encoding the bovine-derived single-chain antibody inhibiting the growth of staphylococcus aureus of claim 1 or 2.
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