CN105641689A - Preparing method and application of dairy cattle staphylococcus aureus beta-hemolysin subunit vaccine - Google Patents
Preparing method and application of dairy cattle staphylococcus aureus beta-hemolysin subunit vaccine Download PDFInfo
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- CN105641689A CN105641689A CN201610068816.7A CN201610068816A CN105641689A CN 105641689 A CN105641689 A CN 105641689A CN 201610068816 A CN201610068816 A CN 201610068816A CN 105641689 A CN105641689 A CN 105641689A
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Abstract
The invention discloses a preparing method and application of a dairy cattle staphylococcus aureus beta-hemolysin subunit vaccine, and aims at providing a preparing method for a subunit vaccine for preventing or treating dairy cattle staphylococcus aureus mastitis. The preparing method comprises the following steps that firstly, a staphylococcus aureus beta-hemolysin protein gene is subjected to site-specific mutagenesis; secondly, the beta-hemolysin protein gene subjected to site-specific mutagenesis is cloned into a pET28a vector; thirdly, the expression vector obtained in the second step is converted into E.coli BL21(DE3), and recombination beta-hemolysin protein is obtained through induction expression; fourthly, the recombination beta-hemolysin protein obtained in the third step is purified through nickel column affinity chromatography; fifthly, the recombination beta-hemolysin protein obtained through purification is fully mixed with a medically-acceptable adjuvant, and the recombination subunit vaccine for dairy cattle staphylococcus aureus mastitis is obtained.
Description
Technical field
The present invention relates to the preparation method of the subunit vaccine of a kind of staphylococcus aureus mastitis in dairy cows and application. Belong to biovaccine preparing technical field.
Background technology
Mammitis of cow (Mastitis) is the most general infectious diseases of adult milk cow, a kind of inflammation that mainly cow mammary gland tissue is subject to infected by microbes and cause, multiple it is born in lactation in postpartum, this disease is extensively present in all over the world, for milk cow common disease, frequently-occurring disease, it it is the disease causing milk-product industry financial loss the most serious. Cause the nearly kind more than 150 of the pathogenic micro-organism of mammitis of cow, main based on streptococcus aureus, intestinal bacteria, streptococcus agalactiae, these three kinds thin microbial mammitis of cow account for more than the 90% of total incidence, wherein especially taking streptococcus aureus as.
The methods for the treatment of of current mammitis of cow mainly adopts antibiotic therapy, microbiotic is used for the treatment of the mammitis of cow history of existing more than 50 year, it plays certain effect in the control of mammitis of cow, but due to not scientifical use microbiotic single, heavy dose of for a long time, cause the elimination of sensitive bacteria, drug tolerant bacteria account for main flow gradually, and especially the resistance problems of streptococcus aureus is day by day serious, result in antibiotic therapy and produces little effect.
Preventing and treating mammitis of cow with vaccine and have good prospect, first, vaccine can prevent milk cow infection pathogen and cause mastitis; Secondly, vaccine contributes to reducing the severity of intramammary infection, controls subclinical type mastitis; 3rd, it may also be useful to vaccine control mastitis there will be no antibiotic remains problem in milk; Being finally easy and simple to handle, expense is cheap. Currently; the vaccine succeeded in developing is little; and mostly number is weak poison live vaccine or inactivated vaccine; in production practice, the control of mastitis there is is certain effect, but the development along with extensive intensive culture; the exquisite weak bacterial strain of people also exists homologous recombination, self virulence returns the potential possibilities such as strong; and inactivated vaccine also exists the deficiencies such as using dosage is big, duration of immunity is short, for improving security and the immune protective efficiency of traditional vaccine, efficient, cheap new generation vaccine development is extremely important.
Streptococcus aureus (Staphylococcusaureus is called for short SA), also claims " S. aureus L-forms ", being the coccus of a kind of Gram-positive, diameter 0.8 ��m, is arranged in thyrsiform under the microscope, and golden yellow pigment can be produced, and therefore gain the name. S. aureus L-forms is one of the main pathogenic fungi causing chronic/recessive mammitis of cow, it is possible to affects the seed output and quality of milk greatly, brings huge financial loss to dairy industry. There are some researches show, the �� hemolysin of streptococcus aureus secretion is the virulence factor causing mammitis of cow.
S. aureus L-forms can produce multiple toxin and enzyme. �� hemolysin (also known as ��-toxin) is exactly one of its toxin, and it has the characteristic such as leucocytotoxicity, hemolytic activity. The single chain polypeptide that �� hemolysin is made up of 330 amino acid, molecular weight is 37��39kDa, and its iso-electric point (pI) is higher than 9. �� hemolysin is that a kind of magnesium relies on neural esterase, there is Phospholipase C (PhospholipaseC, PLC) active, Glycerophosphorylcholine can be decomposed, �� hemolysin relies on this kind of enzymic activity, the phospholipid bilayer of hydrolysis composition cytolemma, thus destroys the integrity of cytolemma, cause lysis, cause haemolysis. After �� hemolysin is carried out rite-directed mutagenesis, mutant just loses hemolytic activity, does not have toxicity, but still retains intact immunogenicity, can directly be used for immune animal, is one of the important candidate albumen of S. aureus L-forms subunit vaccine.
Summary of the invention
It is an object of the invention to provide the preparation method of a kind of milk cow streptococcus aureus beta hemolysis element subunit vaccine and application. Recombinant protein mixes the subunit vaccine of preparation and the natural infection similar process of pathogenic bacterium with suitable adjuvant; cellular immunization can be promoted; inducing immunological memory and cause immunne response widely; produce the reaction of good cross protection, it is a kind of safety and stability more, prepare new generation vaccine simple, that application is convenient, cheap.
Described recombinant beta-hemolysin albumen is from streptococcus aureus, and the nucleotide sequence encoding this albumen is as shown in SEQIDNO.2.
The present invention also provides the preparation method of a kind of milk cow streptococcus aureus beta hemolysis element subunit vaccine, mainly comprises the following steps: 1) rite-directed mutagenesis streptococcus aureus beta hemolysis fibroin gene; 2) by the beta hemolysis fibroin gene clone of rite-directed mutagenesis in pET28a carrier; 3) by step 2) the expression vector Transformed E .coliBL21 (DE3) that obtains, abduction delivering obtains recombinant beta-hemolysin albumen; 4) affinity chromatography purification step 3 is used) recombinant beta-hemolysin albumen of obtaining; 5) staphylococcus aureus mastitis in dairy cows recombinant subunit vaccine is obtained after the recombinant beta obtained by purifying-hemolysin albumen and pharmaceutically acceptable adjuvant (such as ISA206VG adjuvant) are fully mixed even.
Compared with prior art; the advantage that the present invention has and effect are as follows: the protective immunity that namely mammitis of cow subunit vaccine utilizes pathogenic bacterium main is former to be made not containing nucleic acid, the vaccine that can bring out body and produce antibody; it is directly injected body and activating immune system, to reach the object of prevention and therapy disease. Recombinant protein mixes the subunit vaccine of preparation and the natural infection similar process of pathogenic bacterium with suitable adjuvant; cellular immunization can be promoted; inducing immunological memory and cause immunne response widely; produce the reaction of good cross protection; be one more safety and stability, preparation is simple, application is convenient, cheap, and the time saving and energy saving new generation vaccine with significant curative effect.
Accompanying drawing explanation
Fig. 1, beta hemolysis element segmented-PCR product agarose gel electrophoresis result, M:MarkerDL2,000; 1 is �� hemolysin rite-directed mutagenesis fragment 1 (509bp), and 2 is �� hemolysin rite-directed mutagenesis fragment 2 (468bp).
Fig. 2, beta hemolysis element rite-directed mutagenesis over-lap PCR agarose gel electrophoresis result, M:MarkerDL5,000; 1 is �� hemolysin rite-directed mutagenesis overlapping PCR products (936bp).
Fig. 3, beta hemolysis element rite-directed mutagenesis recombinant plasmid enzyme cuts qualification agarose gel electrophoresis result, M:MarkerDL5,000; 1 is pET28a-�� hemolysin rite-directed mutagenesis No. 3 plasmids (NdeI/XhoI), and 2 is pET28a-�� hemolysin rite-directed mutagenesis No. 4 plasmids (NdeI/XhoI).
Fig. 4, beta hemolysis element affinity chromatography purification result, FT:flowthrough; 50mM:50mM imidazoles; 100mM:100mM imidazoles; 150mM:150mM imidazoles.
Fig. 5, recombinant beta-hemolysin protein renaturation result.
Fig. 6, ELISA tire result after detecting beta hemolysis element subunit vaccine immune mouse.
Mouse survival rate experimental result after Fig. 7, Immunization.
Embodiment
The structure of embodiment 1 expression vector pET28a-beta hemolysis element rite-directed mutagenesis
Taking the clinical milk cow staphylococcus aureus gene group being separated to as template, beta hemolysis element being divided into two sections and carries out PCR, result is as shown in Figure 1.
1.1 add sample system for (50 �� l):
1.2PCR amplification program:
1.3 glue reclaim DNA fragmentation:
(1) reaction solution of step 1.2 is carried out 0.8% agarose gel electrophoresis (110V30min);
(2) under ultraviolet lamp, cut glue and reclaim DNA fragmentation in 1.5mlEP pipe;
(3) in the 1.5mlEP pipe in step (2), 500 �� lPCbuffer are added, 50 DEG C, water-bath 10min;
(4) solution in step (3) is moved to adsorption column center, leave standstill 2min, centrifugal, 12,000rpm, 30s;
(5) abandon waste liquid, add 600 �� lPWbuffer to adsorption column center, leave standstill 3min, 12,000rpm, 30s;
(6) repeating step (5);
(7) suction attached column is centrifugal, 12,000rpm, 1min;
(8) 30 �� lddH are added to adsorption column center2O, leaves standstill 3min, centrifugal (12,000rpm, 2min);
(9) collect step (8) DNA sample and carry out electrophoresis.
1.4 over-lap PCR, carry out rite-directed mutagenesis by beta hemolysis element, result as shown in Figure 2:
Add sample system for (50 �� l):
Pcr amplification program:
1.5 double digestions reaction (50 �� l system):
1.5mlEP pipe carries out according to step 1.5 adding sample, mixed even, then these two 50 �� l reaction solutions is placed in 37 DEG C of thermostat water baths, water-bath 3h.
1.6 glue reclaim DNA fragmentation:
(1) reaction solution of step 1.5 is carried out 0.8% agarose gel electrophoresis (110V30min);
(2) under ultraviolet lamp, cut glue and reclaim DNA fragmentation in 1.5mlEP pipe;
(3) in the 1.5mlEP pipe in step (2), 500 �� lPCbuffer are added, 50 DEG C, water-bath 10min;
(4) solution in step (3) is moved to adsorption column center, leave standstill 2min, centrifugal, 12,000rpm, 30s;
(5) abandon waste liquid, add 600 �� lPWbuffer to adsorption column center, leave standstill 3min, 12,000rpm, 30s;
(6) repeating step (5);
(7) suction attached column is centrifugal, 12,000rpm, 1min;
(8) 30 �� lddH are added to adsorption column center2O, leaves standstill 3min, centrifugal (12,000rpm, 2min);
(9) collect step (8) DNA sample and carry out electrophoresis.
1.7 ligations (10 �� l system):
1.5mlEP pipe carries out according to above-mentioned system adding sample, mixed even, then above-mentioned reaction solution is placed in 16 DEG C, takes out after water-bath 16h, 65 DEG C, after water-bath 15min, carry out deactivation, by the preservation of 4 DEG C, sample.
1.8 transformation experiments:
(1) take out the reaction solution of step 1.7, add 100 �� lE.coliDH5 �� competent cells wherein, mixed even;
(2) ice bath 30min;
(3) 42 DEG C, water-bath 100s;
(4) ice bath 2min;
(5) take out, in EP pipe, add 600 �� l LB liquid medium, 37 DEG C, water-bath 1h;
(6) taking out, centrifugal (8,000rpm, 2min), removes 600 �� l, remains the 100 resuspended thalline of �� lLB;
(7) getting bacterium liquid is plated in LK flat board (Kan concentration is 50 �� g/ml), and LK flat board is placed in biochemical constant incubator, cultivates 12h for 37 DEG C.
1.9 recombinant plasmids extract and enzyme cuts qualification:
(1) choose from transformation plate and get mono-clonal to, in 3mlLK liquid nutrient medium, 37 DEG C, 260rpm shakes bacterium and spends the night;
(2) 1ml bacterium liquid is got to, in 1.5mlEP pipe, centrifugal (12,000rpm, 2min), abandons supernatant;
(3) in the EP pipe in step (2), 250 �� lP1buffer are added, resuspended thalline;
(4) in step (3) solution, 250 �� lP2buffer are added, gentle mixed even, leave standstill 2min;
(5) in step (4) solution, 350 �� lP3buffer are added, gentle mixed even;
(6) by step (5) solution, centrifugal (12,000rpm, 10min);
(7) supernatant solution in step (6) is moved to adsorption column center, centrifugal (8,000g, 30s);
(8) abandon waste liquid, add 500 �� lwashbuffer to adsorption column center, centrifugal (9,000g, 30s);
(9) repeating step (8);
(10) suction attached column centrifugal (9,000g, 1min);
(11) adsorption column adds 30 �� lElutionbuffer, leaves standstill 2min, centrifugal (12,000rpm, 2min);
(12) collect step (11) DNA sample and carry out electrophoresis;
(13) as shown in step 1.5, the plasmid extracted is carried out enzyme and cuts qualification, then carry out 0.8% agarose gel electrophoresis.
Recombinant plasmid enzyme is cut qualification result and is seen Fig. 3.
Embodiment 2 transformation of E. coli BL21
Drawing 1 �� l plasmid adds in 100 �� lBL21 competent cells, ice bath 30min;
42 DEG C of heat shock 90s;
Ice bath 2min;
The LB nutrient solution of 900 �� l non-resistants is added in super clean bench;
37 DEG C of 180rpm shake 1h;
Draw that resistance LB of 100 �� l bacterium liquid card-coatings dull and stereotyped, 37 DEG C of incubated overnight.
The a large amount of abduction delivering of embodiment 3
Choose bacterium: choose and get mono-clonal in that resistance LB nutrient solution of 50ml card, 37 DEG C of incubated overnight;
Switching: in 1: 100 ratio switching bacterium liquid to that resistance LB nutrient solution of 500ml card, shaking 3.5L altogether, 37 DEG C of 220rpm cultivate 2-2.5h to OD600Value is to 0.6;
Induction: bacterium liquid OD600Being worth after 0.6, adding 500 �� lIPTG (1M) is 1mmol/L to IPTG final concentration, 37 DEG C of 220rpm inducing culture 4h;
Microorganism collection: the centrifugal 10min of bacterium liquid 6,000rpm, collects thalline; Clean thalline with 40mlPBS, the centrifugal 10min of 6,000rpm, collect thalline, be placed in-20 DEG C of preservations;
Prepared by embodiment 4 beta hemolysis fibroin inclusion body
(1) bacterial cell disruption: with lysate (50mMNaH2PO4, 500mMNaCl, pH8.0) and resuspended thalline, with syringe by even for thalline piping and druming, avoid block throw out to produce; With biomixer Avestin cracking bacterium liquid; Centrifugal thalline after broken, 12,000rpm, 4 DEG C of centrifugal 30min, abandon supernatant, retain throw out;
(2) throw out is heavily floated on (lysate, 100 ��Ms of PMSF, 10mMEDTA, 10mMBenzamidine, 0.01%NaN in 25ml solution3); 12,000rpm, 4 DEG C, centrifugal 20min, abandons supernatant, retains throw out;
(3) repeating step (2) is once;
(4) throw out is resuspended in 25ml solution (lysate, 100 ��Ms of PMSF, 10mMMgCl, 10mMBenzamidene, 0.01%NaN3); 12,000rpm, 4 DEG C, centrifugal 20min, abandons supernatant, retains throw out;
(5) claim weight of precipitate, it is sub-packed in 1.5mlEP pipe by 1g/ pipe ,-20 DEG C of preservations.
Embodiment 5 affinity chromatography purification of Recombinant beta hemolysis fibroin
Get 1g inclusion body and it is added to (50mMNaH in 30ml denaturing soln2PO4, 500mMNaCl, 8M urea, pH8.0), room temperature for overnight; 12,000rpm, 22 DEG C, centrifugal 20min, gets supernatant, draws 20 �� l samples and treats that SDS-PAGE detects;
Post balances: gets 4mlNi-agarose and fills post, balance 10 column volumes with denaturing soln, discharges damping fluid, make liquid level higher than filler 1mm, in order to avoid dry post;
In conjunction with: the filler balanced mixes with sample liquid, rocks 1h on ice;
After completing, mixed solution joins in void column, collects Flowthrough, stays 20 �� l sample detection;
With the solution (50mMNaH of 30 times of column volumes2PO4, 500mMNaCl, 8M urea, 0.1%TritonX114, pH8.0) wash post to effluent liquid can't detect albumen, wash post with the denaturing soln of 10 times of column volumes more subsequently, stay 20 �� l to detect;
Wash-out: add imidazoles in denaturing soln, the imidazole elution of preparation 50mM, 100mM and 150mM, carries out wash-out with 5ml imidazole elution every time, and each concentration carries out 5 wash-outs;
SDS-PAGE analyzes; Result is as shown in Figure 4.
The renaturation of embodiment 6 recombinant betas-hemolysin albumen
Cut and get appropriate length dialysis tubing, with dialyzate (50mMNaH2PO4, 150mMNaCl, pH7.4) infiltrate, first to clamp one end with dialysis clip, then will treat that renaturation sample liquid joins in dialysis tubing, top clamped by dialysis clip, is positioned in 2L dialyzate, 4 DEG C of dialysis 12h;
Abandon dialyzate, then add the fresh dialyzate of 2L, continue dialysis 2h; Dialysis terminates rear 12,000rpm, 4 DEG C, and centrifugal 10min, gets supernatant;
Packing after BCA method mensuration protein concentration is also stored in-80 DEG C; SDS-PAGE analytical results is as shown in Figure 5.
Embodiment 7 recombinant betas-hemolysin protein subunit vaccine preparation
Needing according to experiment, calculate the amount of each composition of vaccine solution, make recombinant beta in vaccine-hemolysin final concentration of protein be 25 �� g/ml, wherein recombinant beta-hemolysin albumen and adjuvant ISA206VG volume ratio are 46: 54;
Being placed in thermostat water bath by mixed for dilution even good recombinant beta-hemolysin protein solution and ISA206VG adjuvant to be heated to 32 DEG C �� 1 DEG C, joined by antigen in adjuvant pipe after temperature-stable, oscillator concussion 10min carries out pre-emulsification;
Vaccine complete for pre-emulsification is positioned in the beaker filling with ice, is fixed on the ultrasonic cell disruption instrument anticipated and carries out emulsification;
After emulsification terminates, observe emulsifying effectiveness: getting part vaccine and be placed in centrifuge tube, the centrifugal 15min of 3,000rpm, vaccine is not stratified is qualified;
Detecting qualified vaccine is dispensed in 15ml centrifuge tube, mark, and sealing film sealing, is placed in 4 DEG C of preservations.
Embodiment 8 mouse immune is tested
Vaccine is taken room temperature (25 DEG C) place, make vaccine temperature return to normal temperature;
To mouse weights, grouping and mark, one group is immunity test group (n=6), immunity ��-appearance sanguinin subunit vaccine; Another group is control group (n=6), immunity PBS, and records numerical value;
Drawing 1ml vaccine with 1ml syringe, left and right back leg respectively injects 50 �� l vaccines.
Challenge viral dosage after embodiment 9 mouse immune
Choose get the mono-bacterium colony of SA (bacterial strain be this laboratory preserve HB0911-3 bacterial strain) in 5ml liquid broth, 220rpm, 37 DEG C shake bacterium and spend the night;
Volume (1ml) by mono-percent will shake the microbionation spent the night in 100ml fresh liquid broth culture, 220rpm, 37 DEG C shake bacterium and spend the night;
Being encased in 500ml centrifugal bottle by 100ml bacterium liquid, the centrifugal 10min of 8,000rpm, sucks substratum, and thalline 100mlPBS is resuspended, repeats above-mentioned steps 3 times, finally resuspended mixed even with 5mlPBS;
Count after bacterium liquid is done 10,000 times of dilutions. Concentration according to bacterium liquid, is diluted to 5 �� 10 by original bacteria liquid8CFU/ml(2MLD50);
Weighing to immune group and control group mice, one group is immunity test group (n=6), and another group records numerical value for control group (n=6);
Drawing bacterium liquid with 1ml syringe, the mouse corresponding according to each concentration bacterium carries out tail vein injection, and injection volume is 200 �� l/20g mouse.
Observe the survival state of mouse continuously, record the death time of each mouse; After attacking poison, mouse survival rate result is as shown in Figure 7.
Embodiment 10ELISA detects beta hemolysis element antibody titer
(1) bag quilt: detection albumen to the 0.5 �� g/ml diluting purifying with coating buffer (50mM carbonate buffer solution, pH9.5), on enzyme plate, every hole adds 100 �� l, and sealing film is sealed rear 4 DEG C of refrigerators placement and spent the night;
(2) wash: after taking out enzyme plate from refrigerator, put into and wash trigger washing, washings PBST;
(3) closing: every hole adds 200 �� l confining liquids (5% skimmed milk), sealing film is sealed latter 37 DEG C and is hatched 2h;
(4) preparation of samples: by known information with needing consumption, with confining liquid, serum is carried out appropriateness and dilute;
(5) wash: with (2);
(6) add sample: add dilute serum, do negative control with confining liquid simultaneously, hatch 1h for 37 DEG C;
(7) wash: with (2);
(8) add two anti-: every hole adds two anti-100 �� l of the HRP mark of appropriateness dilution, hatches 0.5h for 37 DEG C;
(9) wash: with (2);
(10) develop the color: when lucifuge, every hole adds the TMB colour developing liquid of 100 �� l, hatches 10min for 37 DEG C;
(11) terminate: every hole adds the 50 �� l stop buffer (H of 2M2SO4), termination reaction;
(12) detect: measure sample OD value in 450nm wavelength, analytical data;
(13) result analysis: the standard judging antibody positive: P/N >=2.1, OD450 >=0.1.
Claims (4)
1. the preparation method of milk cow Staphylococcus aureus beta hemolysis element subunit vaccine and application, it is characterised in that: the 1) rite-directed mutagenesis of Staphylococcus aureus protective antigen albumen ��-appearance sanguinin and clone; 2) expression and purification of recombinant beta-hemolysin albumen; 3) recombinant beta-hemolysin albumen and pharmaceutically acceptable adjuvant are fully mixed even after obtain staphylococcus aureus mastitis in dairy cows recombinant subunit vaccine.
2. streptococcus aureus protective antigen albumen beta hemolysis according to claim 1 element, it is characterised in that:
A protein that () is made up of the amino acid shown in SEQIDNO.1;
(b) aminoacid sequence in (a) through replacing, disappearance or add an amino acid or several amino acid and there is the streptococcus aureus protective antigen albumen beta hemolysis antigenic protein derivative by (a) of element;
C () recombinant beta-hemolysin protein expression system includes but not limited to intestinal bacteria, yeast, mammalian cell and insect cell etc.
3. the preparation method of milk cow streptococcus aureus beta hemolysis according to claim 1 element subunit vaccine, it is characterised in that comprise the steps:
1) rite-directed mutagenesis streptococcus aureus beta hemolysis fibroin gene;
2) by the beta hemolysis fibroin gene clone of rite-directed mutagenesis in pET28a carrier;
3) by step 2) the expression vector Transformed E .coliBL21 (DE3) that obtains, abduction delivering obtains recombinant beta-hemolysin albumen;
4) affinity chromatography purification step 3 is used) recombinant beta-hemolysin albumen of obtaining;
5) staphylococcus aureus mastitis in dairy cows recombinant subunit vaccine is obtained after the recombinant beta obtained by purifying-hemolysin albumen and pharmaceutically acceptable adjuvant (such as ISA206VG adjuvant) are fully mixed even.
4. subunit vaccine according to claim 1 is in the application prevented or in treatment staphylococcus aureus mastitis in dairy cows.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107224576A (en) * | 2017-03-06 | 2017-10-03 | 浙江海隆生物科技有限公司 | Vaccine of staphylococcus aureus mastitis in dairy cows subunit and its preparation method and application |
| CN115819624A (en) * | 2022-12-16 | 2023-03-21 | 广州源创生物医药科技有限公司 | Recombinant fusion protein and preparation method and application thereof |
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