CN105641689B

CN105641689B - Preparation method and application of beta-hemolysin subunit vaccine of staphylococcus aureus of dairy cows

Info

Publication number: CN105641689B
Application number: CN201610068816.7A
Authority: CN
Inventors: 钱泓; 吴有强; 宣春玲; 查银河; 贾宝琴; 曹珊珊; 陈藻
Original assignee: Zhejiang Hailong Biotechnology Co ltd
Current assignee: Zhejiang Hailong Biotechnology Co ltd
Priority date: 2016-01-21
Filing date: 2016-01-21
Publication date: 2024-09-10
Anticipated expiration: 2036-01-21
Also published as: CN105641689A

Abstract

The invention discloses a preparation method and application of a beta-hemolysin subunit vaccine of staphylococcus aureus of dairy cows. Aims at providing a preparation method of subunit vaccine for preventing or treating staphylococcus aureus mastitis of dairy cows, which comprises the following steps: 1) Site-directed mutagenesis of a staphylococcus aureus beta-hemolysin protein gene; 2) Cloning the site-directed mutant beta-hemolysin protein gene into a pET28a vector; 3) Transforming the expression vector obtained in the step 2) into E.coli BL21 (DE 3), and carrying out induced expression to obtain recombinant beta-hemolysin protein; 4) Purifying the recombinant beta-hemolysin protein obtained in the step 3) by using nickel column affinity chromatography; 5) And fully and uniformly mixing the purified recombinant beta-hemolysin protein with a pharmaceutically acceptable adjuvant to obtain the recombinant subunit vaccine for the staphylococcus aureus mastitis of the dairy cows.

Description

Preparation method and application of beta-hemolysin subunit vaccine of staphylococcus aureus of dairy cows

Technical Field

The invention relates to a preparation method and application of subunit vaccine for dairy cow staphylococcus aureus mastitis. Belongs to the technical field of biological vaccine preparation.

Background

Dairy cow Mastitis (Mastis) is the most common infectious disease of adult cows, mainly an inflammation caused by microbial infection of cow mammary tissue, and is frequently generated in the postnatal lactation period, and the disease is widely existing in various places in the world and is a common disease and frequently-occurring disease of cows, and is the disease causing the most serious economic loss of dairy industry. The pathogenic microorganisms causing cow mastitis are about 150, mainly staphylococcus aureus, escherichia coli and streptococcus agalactiae, and the cow mastitis caused by the three bacteria account for more than 90% of the total incidence rate, wherein staphylococcus aureus is the most important.

The current treatment method of the dairy cow mastitis mainly adopts antibiotic treatment, the antibiotic has been used for treating the dairy cow mastitis for more than 50 years, and plays a certain role in preventing and treating the dairy cow mastitis, but the long-term single and large-dose unscientific use of the antibiotic causes elimination of sensitive bacteria, drug-resistant bacteria gradually occupy the main stream, and especially the drug-resistant problem of staphylococcus aureus is increasingly serious, so that the treatment effect of the antibiotic is very little.

The vaccine has good prospect for preventing and treating the cow mastitis, firstly, the vaccine can prevent the cow from infecting pathogenic bacteria to cause the cow mastitis; secondly, the vaccine helps to reduce the severity of breast infection, controlling subclinical mastitis; thirdly, the vaccine can be used for preventing and treating the mammitis without the problem of antibiotic residues in milk; finally, the operation is simple and convenient, and the cost is low. At present, the developed vaccine is few, and the most number is attenuated live vaccine or inactivated vaccine, and has a certain effect on preventing and treating mammitis in production practice, however, along with the development of large-scale intensive culture, the artificial attenuated strain has potential possibility of homologous recombination, self virulence reversion and the like, and the inactivated vaccine also has the defects of large dosage, short immunization period and the like, so that the development of a novel vaccine with high efficiency and low cost is extremely important for improving the safety and the immunity protection of the traditional vaccine.

Staphylococcus aureus (Staphylococcus aureus, abbreviated as SA), also known as "staphylococcus aureus", is a gram-positive coccus of 0.8 μm diameter, which is arranged in a cluster under a microscope and produces a golden yellow pigment, and is therefore known. The staphylococcus aureus is one of main pathogenic bacteria causing chronic/recessive cow mastitis, can greatly influence the yield and quality of milk, and brings great economic loss to the dairy industry. Studies have shown that beta-hemolysin secreted by Staphylococcus aureus is a causative agent of bovine mastitis.

The staphylococcus aureus can produce a variety of toxins and enzymes. Beta hemolysin (also known as beta-toxin) is one of its toxins, and has characteristics of leukocyte cytotoxicity, hemolytic activity, and the like. Beta hemolysin is a single-chain polypeptide consisting of 330 amino acids, has a molecular weight of 37-39 kDa and an isoelectric point (pI) higher than 9. Beta hemolysin is a magnesium-dependent neuroesterase with Phospholipase C (PLC) activity, which can decompose glycerophosphorylcholine, by which beta hemolysin hydrolyzes phospholipid bilayer constituting cell membrane, thus destroying the integrity of cell membrane, leading to cell lysis, causing hemolysis. After site-directed mutagenesis is carried out on the beta hemolysin, the mutant loses hemolytic activity, has no toxicity, still retains perfect immunogenicity, can be directly used for immunizing animals, and is one of important candidate proteins of the staphylococcus aureus subunit vaccine.

Disclosure of Invention

The invention aims to provide a preparation method and application of a beta-hemolysin subunit vaccine of staphylococcus aureus of dairy cows. The subunit vaccine prepared by mixing the recombinant protein and a proper adjuvant is similar to the natural infection process of pathogenic bacteria, can promote cellular immunity, induce immune memory and cause wide immune response, generates good cross protection reaction, and is a novel vaccine which is safer and more stable, simple to prepare, convenient to use and low in cost.

The recombinant beta-hemolysin protein is derived from staphylococcus aureus, and the nucleotide sequence of the encoded protein is shown as SEQ ID NO. 2.

The invention also provides a preparation method of the beta-hemolysin subunit vaccine of the staphylococcus aureus of the dairy cows, which mainly comprises the following steps: 1) Site-directed mutagenesis of a staphylococcus aureus beta-hemolysin protein gene; 2) Cloning the site-directed mutant beta-hemolysin protein gene into a pET28a vector; 3) Transforming the expression vector obtained in the step 2) into E.coli BL21 (DE 3), and carrying out induced expression to obtain recombinant beta-hemolysin protein; 4) Purifying the recombinant beta-hemolysin protein obtained in the step 3) by using nickel column affinity chromatography; 5) And fully and uniformly mixing the purified recombinant beta-hemolysin protein with a pharmaceutically acceptable adjuvant (such as an ISA 206VG adjuvant) to obtain the recombinant subunit vaccine for the staphylococcus aureus mastitis of the dairy cows.

Compared with the prior art, the invention has the following advantages and effects: the cow mammitis subunit vaccine is prepared with the main protective immunogen of pathogenic bacteria, and has no nucleic acid and capacity of inducing body to produce antibody, and the vaccine is injected directly into body to activate immune system for preventing and treating diseases. The subunit vaccine prepared by mixing the recombinant protein and a proper adjuvant is similar to the natural infection process of pathogenic bacteria, can promote cellular immunity, induce immune memory and cause wide immune response, generates good cross protection reaction, and is a novel vaccine which is safer and more stable, simple to prepare, convenient to use, low in price, time-saving and labor-saving and has obvious curative effect.

Drawings

FIG. 1, agarose gel electrophoresis results of beta-hemolysin fragment PCR products, M: marker DL2,000;1 is a site-directed mutant fragment of beta-hemolysin 1 (509 bp), and 2 is a site-directed mutant fragment of beta-hemolysin 2 (468 bp).

FIG. 2, beta-hemolysin site-directed mutagenesis overlap PCR agarose gel electrophoresis results, M: marker DL5,000;1 is the site-directed mutagenesis overlap PCR product of beta-hemolysin (936 bp).

FIG. 3, results of agarose gel electrophoresis of site-directed mutagenesis recombinant plasmid restriction enzyme digestion identification of beta-hemolysin, M: marker DL5,000;1 is pET28 a-beta hemolysin site-directed mutagenesis plasmid No. 3 (NdeI/XhoI), and 2 is pET28 a-beta hemolysin site-directed mutagenesis plasmid No. 4 (NdeI/XhoI).

FIG. 4, beta-hemolysin nickel column affinity chromatography purification results, FT: flow through;50mM:50mM imidazole; 100mM:100mM imidazole; 150mM:150mM imidazole.

FIG. 5, results of renaturation of recombinant beta-hemolysin protein.

FIG. 6 results of ELISA detection of titers after immunization of mice with the beta-hemolysin subunit vaccine.

FIG. 7 shows the results of experiments on survival rate of mice after immune challenge.

Detailed Description

EXAMPLE 1 construction of expression vector pET28 a-beta-hemolysin site-directed mutagenesis

The result of PCR was carried out by dividing beta-hemolysin into two segments using the clinically isolated staphylococcus aureus genome of cows as a template, and is shown in FIG. 1.

1.1 The loading system was (50 μl):

1.2 PCR amplification procedure:

1.3 gel recovery of DNA fragments:

(1) Subjecting the reaction solution of step 1.2 to 0.8% agarose gel electrophoresis (110V for 30 min);

(2) Cutting gel under ultraviolet lamp to recover DNA fragment in 1.5ml EP tube;

(3) Adding 500. Mu.l of PC buffer into the 1.5ml EP tube in the step (2), and carrying out water bath at 50 ℃ for 10min;

(4) Transferring the solution in the step (3) to the center of an adsorption column, standing for 2min, centrifuging at 12,000rpm for 30s;

(5) Discarding the waste liquid, adding 600 μl PW buffer into the center of the adsorption column, standing for 3min, and at 12,000rpm for 30s;

(6) Repeating step (5);

(7) Centrifuging the empty adsorption column at 12,000rpm and 1min;

(8) Adding 30 μl of ddH ₂ O to the center of the adsorption column, standing for 3min, and centrifuging (12,000 rpm,2 min);

(9) And (3) collecting the DNA sample in the step (8) for electrophoresis.

1.4 Overlap PCR, site-directed mutagenesis of β -hemolysin was performed and the results are shown in FIG. 2:

the loading system was (50 μl):

PCR amplification procedure:

1.5 double cleavage reaction (50. Mu.l system):

Sample addition and mixing were performed according to step 1.5 in a 1.5ml EP tube, and then the two 50. Mu.l reaction solutions were placed in a 37℃thermostat water bath for 3h.

1.6 Gel recovery of DNA fragments:

(1) Subjecting the reaction solution of step 1.5 to 0.8% agarose gel electrophoresis (110V 30 min);

(6) Repeating step (5);

(7) Centrifuging the empty adsorption column at 12,000rpm and 1min;

(9) And (3) collecting the DNA sample in the step (8) for electrophoresis.

1.7 Ligation (10. Mu.l system):

sample addition and uniform mixing are carried out in a 1.5ml EP tube according to the system, the reaction liquid is placed at 16 ℃ for 16 hours in a water bath, then taken out, inactivated after 15 minutes in the water bath at 65 ℃, and the sample is stored at 4 ℃.

1.8 Transformation experiments:

(1) Taking out the reaction solution in the step 1.7, adding 100 mu l of E.coli DH5 beta competent cells into the reaction solution, and uniformly mixing the mixture;

(2) Ice bath for 30min;

(3) Water bath at 42 ℃ for 100s;

(4) Ice bath for 2min;

(5) Taking out, adding 600 μl of liquid LB culture medium into the EP tube, and water-bathing at 37deg.C for 1 hr;

(6) Remove, centrifuge (8,000 rpm,2 min), remove 600. Mu.l, and leave 100. Mu.l LB resuspended cells;

(7) Bacterial liquid was plated on LK plate (Kan concentration 50. Mu.g/ml), and LK plate was placed in biochemical incubator at 37℃for 12 hours.

1.9 Recombinant plasmid extraction and enzyme digestion identification:

(1) Monoclonals were picked from the transformation plate into 3ml LK liquid medium, shaking at 37℃and 260rpm overnight;

(2) Taking 1ml of bacterial liquid into a 1.5ml EP tube, centrifuging (12,000 rpm,2 min), and discarding the supernatant;

(3) Adding 250 mu l P to 1buffer into the EP tube in the step (2) to resuspend the thalli;

(4) Adding 250 mu l P to 2buffer into the solution in the step (3), mixing gently, and standing for 2min;

(5) Adding 350 mu l P buffer into the solution in the step (4), and gently mixing;

(6) Centrifuging (12,000 rpm,10 min) the solution of step (5);

(7) Transferring the supernatant solution in the step (6) to the center of an adsorption column, and centrifuging (8,000 g,30 s);

(8) Discarding the waste liquid, adding 500 μl of wash buffer into the center of the adsorption column, and centrifuging (9,000 g,30 s);

(9) Repeating step (8);

(10) Centrifuging (9,000 g,1 min) with a hollow adsorption column;

(11) Adding 30 μl of the adsorption column into the adsorption buffer, standing for 2min, and centrifuging (12,000 rpm,2 min);

(12) Collecting the DNA sample in the step (11) for electrophoresis;

(13) The extracted plasmid was identified by digestion and then subjected to 0.8% agarose gel electrophoresis as shown in step 1.5.

The result of the enzyme digestion identification of the recombinant plasmid is shown in figure 3.

Example 2 transformation of E.coli BL21

Mu.l of plasmid was aspirated and added to 100. Mu.l BL21 competent cells, ice-bath for 30min;

heat shock at 42 ℃ for 90s;

Ice bath for 2min;

adding 900 mu l of non-resistant LB culture solution into the super clean bench;

shaking at 180rpm at 37 ℃ for 1h;

Mu.l of the bacterial liquid was pipetted onto a card-resistant LB plate and incubated overnight at 37 ℃.

Example 3 massive induction of expression

Selecting: selecting a monoclonal antibody into 50ml of kana-resistant LB culture solution, and culturing at 37 ℃ overnight;

And (3) switching: transferring the bacterial liquid to 500ml of kana resistance LB culture liquid according to the ratio of 1:100, shaking 3.5L altogether, and culturing at 37 ℃ and 220rpm for 2-2.5h until the OD ₆₀₀ value reaches 0.6;

Induction: after the bacterial liquid OD ₆₀₀ is 0.6, 500 μl of IPTG (1M) is added until the final concentration of the IPTG is 1mmol/L, and the culture is induced for 4h at 37 ℃ at 220 rpm;

And (3) thallus collection: centrifuging the bacterial liquid at 6,000rpm for 10min, and collecting bacterial cells; washing the thallus with 40ml PBS, centrifuging at 6,000rpm for 10min, collecting thallus, and storing at-20deg.C;

EXAMPLE 4 preparation of beta-hemolysin protein inclusion bodies

(1) And (3) thallus crushing: re-suspending the thallus with lysate (50 mM NaH ₂PO₄, 500mM NaCl,pH 8.0), blowing the thallus with a syringe uniformly, and avoiding generation of massive precipitate; cracking the bacterial liquid by using an Avestin cell breaker; after crushing, centrifuging the thalli at 12,000rpm and at 4 ℃ for 30min, discarding the supernatant, and reserving sediment;

(2) The precipitate was resuspended in 25ml of solution (lysate, 100. Mu.M PMSF,10mM EDTA,10mM Benzamidine,0.01%NaN ₃); centrifuging at 12,000rpm and 4 ℃ for 20min, discarding the supernatant, and retaining the precipitate;

(3) Repeating the step (2) once;

(4) The pellet was resuspended in 25ml of solution (lysate, 100. Mu.M PMSF,10mM MgCl,10mM Benzamidene,0.01%NaN ₃); centrifuging at 12,000rpm and 4 ℃ for 20min, discarding the supernatant, and retaining the precipitate;

(5) The precipitate was weighed and dispensed into 1.5ml EP tubes at 1 g/tube and stored at-20 ℃.

EXAMPLE 5 Nickel column affinity chromatography purification of recombinant beta-hemolysin protein

1G of inclusion bodies were added to 30ml of a denaturing solution (50 mM NaH ₂PO₄, 500mM NaCl,8M urea, pH 8.0) and stirred overnight at room temperature; centrifuging at 12,000rpm and 22 ℃ for 20min, taking supernatant, and sucking 20 μl of sample for SDS-PAGE detection;

Column balance: loading 4ml Ni-agarose into column, balancing 10 column volumes with denatured solution, discharging buffer solution to make liquid level higher than filler by 1mm to avoid drying column;

Combining: mixing the well-balanced filler with the sample liquid, and shaking on ice for 1h;

After the combination is completed, adding the mixed solution into an empty column, collecting Flow through, and reserving 20 μl of sample for detection;

The column was washed with 30 column volumes of solution (50 mM NaH ₂PO₄, 500mM NaCl,8M urea, 0.1%Triton X114,pH 8.0) until no protein was detected in the effluent, followed by 10 column volumes of denaturing solution to leave 20. Mu.l for detection;

Eluting: imidazole is added into the denatured solution to prepare 50mM, 100mM and 150mM imidazole eluents, each time, 5ml imidazole eluents are used for eluting, and each concentration is used for 5 times of elution;

SDS-PAGE analysis; the results are shown in FIG. 4.

EXAMPLE 6 renaturation of recombinant beta-hemolysin protein

Cutting a dialysis bag with proper length, soaking with a dialysis solution (50 mM NaH ₂PO₄, 150mM NaCl,pH 7.4), clamping one end with a dialysis clamp, adding the sample solution to be renatured into the dialysis bag, clamping the top end with the dialysis clamp, and dialyzing at 4 ℃ for 12h;

Discarding the dialysate, adding 2L of fresh dialysate, and continuing dialysis for 2 hours; centrifuging at 12,000rpm and 4deg.C for 10min after dialysis, and collecting supernatant;

The BCA method is used for measuring the protein concentration, and then split charging and storing at-80 ℃; SDS-PAGE analysis results are shown in FIG. 5.

EXAMPLE 7 preparation of recombinant beta-hemolysin protein subunit vaccine

According to experimental requirements, calculating the amounts of all components of the vaccine solution to ensure that the final concentration of the recombinant beta-hemolysin protein in the vaccine is 25 mug/ml, wherein the volume ratio of the recombinant beta-hemolysin protein to the adjuvant ISA 206VG is 46:54;

Placing the diluted and mixed recombinant beta-hemolysin protein solution and ISA 206VG adjuvant in a constant-temperature water bath kettle, heating to 32+/-1 ℃, adding an antigen into an adjuvant pipe after the temperature is stable, and vibrating for 10min by a vibrator to pre-emulsify;

Placing the pre-emulsified vaccine in a beaker filled with ice, and fixing the vaccine on a pre-treated ultrasonic cell disruption instrument for emulsification;

After emulsification is finished, observing the emulsification effect: placing part of vaccine into a centrifuge tube, centrifuging at 3,000rpm for 15min, and judging that the vaccine is qualified without layering;

Packaging the vaccine qualified in detection into 15ml centrifuge tube, marking, sealing with sealing film, and storing at 4deg.C.

Example 8 mouse immunization experiment

Placing the vaccine at room temperature (25deg.C), and recovering the vaccine temperature to normal temperature;

Mice were weighed, grouped and labeled, one group was an immunoassay group (n=6), immunized with β -angiostatin subunit vaccine; the other group was a control group (n=6), PBS was immunized, and the values were recorded;

1ml of the vaccine was aspirated with a 1ml syringe and 50. Mu.l of each of the left and right hind legs was injected.

Example 9 toxicity test after mice immunization

SA single colony (strain is HB0911-3 strain preserved in the laboratory) is selected and placed in 5ml of liquid broth culture medium, and the strain is shaken at 220rpm and 37 ℃ for overnight;

Inoculating the bacteria at one percent volume (1 ml) overnight in 100ml fresh liquid broth medium, shaking at 220rpm at 37 ℃ overnight;

filling 100ml of bacterial liquid into a 500ml centrifugal bottle, centrifuging at 8,000rpm for 10min, sucking off the culture medium, re-suspending the bacterial cells with 100ml of PBS, repeating the steps for 3 times, and finally re-suspending the bacterial cells with 5ml of PBS uniformly;

The bacterial liquid was diluted 10,000 times and counted. Diluting the original bacterial liquid to 5X 10 ⁸CFU/ml(2M LD₅₀ according to the concentration of the bacterial liquid);

mice in the immunized group and the control group were weighed, one group was the immune test group (n=6), and the other group was the control group (n=6), and the values were recorded;

the bacterial solution was aspirated by a 1ml syringe, and tail vein injection was performed on mice corresponding to each concentration of bacteria, with an injection amount of 200. Mu.l/20 g mice.

Continuously observing the survival condition of the mice, and recording the death time of each mouse; the survival rate results of the mice after challenge are shown in figure 7.

EXAMPLE 10ELISA detection of beta-hemolysin antibody titres

(1) Coating: diluting the purified protein for detection with coating solution (50 mM carbonate buffer solution, pH 9.5) to 0.5 μg/ml, adding 100 μl per well on the ELISA plate, sealing with sealing film, and standing overnight at 4deg.C in refrigerator;

(2) Washing: taking out the ELISA plate from the refrigerator, putting the ELISA plate into a plate washer for washing, and using PBST for washing liquid;

(3) Closing: 200 μl of sealing solution (5% skimmed milk) is added into each hole, and the mixture is incubated for 2h at 37 ℃ after sealing the sealing film;

(4) Sample preparation: moderately diluting serum with a sealing liquid according to known information and required dosage;

(5) Washing: and (2);

(6) Sample adding: adding diluted serum, and simultaneously taking a blocking solution as a negative control, and incubating for 1h at 37 ℃;

(7) Washing: and (2);

(8) Adding a secondary antibody: add 100. Mu.l of appropriately diluted HRP-labeled secondary antibody per well and incubate at 37℃for 0.5h;

(9) Washing: and (2);

(10) Color development: adding 100 μl of TMB color developing solution into each well under dark condition, and incubating at 37deg.C for 10min;

(11) And (3) terminating: mu.l of stop solution (2M H ₂SO₄) was added to each well to stop the reaction;

(12) And (3) detection: measuring the OD value of the sample at the wavelength of 450nm, and analyzing the data;

(13) Analysis of results: criteria for judging antibody positivity: P/N is more than or equal to 2.1, and OD450 is more than or equal to 0.1.

Claims

1. A preparation method of a staphylococcus aureus beta-hemolysin subunit vaccine for dairy cows is characterized by comprising the following steps:

1) The amino acid sequence of the staphylococcus aureus protective antigen protein beta-hemolysin is shown as SEQ ID NO.1, and the gene sequence of the beta-hemolysin protein with the site-directed mutation is cloned into a pET28a vector as shown as SEQ ID NO. 2;

2) Converting the expression vector obtained in the step 1) into competent cells, and carrying out induced expression and purification to obtain recombinant beta-hemolysin protein; and

3) And fully and uniformly mixing the recombinant beta-hemolysin protein with the ISA206VG adjuvant to obtain the recombinant subunit vaccine for the staphylococcus aureus and the mastitis of the dairy cows.

2. The method of claim 1, wherein the competent cell is e.coli BL21 (DE 3).

3. The method of claim 1, wherein the purification in step 2) is a nickel column affinity chromatography purification.