CN102993308A

CN102993308A - Methicillin-resistant staphylococcus aureus (MRSA) vaccine recombinant protein antigen HI2 and preparation method and application thereof

Info

Publication number: CN102993308A
Application number: CN2012103795088A
Authority: CN
Inventors: 邹全明; 樊绍文; 卢陆; 曾浩; 冯强; 左钱飞; 章金勇; 顾江; 鲁东水
Original assignee: CHONGQING YUANLUN BIO-TECHNOLOGY Co Ltd; Third Military Medical University TMMU
Current assignee: Third Military Medical University TMMU; Chengdu Olymvax Biopharmaceuticals Inc
Priority date: 2012-09-29
Filing date: 2012-09-29
Publication date: 2013-03-27
Anticipated expiration: 2032-09-29
Also published as: CN102993308B

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 fusion recombinant protein HI2 that a kind of methicillin-resistant staphylococcus aureus alpha hemolysin non-toxic mutant and iron surface determine albumen (IsdB) active fragments, the invention further relates to preparation method and the application in preparation vaccine and detection kit thereof of this recombinant protein.

Background technology

Anti-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 widely disease, such 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 since the multiple antibiotic resistance of this bacterium so that situation is difficult to control more, the selection for the treatment of means is fewer and feweri.

The immunology means are had great expectations to control streptococcus aureus especially infection and the propagation of methicillin-resistant staphylococcus grape ball.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 consist of 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 at phospholipid bilayer mutually and is formed the hole.Cause subsequently cytolemma to destroy, ion oozes out etc. in the born of the same parents.Its toxicity is very strong, can cause a disease in the very low concentrations situation.Therefore, inducing body to produce Effective Anti Hla antibody should be an importance of control infection of staphylococcus aureus.Nontoxic Hla(H35L after we will suddenly change in this invention) 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 the vital role in mid-term in the MRSA field planting, it also is MRSA obtains iron from the host a main tool 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 in this invention with I2 as another component of candidate of vaccine and and Hla(H35L) merge.

Summary of the invention

High drug-resistance for 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 the 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, and not only the expression amount height is convenient to separation and purification but also highly effective and safe.But animal experiment proof effective stimulus body produces higher humoral immunoresponse(HI) and good immanoprotection action.

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; Have the dna sequence dna of SEQ ID NO:1 and the aminoacid sequence of SEQ IDNO:2.

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

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

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

Preparation method's albumen of above-mentioned recombination fusion protein may further comprise the steps:

1) full gene synthesizes (synthetic by Shanghai JaRa Bioisystech Co., Ltd) with the dna sequence dna of acquisition coding HI2 and is cloned into expression vector;

2) then expressed carrier is converted into Host Strains, the Host Strains behind the Induction Transformation is expressed the 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 made up in 1987 by Smith and Johnson, its principal feature is the glutathione-S-transferase (GST) that to be connected to a molecular weight on the carrier be 26kDa, just contain a GST label in the expressed fusion rotein, this label is the mark of protein purification.Compare with other fusion vectors, the pGEX serial carrier has the adding that purification condition gentleness, step simply, do not need denaturing agent, thereby makes the albumen behind the purifying can keep to greatest extent its space conformation and immunogenicity.

The application of aforesaid recombinant protein in the subunit vaccine of preparation methicillin-resistant staphylococcus aureus resistance.

The application of above-mentioned recombinant protein in preparation methicillin-resistant staphylococcus aureus detection kit.

In view of Hla has extremely strong toxicity, the present invention suddenlys change at its avtive spot H35, substitutes with Leu, i.e. Hla (H35L).The 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 in the hope of 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.The HI2 recombinant protein can be directly and adjuvant (such 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, be applicable to the injecting immune inoculation.

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

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;

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

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

Utilize the subunit vaccine of HI2 recombinant protein preparation 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 through the experimentation on animals confirmation, 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 development and the application for control vaccine and diagnostic kit has 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.

Description of 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 fragment that the qualification result of recombinant expression plasmid pGEX-6p-2-HI2 after enzyme is cut, enzyme are 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 behind the PP enzyme, this albumen theoretical molecular is 47.7kD, finds out with theory expectation from figure to conform to

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 U.S. GE Healthcare company product, pET22b are that U.S. Merck company product, coli strain XL-1blue are U.S. Agilent company 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 U.S. Omega company product that plasmid extraction kit and gel reclaim test kit;

It is a day root 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 U.S. GE Healthcare company product.

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

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

1, transforms

Get 3 pipe intestinal bacteria XL1blue competent cells from-80 ℃ of refrigerators, the first pipe adds the 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 ℃ of metal baths, rapidly ice bath 2min.Add the blank substratum of 600 μ lLB, mixing places 37 ℃ of shaking table 220rp jolting 1h.

Each pipe discards 300 μ l supernatants with the centrifugal 3min. of 5000rpm room temperature, and resuspended thalline is got 200 μ l and coated Amp resistance LB flat board again.Flat-plate inverted places 37 ℃ of incubators to cultivate 24h.

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

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

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

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

The double digestion reaction system:

37 ℃ of enzymes are cut 2h;

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

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

1. target protein abduction delivering

1) gets 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 ℃ 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 ℃ of refrigerators for subsequent use), cultivate 2 ~ 3h for 37 ℃, 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, place again 30 ℃ of 3h of shaking table abduction delivering, 25 ℃ of 5h, 16 ℃ of abduction deliverings that spend the night.

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

2. processing supernatant

Get Glutathione Sepharose 4B 20 μ l, after PBS washing 3 times, ready supernatant is added among the Glutathione Sepharose 4B, room temperature is in conjunction with 1h.Behind the centrifugal 3min, using PBS-0.25% polysorbas20 washing 2 times with 14000rpm under 4 ℃, 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 pours into 5% concentrated glue in the glue version, and is at adding distilled water that glue laminated is flat, room temperature is placed 30min solidifies it, and the distilled water on upper strata is done, and pours into 10% separation gel again, plugs immediately comb, and it is for subsequent use that room temperature placement 30min solidifies it.

5. the upper cleer and peaceful precipitation that will handle well is not got 10 μ L loadings, carries out the SDS-PAGE electrophoresis.The voltage 80v of elder generation electrophoresis 30min, transfer to again 180v, behind electrophoresis 1 ~ 2h, glue is taken out, place the vibration dyeing of coomassie brilliant blue staining liquid, place again destainer vibration decolouring after, observations under the imaging system, PGEX-6P-2-HI2/XL-1blue is soluble proteins and no significant difference at 16 ℃, 25 ℃, 30 ℃.

The preparation of embodiment 4:HI2 antigen

1. amplification culture is obtained albumen

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

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

In the protein-bonded Glutathione Sepharose of the about 800 μ L of remainder 4B, add 800 μ L PBS and 120 μ L PreScission protease(PP enzymes), after room temperature vertical rotary enzyme is cut 5h, behind the centrifugal absorption supernatant, respectively with 800 μ L PBS washing 3 times, after getting 10 μ L sample denaturing treatment after each, loading 5 μ L carry out protein electrophoresis (method is the same), be observations under the phase system, enzyme is cut front gst fusion protein molecular weight about 96kDa, 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, sees Fig. 2.

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

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

Place 37 ℃ to hatch 24 hours in the MH flat board inoculation; Picking list bacterium colony is inoculated in the MH liquid nutrient medium on flat board, places 37 ℃ of constant-temperature table concussions to cultivate the centrifugal 10min collection of 6000rpm thalline after 6 hours, washs thalline 2 times with physiological saline; Again bacterium liquid is carried out 10 times and 1.25 times of dilutions, and under the ultraviolet spectrometry system, measure the absorbancy at the 600nm place (OD600) of each bacterium liquid, and get each dilution bacterium liquid 100 μ L and coat the MH flat board, place 37 ℃ to hatch after 24 hours and count bacterium colony; The quantitative curve of OD600 value drawing standard according to each flat-plate bacterial colony number and bacterium liquid.

The result: the 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 places 37 ℃ to hatch 24 hours in the MH flat board inoculation; Picking list bacterium colony is inoculated in the MH liquid nutrient medium on flat board, and place 37 ℃ of constant-temperature table concussions to cultivate after 6 hours and collect thalline, and utilize the typical curve formula to carry out quantitatively, be 2.0 * 10 with bacterium liquid dilution (or concentrated) again ¹⁰CFU/mL, 1.5 * 10 ¹⁰CFU/mL, 1.25 * 10 ¹⁰CFU/mL, 1.0 * 10 ¹⁰CFU/mL different concns group is that the BALB/C mice (100 μ l/ only) of 18 ~ 20g is carried out systemic infection with each group bacterium liquid by 6 ~ 8 ages in week of tail vein injection, body weight again, and the physiological saline control group is set simultaneously, observes and also adds up the mortality ratio of respectively organizing mouse in 7 days;

2. timing adopts colony counting method that the bacteria planting amount is detected every 24 hours (to infecting rear 7 days) after infecting: choose at random 3 mouse from each infected group and control group, utilize the eyeball excise method, get mouse blood sample 0.5 ~ 1mL, get 20 μ L blood samples and be used for afterwards bacterial count with 10 times of 180 μ L heparin dilutions, get 50 μ L and be applied to flat board, place 37 ℃, counting clone number behind the 24h; Take behind the blood sample mouse put to death put into 75% alcohol soaking disinfection after, its four limbs are fixed in taking-up, with its dissection, take out spleen, kidney, liver, place the aseptic PBS of 2mL, in the glass homogenizer of cleaning, carry out homogenate, get the 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 the solid medium, places 37 ℃, 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 ⁹Mouse death rate is that mortality ratio is 100% in 90%, the 72h in the CFU dosage group 48h; 1.25 * 10 ⁹Mouse death rate is that mortality ratio is that mouse death rate is that mortality ratio is that mouse death rate is 100% in 90%, the 120h in 80%, the 96h in the CFU dosage group 48h; 1.0 * 10 ⁹Mouse death rate is that mortality ratio is that mouse death rate is that 20%, 7 day (d) interior mortality ratio is that mouse death rate is 70% in 10%, the 72h in the CFU dosage group 48h; This shows that the MRSA-252 minimum lethal dose is about 1.25 * 10 ⁹CFU, sublethal dose are 1.0 ~ 1.25 * 10 ⁹CFU.

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

After infecting Bacteria in Blood when 48h, reach peak value, maximum field planting amount reaches 8.0 * 10 ⁹CFU/ml, the Bacteria in Blood amount begins to reduce when 72h, does not detect bacterium in the blood during to 96h; The bacterium that infects field planting in rear spleen, kidney, the liver all reached peak value at 72 o'clock, maximum field planting amount all reaches 8.0 * 10 ⁹CFU/ml; Bacteria planting detected result in the blood of control group mice, spleen, kidney, the liver is zero.

Above result has 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 the successful development of the single subunit vaccine of MRSA and many subunits of MRSA fusion bacterin and MRSA 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 ₃With 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) for the second time immunity was carried out the immunity 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) for the third time immunity was carried out for the third time immunity on the 21st day, and immune component is the same, and injection volume proteantigen amount is identical with for the second time immunity, and immunization route is the same;

Rear the 7th and 14 day of 2 for the third time immunity, the blood of collection BALB/C mice, behind ELISA detection mouse immune, IgG, IgG1, IgG2 _aThe humoral response level.

1) preparation liquid

1. the preparation of coating buffer: take by weighing NaHCO ₃1.6g, Na ₂CO ₃2.9g, be dissolved in 1L ddH ₂O transfers to 9.6 with the PH meter 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 1L ddH ₂O adds 500 μ L Tween 20 again, with the PH meter pH is transferred to 7.4 again;

4. the preparation of washings: take by weighing 2.42g Tris and be dissolved in 1L ddH ₂O adds 500 μ L Tween 20 again, with the PH meter pH is transferred to 7.4 again;

5. nitrite ion (TMB) is sky root company product;

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

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

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

2. coated: the recombinant protein diluent is added enzyme plate, 200 μ L/ holes, 4 ℃ are spent the night and wrap with preservative film after empty the doing with washings washing 3 times afterwards, place 4 ℃ of refrigerators for subsequent use;

3. sealing: enzyme plate adds confining liquid 100 μ L/ holes, places 37 ℃ of incubators 2 hours, washs 3 times;

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

5. get the good enzyme plate of sealing, add successively dilute serum, 100 μ L/ holes,, place 37 ℃ of incubator 30min, wash empty doing 3 times;

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

7. add dilution antibody working fluid, 100 μ L/ holes place 37 ℃ of incubator 1h, wash three times, empty doing;

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

9. add stop buffer (2M H ₂SO ₄), place immediately on the microplate reader and measure the OD value with 450nm wavelength place;

10. the result judges: A _Sample/ A _NegativeZhi＞=2.1 positive (negative control is that serum 1:1000 doubly dilutes before the mouse immune).

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

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

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

Attack malicious immune protective effect after the table 1 injecting immune BALB/c mouse

The result shows that 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 the MRSA viable bacteria than other each groups.

By above embodiment, those skilled in the art utilize ordinary skill knowledge can use apparently the prepared recombinant protein of the present invention and other related reagents, preparation related kits such as coated reagent, detection antibody, developer, terminator, for example detection kit is used for diagnosis and whether infects golden yellow staphylococcus, determines prognosis etc.

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

The above is preferred embodiment of the present invention only, is not to limit practical range of the present invention; If do not break away from the spirit and scope of the present invention, the present invention is made amendment or is equal to replacement, all should be encompassed in the middle of the protection domain of claim of the present invention.

Claims

1. 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 forms by the connection peptides fusion, the dna sequence dna of this recombination fusion protein is shown in SEQ ID NO:1, and aminoacid sequence is shown in SEQ ID NO:2.

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

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

4. recombination fusion protein according to claim 1 is characterized in that: described Linker structure is-(Linker) n-, and its Linker is GGGGS or GGSGG or YAPVDV, preferred GGGGS; N is 1,2,3,4, preferred 1.

5. the preparation method of recombinant protein claimed in claim 1 is characterized in that, may further comprise the steps:

(1) by doing template with the methicillin-resistant staphylococcus aureus genome, carries out the goal gene that pcr amplification obtains purpose coding HI2 with special primer; Or the rear synthetic goal gene that obtains of full gene of codon optimization.

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

(3) Host Strains behind the Induction Transformation is expressed the HI2 recombinant protein;

(4) purification of recombinant proteins.

6. an expression vector is characterized in that: the nucleotide sequence that comprises the described recombinant protein of coding claim 1.

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

8. an expression is such as the Host Strains of expression vector as described in claim 6 or 7.

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, is preferably E.colistrain XL1 blue.

10. the application of HI2 recombination fusion protein claimed in claim 1 in the subunit vaccine of preparation methicillin-resistant staphylococcus aureus resistance.