CN102993308B - Methicillin-resistant staphylococcus aureus (MRSA) vaccine recombinant protein antigen HI2 and preparation method and application thereof - Google Patents

Abstract

The invention discloses a methicillin-resistant staphylococcus aureus (MRSA) vaccine recombinant protein antigen HI2, a preparation method and application thereof. The recombinant protein comprises a staphylococcus aureus (alpha) hemolysin (Hla) fragment and a ferrum surface determination protein (IsdB) fragment, wherein the Hla and the IsdB fragments are fused by a connecting peptide. The protein can be applied to preparation of a subunit vaccine and relative detection kits for resisting infection from methicillin-resistant staphylococcus aureus. According to the preparation method, the fused protein can be subjected to recombinant expression through the genetic engineering technology, therefore high expression quantity is gained, the separation and purification are convenient to carry out, and high efficiency and safety are ensured. As the animal experiment shown, the antigen can effectively stimulate an organism to perform higher humoral immune response and excellent immunoprotection.

Description

Methicillin-resistant staphylococcus aureus (MRSA) vaccine recombinant protein antigen HI2 and preparation method and application

Technical field

The invention belongs to biological technical field, be particularly related to the restructuring that a kind of methicillin-resistant staphylococcus aureus alpha hemolysin non-toxic mutant and iron surface determine albumen (IsdB) active fragments and merge recombinant protein HI2, the invention further relates to the preparation method of this recombinant protein and in the application of preparing in vaccine and detection kit.

Background technology

Resistance to X-1497 Golden yellow staphylococcus (methicillin-resistant Staphylococcus aureus, MRSA) be the gram positive coccus that can infect any one position of human body, can cause human body to produce disease widely, as microbemia, endocarditis, folliculitis, malignant boil, furuncle, carbuncle, pustulosis, honeycomb (group) is knitted inflammation, botryomycosis, toxic shock syndrome, cental system infects, infectious ocular inflammatory disease, osteomyelitis, bone and the infection of joint, respiratory system infection etc., bring great threat to human health.And be more difficult to control because the multiple antibiotic resistance of this bacterium makes situation, the selection for the treatment of means is fewer and feweri.

Immunology means are had great expectations to control especially infection and the propagation of methicillin-resistant staphylococcus grape ball of streptococcus aureus.Immunization strategy can be divided into active immunity and passive immunization, and passive immunization carries out with the antibody of target streptococcus aureus, and active immunity is by selecting suitable antigen to impel body to produce immune response.Suitable polysaccharide and polypeptide can form as antigen the active principle of vaccine.

Hla is that a perforation of all expressing on most of clinical separation strains forms toxin.It is relevant to various diseases pneumonia, septicemia sacroiliitis and brain running sore etc.The about 33kDa of Hla monomer, with heptamer form performance function, the 110-150 amino acid of each monomer is drawn close mutually and on phospholipid bilayer, is formed hole.Cause subsequently cytolemma to destroy, ion oozes out etc. in born of the same parents.Its toxicity is very strong, in very low concentrations situation, can cause a disease.Therefore, induction body generation Effective Anti Hla antibody should be an importance controlling infection of staphylococcus aureus.We are in this invention using nontoxic Hla(H35L after sudden change) as one of component of candidate of vaccine.

Methicillin-resistant staphylococcus aureus iron ion surface determines that protein I sdB is a kind of Outer membrane protein antigen, IsdB is not only important adventitia holdfast albumen of MRSA, adhere to vital role in mid-term in MRSA field planting, it is also MRSA obtains an iron main tool from host simultaneously.IsdB albumen is transmembrane protein, and its extracellular region correspondence 151 ~ 277 aminoacid sequences (I1) and two Structure and function domains of 338 ~ 466 aminoacid sequences (I2) are being caught oxyphorase performance Main Function.We are another component of candidate using I2 as vaccine and Hla(H35L in this invention) merge.

Summary of the invention

For the high drug-resistance of methicillin-resistant staphylococcus aureus, the invention provides the restructuring fusion recombinant protein that a kind of methicillin-resistant staphylococcus aureus alpha hemolysin non-toxic mutant and iron surface determine protein B active fragments, it can be applicable to prepare the subunit vaccine of methicillin-resistant staphylococcus aureus resistance infection and relevant detection kit.The present invention adopts the recombinant expressed described fusion rotein of genetic engineering technique, not only high separation and purification but also the highly effective and safe be convenient to of expression amount.Animal experiment proves to produce higher humoral immunoresponse(HI) and good immanoprotection action by effective stimulus body.

Recombination fusion protein determines that by streptococcus aureus alpha hemolysin (Hla) and iron surface protein B (IsdB) fragment (I2) forms, and wherein, Hla and I2 merge by connection peptides.Described recombination fusion protein has the constructional feature of Hla-Linker-I2, is called for short HI2; There is the DNA sequence dna of SEQ ID NO:1 and the aminoacid sequence of SEQ IDNO:2.

Described connection peptides is Linker, and this connection peptides is-(Linker) n-, and wherein Linker is GGGGS or GGSGG or YAPVDV, and n is 1-4, and preferably Linker is GGGGS, and n is 1.

Described Hla is the non-toxic mutant of H35L, and its DNA sequence dna is as shown in SEQ ID NO:3, and its aminoacid sequence is as shown in SEQ ID NO:4

Described I2 is that streptococcus aureus iron surface determines protein B film outskirt the second structural domain, i.e. the polypeptide of 484-559 amino acids composition, and its DNA sequence dna is as shown in SEQ ID NO:5, and aminoacid sequence is as shown in SEQ ID NO:6.

Preparation method's albumen of above-mentioned recombination fusion protein, comprises the following steps:

1) full gene synthetic (synthetic by Shanghai JaRa Bioisystech Co., Ltd) is to obtain the DNA sequence dna of coding HI2 and to be cloned into expression vector;

2) then expressed carrier is converted into Host Strains, the Host Strains after Induction Transformation is expressed HI2 recombinant protein.

Described expression vector is pGex serial carrier, pET serial carrier, is preferably pGex-6P-2.

Express the Host Strains of above-mentioned expression vector.Described Host Strains is E.colistrain XL1 blue, BL21 series or HMS174 series, is preferably E.colistrain XL1 blue.

The present invention preferably adopts pGEX-6p-2 vector construction recombinant expression vector, express recombinant protein HI2, pGEX is the carrier of the expressed fusion protein that built in 1987 by Smith and Johnson, its principal feature is on carrier, to be connected to the glutathione-S-transferase that a molecular weight is 26kDa (GST), in expressed fusion rotein, just contain a GST label, this label is the mark of protein purification.Compared with other fusion vectors, pGEX serial carrier has purification condition gentleness, step simply, does not need adding of denaturing agent, thereby makes the albumen after purifying can keep to greatest extent its space conformation and immunogenicity.

The application of recombinant protein as above in the subunit vaccine of preparing methicillin-resistant staphylococcus aureus resistance.

Above-mentioned recombinant protein is in the application of preparing in methicillin-resistant staphylococcus aureus detection kit.

In view of Hla has extremely strong toxicity, the present invention suddenlys change at its avtive spot H35, alternative with Leu, i.e. Hla (H35L).IsdB molecule is larger, and we select has good immunogenic second structural domain IsdB (338-466) and Hla (H35L) fusion, is convenient to expression and purification to reaching, and has better immunogenic effect.

The present invention also provides a kind of Host Strains, and its importing has the recombinant expression vector of above-mentioned structure.

The present invention adopts this recombination fusion protein of genetic engineering technique clonal expression, and expression amount is high, is convenient to separation and purification, and highly effective and safe.HI2 recombinant protein can be directly and adjuvant (as aluminum hydroxide adjuvant, aluminum phosphate adjuvant, Stearinsaeure aluminium adjuvant, MF59, complete Freund's adjuvant, complete Freund's adjuvant, mycobacterium bacille Calmette-Guerin vaccine adjuvant etc.) be used in conjunction with, be applicable to injecting immune inoculation.

We determine according to the newest fruits of staphylococcus aureus gene group and proteomics the vaccine building based on alpha hemolysin (Hla) and iron surface decision albumen (IsdB).

The expression method of genetically engineered recombinant protein of the present invention has the following advantages:

1) HI2 expression of recombinant proteins plasmid abduction delivering in prokaryotic expression system-intestinal bacteria;

2), while selecting pGEX carrier series, HI2 recombinant protein is with fusion protein form expression; On expression vector, be connected to the glutathione-S-transferase that a molecular weight is 26kDa (GST), in expressed fusion rotein, just contain a GST label, this label just becomes the mark of protein purification, make purification condition gentleness, step simply, not need adding of denaturing agent, thereby the albumen after purifying can keep its space conformation and immunogenicity to greatest extent; Purifying HI2 recombinant protein purity is out greater than 95%;

3) HI2 recombinant protein all can produce specific antibody by induced animal.

Utilize subunit vaccine prepared by HI2 recombinant protein of the present invention to carry out immunization by subcutaneous (muscle) injecting pathway, excitating organism produces high titre IgG antibody and cellullar immunologic response.And confirm through experimentation on animals, described genetically engineered recombinant multivalent vaccine has the immune protective effect that good anti-MRSA infects.For further combined vaccine and the fusion bacterin research of many subunits lay the first stone, simultaneously for development and the application of control vaccine and diagnostic kit have important effect.

In order to make the object of the invention, technical scheme and advantage clearer, below in conjunction with drawings and Examples, the present invention is further elaborated.Should be appreciated that specific embodiment described herein, only in order to explain the present invention, is not intended to limit the present invention.

Brief description of the drawings

Fig. 1 is that the enzyme of expression vector pGEX-6p-2-HI2 is cut qualification result:

Swimming lane 1: nucleic acid (DNA) molecular weight standard (Marker);

Swimming lane 2: the qualification result of recombinant expression plasmid pGEX-6p-2-HI2 after enzyme is cut, the fragment that enzyme is cut rear separation is 4.8kbp and 1.3kb;

Fig. 2 HI2 protein purification result

Swimming lane 1: molecular weight of albumen standard (Marker);

Swimming lane 2:GST-HI2 albumen

Swimming lane 3: the HI2 albumen that purifying obtains after PP enzyme, this albumen theoretical molecular is 47.7kD, finds out with theory expectation and conforms to from figure

Embodiment

Bacterial strain used in the present invention and all ingredients are as follows:

1. bacterial strain

The strain of streptococcus aureus MRSA-252 international standard is provided by U.S. ATCC;

2. reagent

Plasmid pGEX-6p-2 is that GE Healthcare company of U.S. product, pET22b are that Merck company of U.S. product, coli strain XL-1blue are Agilent company of U.S. product, is applicant microorganism teaching and research room and preserves.

PrimeSTAR HS DNA Polymerase, DNA Marker, restriction enzyme BamH I and Not I, albumen Marker, T4 ligase enzyme solution I are Dalian TakaRa company product;

It is Omega company of U.S. product that plasmid extraction kit and gel reclaim test kit;

It is Tian Gen company product that bacterial genomes is extracted test kit, ultra-thin recovery test kit and nitrite ion;

PreScission protease, gsh-sepharose Glutathione Sepharose 4B are GE Healthcare company of U.S. product.

Embodiment 1: the gene of coding HI2 is synthesized and is cloned on pGEX-6P-2 and pET22b by Shanghai JaRa Bioisystech Co., Ltd.

Embodiment 2: recombinant plasmid transformed expressive host bacterium and qualification.

1, transform

Get 3 pipe intestinal bacteria XL1blue competent cells from-80 DEG C of refrigerators, the first pipe adds pGEX-6P-2 plasmid, makes positive control; The second pipe adds synthetic pGEX-6P-2-HI2; The 3rd pipe does not add foreign DNA, makes negative control.Ice bath 50min, thermal shock 90s in 42 DEG C of metal baths, rapidly ice bath 2min.Add the blank substratum of 600 μ lLB, mix, be placed in 37 DEG C of shaking table 220rp jolting 1h.

Each pipe, with the centrifugal 3min. of 5000rpm room temperature, discards 300 μ l supernatants, more resuspended thalline, gets 200 μ l and coats Amp resistance LB flat board.Flat-plate inverted is placed in 37 DEG C of incubators and cultivates 24h.

2, screening, the qualification of the positive recombinant plasmid of pGEX-6p-2-HI2

1. negative control flat board does not have bacterium colony to occur; Positive control flat board covers with bacterium colony, illustrates that competent cell making is correct, credible result.Picking transforms the dull and stereotyped upper good bacterium colony of separating, and is inoculated in Amp resistance LB substratum, and 37 DEG C of shaking culture are spent the night;

2. plasmid extraction: carry out with reference to plasmid extraction kit specification sheets;

3. plasmid DNA is carried out BamH I and Not I double digestion;

Double digestion reaction system:

37 DEG C of enzymes are cut 2h;

4. 1% agarose gel electrophoresis detects double digestion result, and result is as Fig. 1, and swimming lane 2, for 4.8kb and 1.3kb two fragments that recombinant plasmid forms after by double digestion, illustrate that pGEX-6p-2-HI2 recombinant plasmid transformed successfully.

Embodiment 3: the qualification of recombination fusion protein HI2 abduction delivering, purifying and expression-form in prokaryotic expression system-intestinal bacteria

1. target protein abduction delivering

1) get double digestion and identify that correct pGEX-6P-2-HI2/XL-1blue bacterium liquid 100 μ L add in the TB substratum of 10mLAmp resistance, 37 DEG C of incubated overnight of 80rpm, the bacterium liquid 400 μ L that get respectively incubated overnight add in the TB substratum of 20mLAmp resistance (remaining bacterium liquid is kept in 4 DEG C of refrigerators for subsequent use), cultivate 2 ~ 3h for 37 DEG C, rotating speed 200rpm, when re-activation is 0.8-1.0 to OD600, add IPTG 4 μ L, making its final concentration is 200 μ M, be placed in again 30 DEG C of 3h of shaking table abduction delivering, 25 DEG C of 5h, 16 DEG C of abduction deliverings that spend the night.

2) the bacterium liquid after abduction delivering is taken out, with the centrifugal 5min of 12000rpm, supernatant discarded, adds 1mLlysis buffer to mix, ultrasonic degradation 3min (ultrasonic 6 times 30s/ time), then 4 DEG C of centrifugal 15min of 14000rpm, cleer and peaceful precipitation in separation.

2. process supernatant

Get Glutathione Sepharose 4B 20 μ l, with after PBS washing 3 times, ready supernatant is added in Glutathione Sepharose 4B, room temperature is in conjunction with 1h.At 4 DEG C, with after the centrifugal 3min of 14000rpm, use PBS-0.25% polysorbas20 washing 2 times, PBS washs once.Glutathione Sepharose 4B after combination adds 20 μ L 2 × protein loading buffer, boils 5min, the centrifugal 3min of 14000rpom.

3. process precipitation

In precipitation, add the resuspended thalline of 500 μ L lysis buffer, get the resuspended bacterium liquid of 80 μ L and add 20 μ L 5 × protein loading buffer, boil 5min, the centrifugal 3min of 14000rpm.

4.SDS-PAGE electrophoresis, concentrates glue by 5% and pours in glue version, is adding distilled water that glue laminated is flat, and room temperature is placed 30min solidifies it, the distilled water on upper strata is fallen to do, then pour into 10% separation gel, plugs immediately comb, and it is for subsequent use that room temperature placement 30min solidifies it.

5. the upper cleer and peaceful precipitation of handling well is got respectively to 10 μ L loadings, carry out SDS-PAGE electrophoresis.The first 80v electrophoresis of voltage 30min, be adjusted to again 180v, after electrophoresis 1 ~ 2h, glue is taken out, be placed in coomassie brilliant blue staining liquid vibration dyeing, then be placed in after destainer vibration decolouring observations under imaging system, PGEX-6P-2-HI2/XL-1blue is soluble proteins and no significant difference at 16 DEG C, 25 DEG C, 30 DEG C.

The preparation of embodiment 4:HI2 antigen

1. amplification culture is obtained albumen

Go bail for and exist in 4 DEG C of refrigerators pGEX-6P-2-HI2/XL-1blue bacterium liquid 400 μ L for subsequent use to join 20mL once to activate containing in the TB substratum of Amp resistance, after 37 DEG C of cultivation 5 ~ 6h of 200rpm, getting bacterium liquid that 8mL once activates joins 400mL and carries out re-activation containing in the TB substratum of Amp resistance, 37 DEG C are cultivated 3 ~ 4h to OD600 is 1.0 o'clock, adding 80 μ L IPTG(final concentrations is 200 μ M) be placed in 16 DEG C of shaking tables spend the night induction after, the centrifugal 15min of 12000rpm collects thalline, add again after the resuspended thalline of 20mL lysis buffer, bacterium liquid is carried out to ultrasonic degradation 3min (200V), collecting supernatant and 800 μ L processes for Glutathione Sepharose 4B gel beads (beads) combination in conjunction with gst fusion protein, carry out again SDS-PAGE gel electrophoresis.

2. use enzyme blanking method, target protein and GST label are separated, obtain HI2

In the protein-bonded Glutathione Sepharose of remainder approximately 800 μ L 4B, add 800 μ L PBS and 120 μ L PreScission protease(PP enzymes), room temperature vertical rotary enzyme is cut after 5h, after centrifugal absorption supernatant, respectively with 800 μ L PBS washing 3 times, after each, get after 10 μ L sample denaturing treatment, loading 5 μ L carry out protein electrophoresis (method is the same), be observations under phase system, enzyme is cut front gst fusion protein molecular weight in 96kDa left and right, the HI2 molecular weight of albumen that enzyme scales off 43 and 55kDa between, 47.7kDa is consistent with expection molecular weight of albumen size, see Fig. 2.

3.BCA method is measured protein content, and maximum concentration is 2.0mg/mL.

Embodiment 5: infect the foundation with staphylococcus aureus strains (international standard strain MRSA-252) standard quantitative curve

Inoculation is placed in to 37 DEG C in MH flat board hatches 24 hours; On flat board, picking list bacterium colony, is inoculated in MH liquid nutrient medium, is placed in the concussion of 37 DEG C of constant-temperature tables and cultivates the centrifugal 10min of 6000rpm after 6 hours and collect thalline, with physiological saline washing thalline 2 times; Again bacterium liquid is carried out to 10 times and 1.25 times of dilutions, and under ultraviolet spectrometry system, measure the absorbancy at 600nm place (OD600) of each bacterium liquid, and get each dilution bacterium liquid 100 μ L and coat MH flat board, be placed in 37 DEG C and hatch after 24 hours and count bacterium colony; According to the quantitative curve of OD600 value drawing standard of each flat-plate bacterial colony number and bacterium liquid.

Result: typical curve formula is Y=2.3065X+0.0051(10 ⁹cFU/ml), relation conefficient is 0.9999.

Embodiment 6: the structure of pyemia animal model

1 is placed in 37 DEG C by inoculation in MH flat board hatches 24 hours; On flat board, picking list bacterium colony, is inoculated in MH liquid nutrient medium, is placed in 37 DEG C of constant-temperature table concussion cultivations and collects thalline after 6 hours, and utilize typical curve formula to carry out quantitatively, then be 2.0 × 10 by bacterium liquid dilution (or concentrated) ¹⁰cFU/mL, 1.5 × 10 ¹⁰cFU/mL, 1.25 × 10 ¹⁰cFU/mL, 1.0 × 10 ¹⁰cFU/mL different concns group, then the BALB/C mice that is 18 ~ 20g by 6 ~ 8 week age of tail vein injection, body weight with each group of bacterium liquid (100 μ l/ are only) carries out systemic infection, physiological saline control group is set simultaneously, the mortality ratio of observing 7 days and adding up each group of mouse;

2. after infecting, timing adopts colony counting method to detect bacteria planting amount every 24 hours (to infecting latter 7 days): from each infected group and control group, choose at random 3 mouse, utilize eyeball excise method, get mouse blood sample 0.5 ~ 1mL, get after 10 times of 20 μ L blood samples, 180 μ L heparin dilutions for bacterial count, get 50 μ L and be applied to flat board, be placed in 37 DEG C, counting clone number after 24h; After taking blood sample, mouse is put to death and put into after 75% alcohol soaking disinfection, take out its four limbs are fixed, by its dissection, take out spleen, kidney, liver, be placed in the aseptic PBS of 2mL, in clean glass homogenizer, carry out homogenate, get 1mL homogenate and dilute according to 1:10,1:100,1:1000 ratio; Every extent of dilution is got 100 μ L and is coated gently on solid medium, is placed in 37 DEG C, cultivates 24h, does enumeration.

The results are shown in table 1:

Determining of table 1MRSA-252 minimum lethal dose and sublethal dose

2.0 × 10 ⁹12 hours (h) interior mouse death rates of CFU dosage group are 100%; 1.5 × 10 ⁹in CFU dosage group 48h, mouse death rate is that in 90%, 72h, mortality ratio is 100%; 1.25 × 10 ⁹in CFU dosage group 48h, mouse death rate is that in 80%, 96h, mortality ratio is that mouse death rate is that in 90%, 120h, mortality ratio is that mouse death rate is 100%; 1.0 × 10 ⁹in CFU dosage group 48h, mouse death rate is that in 10%, 72h, mortality ratio is that mouse death rate is that 20%, 7 day (d) interior mortality ratio is that mouse death rate is 70%; MRSA-252 minimum lethal dose is about 1.25 × 10 as can be seen here ⁹cFU, sublethal dose is 1.0 ~ 1.25 × 10 ⁹cFU.

Field planting amount after 3.MRSA-252 infection BALB/C mice in blood and each internal organs:

After infecting Bacteria in Blood in the time of 48h, reach peak value, maximum field planting amount reaches 8.0 × 10 ⁹cFU/ml, in the time of 72h, Bacteria in Blood amount starts to reduce, and during to 96h, in blood, does not detect bacterium; After infecting, in spleen, kidney, liver, the bacterium of field planting all reached peak value at 72 o'clock, and maximum field planting amount all reaches 8.0 × 10 ⁹cFU/ml; Bacteria planting detected result in the blood of control group mice, spleen, kidney, liver is zero.

Above result has been carried out the evaluation of animal model for survival rate and blood, spleen, kidney, the liver major organs bacteria planting amount of mouse, for the pathogenetic research that successful development and the MRSA of the single subunit vaccine of MRSA and many subunits of MRSA fusion bacterin infect is laid a good foundation.

Embodiment 7: the detection of immune animal and antibody

1. immune animal

1) first immunisation, with PBS dilution HI2 proteantigen, adding concentration is the Al (OH) of 1mg/mL ₃; With No. 5 half mould syringe needles, bilateral inguinal region, vola and back are subcutaneous to an injection, and every BALB/C mice injection volume is 100 μ L, and positive controls, negative control group and blank group are set;

2) immunity for the second time, carries out immunity for the second time on the 14th day, and immune component is the same, and injection volume proteantigen amount is 1/2 of first immunisation, and immunization route is the same;

3) immunity for the third time, carries out immunity for the third time on the 21st day, and immune component is the same, and injection volume proteantigen amount is with immune identical for the second time, and immunization route is the same;

After 2 immunity for the third time the 7th and 14 days, gather the blood of BALB/C mice, detect after mouse immune IgG, IgG1, IgG2 with ELISA _ahumoral response level.

1) prepare liquid

1. the preparation of coating buffer: take NaHCO ₃1.6g, Na ₂cO ₃2.9g, is dissolved in 1L ddH ₂o, counts pH is adjusted to 9.6 with PH;

2. the preparation of confining liquid: 1g bovine serum V, is dissolved in 100mL antibody diluent (1:100);

3. the preparation of antibody diluent: phosphoric acid salt is dissolved in to 1L ddH ₂o, then add 500 μ L Tween 20, then count pH is adjusted to 7.4 with PH;

4. the preparation of washings: take 2.42g Tris and be dissolved in 1L ddH ₂o, then add 500 μ L Tween 20, then count pH is adjusted to 7.4 with PH;

5. nitrite ion (TMB) is Tian Gen company product;

6. stop buffer (2M H ₂sO ₄) preparation: the 22.2mL vitriol oil is poured into 177.8mL ddH ₂in O.

2) ELISA detects the antibody titer that FnBA1 recombinant protein immune mouse produces

1. with coating buffer by the HI2 recombinant protein dilution after purifying be: 1ug/mL, 5ug/mL, 10ug/mL;

2. coated: recombinant protein diluent is added to enzyme plate, 200 μ L/ holes, 4 DEG C spend the night after with washings washing 3 times, emptyly wrap with preservative film after dry, be placed in 4 DEG C of refrigerators for subsequent use;

3. sealing: enzyme plate adds confining liquid 100 μ L/ holes, is placed in 37 DEG C of incubators 2 hours, washs 3 times;

4. serum is carried out to the doubling dilutions such as 1:100,1:500,1:1000,1:2000,1:4000,1:8000;

5. get the enzyme plate having sealed, add successively dilute serum, 100 μ L/ holes,, be placed in 37 DEG C of incubator 30min, wash 3 times, empty dry;

6. the goat anti-mouse igg, IgG1, the IgG2a antibody that add HRP mark are preserved to liquid, dilution 1:5000, makes antibody working fluid;

7. add dilution antibody working fluid, 100 μ L/ holes, are placed in 37 DEG C of incubator 1h, wash three times, empty dry;

8. add substrate nitrite ion (TMB) 100 μ L/ holes, room temperature lucifuge reaction 5min;

9. add stop buffer (2M H ₂sO ₄), be placed in immediately in microplate reader and measure OD value with 450nm wavelength place;

10. result judgement: A _sample/ A _negativezhi≤2.1 positive (negative control is that before mouse immune, serum 1:1000 doubly dilutes).

Result: the antibody titer that detects the generation of HI2 proteantigen immune mouse reaches 1:512000; After immunity, the antibody positive rate of the 7th day reaches 90%, illustrates that the HI2 recombinant protein that the present invention builds can make generation antibody in immune mouse body.

Embodiment 8: determine the malicious protection of attacking of HI2 recombinant protein immune animal by immune mouse

With the immunization protocol of embodiment 5, the MRSA tail vein injection that adopts lethal dose on the 10th day after immune 4 times is attacked poison experiment to immune mouse and control mice, observe the death condition of each group of mouse, after the observation period of 10 days, calculate the death/survival rate of immune mouse, as table 1.

After table 1 injecting immune BALB/c mouse, attack malicious immune protective effect

Result demonstration, the immune protective rate of HI2+ adjuvant group reaches 80%, turns out to be an effective antigen.

Conclusion: the fusion bacterin antigen HI2 of many subunits of genetically engineered immunity BALB/c mouse can produce the effective provide protection of attacking for MRSA viable bacteria compared with other each group.

By above embodiment, those skilled in the art utilize ordinary skill knowledge can apply apparently the prepared recombinant protein of the present invention and other related reagents, for example coated reagent, detection antibody, developer, terminator etc. are prepared related kit, whether for example detection kit, infect golden yellow staphylococcus, determine prognosis etc. for diagnosis.

Those skilled in the art can be by HI2 recombinant protein of the present invention for other any applicable purposes.

The foregoing is only preferred embodiment of the present invention, be not used for limiting practical range of the present invention; If do not depart from the spirit and scope of the present invention, the present invention is modified or is equal to replacement, all should be encompassed in the middle of the protection domain of the claims in the present invention.

Claims (11)

1. a recombination fusion protein, it is characterized in that: this albumen determines that by streptococcus aureus alpha hemolysin (Hla) and iron surface protein B (IsdB) fragment is merged and formed by connection peptides, the DNA sequence dna of this recombination fusion protein is as shown in SEQ ID NO:1, and aminoacid sequence is as shown in SEQ ID NO:2.

2. recombination fusion protein according to claim 1, is characterized in that: the non-toxic mutant that described Hla is H35L, and its DNA sequence dna is as shown in SEQ ID NO:3, and aminoacid sequence is as shown in SEQ ID NO:4.

3. recombination fusion protein according to claim 1, is characterized in that: described IsdB is that streptococcus aureus iron surface determines albumen ad hoc structure territory, and its DNA sequence dna is as shown in SEQ ID NO:5, and aminoacid sequence is as shown in SEQ ID NO:6.

4. the preparation method of recombination fusion protein claimed in claim 1, is characterized in that, comprises the following steps:

(1), by doing template with methicillin-resistant staphylococcus aureus genome, carry out the goal gene of pcr amplification acquisition coding recombination fusion protein claimed in claim 1 with special primer; Or the codon optimization synthetic goal gene that obtains of full gene afterwards;

(2) gene fragment clone step (1) being obtained, to expression vector, is then converted into Host Strains;

(3) Host Strains after Induction Transformation is expressed recombination fusion protein claimed in claim 1;

(4) purification of recombinant proteins.

5. an expression vector, is characterized in that: comprise the nucleotide sequence of recombination fusion protein described in coding claim 1.

6. expression vector according to claim 5, is characterized in that, described expression vector is pGex serial carrier, pET serial carrier.

7. expression vector according to claim 6, is characterized in that, described expression vector is pGex-6P-2.

An expression as arbitrary in claim 5-7 as described in the Host Strains of expression vector.

9. Host Strains as claimed in claim 8, is characterized in that, described Host Strains is E.colistrain XL1 blue, BL21 series or HMS174 series.

10. Host Strains according to claim 9, is characterized in that, described Host Strains is E.colistrain XL1 blue.

11. application of recombination fusion protein claimed in claim 1 in the subunit vaccine of preparing methicillin-resistant staphylococcus aureus resistance.