CN102977214B - Recombinant protein HF2 used for methicillin-resistant staphylococcus aureus (MRSA) vaccine, and preparation method and application thereof - Google Patents

Abstract

The invention discloses a recombinant fusion protein HF2 which is composed of staphylococcus aureus alpha hemolysin (H1a) and a fibronectin binding protein (FNBA) segment, wherein the H1a and the FNBA are fused through a linker peptide. The protein can be used for preparing subunit vaccines and relevant detection kits capable of resisting infections of the methicillin-resistant staphylococcus aureus (MRSA). A gene engineering technology is used to recombine and express the fused protein. The method has high expression level, facilitates separation and purification and has high efficiency and safety. Animal experiment results show that the recombinant fusion protein HF2 can effectively stimulate a body to produce relatively high humoral immune response and good immune protection effect.

Description

Recombinant protein HF2 and preparation method and application for methicillin-resistant staphylococcus aureus (MRSA) vaccine

Technical field

The invention belongs to biological technical field, be particularly related to the recombinant protein HF2 of a kind of methicillin-resistant staphylococcus aureus alpha hemolysin non-toxic mutant and fibronectin binding protein A active fragments, the invention further relates to preparation method and the application in preparing vaccine and detection kit thereof of this recombinant protein.

Background technology

Streptococcus aureus can cause human body to produce disease widely, as microbemia, and 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 because the multiple antibiotic resistance of this bacterium makes situation more be difficult to control, the selection for the treatment of means is fewer and feweri.

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 form as antigen the active principle of vaccine.If polysaccharide CP5，CP8Bei Nabi company is as vaccine component; Protein I sdBBei Merck company is as vaccine component.

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, in born of the same parents, ion oozes out etc.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.

Fibronectin is a relevant multifunctional receptor in surface in conjunction with albumen (FNBA), and special Reversible binding is in people's fibronectin, fibrin, fibrinogen etc.This is combined in body operation and injured situation bacterium is effectively adhered to, and invades field planting thereupon.FNBP is the acidic protein of one 929 about 106kDa of amino acid.Induction body produces its antibody and can stop SA to adhere to people's tissue, and then prevents infections.Because FNBA molecular weight is large, be difficult to carry out with recombinant bacterial strain the expression of intact proteins, we are using FNBA mature peptide 464-904 amino acid as one of candidate vaccine.

Summary of the invention

High drug-resistance for methicillin-resistant staphylococcus aureus, recombinant protein is merged in the restructuring that the invention provides a kind of methicillin-resistant staphylococcus aureus alpha hemolysin non-toxic mutant and fibronectin binding protein A active fragments, and 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 proof can produce higher humoral immunoresponse(HI) and good immanoprotection action by effective stimulus body.

Recombination fusion protein is combined albumen (FNBA) fragment by streptococcus aureus alpha hemolysin (Hla) and is formed with fibronectin, wherein, Hla and FNBA merge by connection peptides.Described recombination fusion protein has the constructional feature of Hla-Linker-FNBA, is called for short HF2; There is the DNA sequence dna of SEQ ID NO:1 and the aminoacid sequence of SEQ ID NO: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 preferably Linker is GGGGS, and n is 1.

Described Hla is the mutant Hla (delta 110-150) of 110-150 disappearance and the streptococcus aureus alpha hemolysin holotoxin Hla of Hla (H35L, delta110-150).

Described Hla is the streptococcus aureus alpha hemolysin holotoxin Hla of the mutant Hla (H35L, delta110-150) of 110-150 disappearance preferably, and its aminoacid sequence is as shown in SEQ ID NO:4.

Described FNBA be streptococcus aureus fibronectin in conjunction with protein film outskirt, refer to that particularly streptococcus aureus fibronectin is in conjunction with protein film outskirt, its 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 is synthetic to obtain the DNA sequence dna of coding Hla (H35L delta110-150);

2) use forward and the reverse primer of nucleotide sequence as shown in SEQ ID NO:7 and SEQ ID NO:8, amplify the Hla part of recombination fusion protein;

3) with nucleotide sequence if DNA sequence dna is forward and the reverse primer as shown in SEQ ID NO:9 and SEQ ID NO:10, from MRSA genome amplification, go out the FNBA part of recombination fusion protein;

4) with Hla and FNBA, do template, forward and reverse primer with nucleotide sequence as shown in SEQ ID NO:7 and SEQ ID NO:10 amplify recombination fusion protein HF2 fragment;

5) by obtained HF2 gene fragment clone to expression vector, be then converted into Host Strains, Host Strains after Induction Transformation is expressed HF2 recombinant protein;

6) purification of recombinant proteins.

Express the expression vector of HF2 recombinant protein, the nucleotide sequence SEQ ID NO:2 that it comprises the described HF2 recombinant protein of encoding.

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, the present invention preferably adopts pGEX-6p-2 carrier to build recombinant expression vector, express recombinant protein HF2, 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.Compare 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 the recombinant protein of the above in preparing the subunit vaccine of methicillin-resistant staphylococcus aureus resistance.

The application of above-mentioned recombinant protein in preparing 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, i.e. Hla (H35L) with Leu.In order further to reduce the molecular weight of the Hla former protein structure of trying one's best simultaneously, the present invention can form the 110-150 amino acids of perforation structure and remove, and form Hla (H35L delta110-150), and its aminoacid sequence is SEQ ID NO:4.

FnBA albumen is twice transmembrane protein, the aminoacid sequence of the corresponding 28-943 in its extracellular region, and it adheres to the main protein by extracellular region and plays a role, in order to keep the structure and function of FNBA not have greatly changed on to greatest extent, mature peptide 464-904 amino acid for the present invention, be FNBA2, its sequence is SEQ ID NO:6, be connected with small peptide GGGGS with Hla (H35L delta110-150), form last fusion rotein name HF2, its aminoacid sequence is SEQ ID NO:2.

Therefore, 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.HF2 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 are according to the newest fruits of staphylococcus aureus gene group and proteomics, determine and build the vaccine in conjunction with albumen (FNBPA) based on alpha hemolysin (Hla) and fibronectin.

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

1) HF2 expression of recombinant proteins plasmid abduction delivering in prokaryotic expression system-intestinal bacteria;

2), while selecting pGEX carrier series, HF2 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 HF2 recombinant protein purity is out greater than 95%;

3) HF2 recombinant protein all can produce specific antibody by induced animal.

Utilize subunit vaccine prepared by HF2 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 He Duo subunit fusion bacterin research lays the first stone, development and the application for control vaccine and diagnostic kit simultaneously 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.

Accompanying drawing explanation

Fig. 1 is the pcr amplification result of HF2 gene fragment, wherein

Swimming lane 1: goal gene fragment HF2 (2.1kb);

Swimming lane 2: nucleic acid (DNA) molecular weight standard (Marker)

Fig. 2 is that the enzyme of expression vector pGEX-6p-2-HF2 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-HF2 after enzyme is cut, the fragment that enzyme is cut rear separation is 4.8kbp and 2.1kb;

Fig. 3 HF2 protein purification result

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

Swimming lane 2:HF2 albumen

Fig. 4 recombinant expression vector checks order afterwards and the nucleotide sequence comparison result of HF2.

Embodiment

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

1. bacterial strain

Streptococcus aureus MRSA-252 international standard Zhu You U.S. ATCC provides;

2. reagent

Plasmid pGEX-6p-2 is that U.S. GE Healthcare 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;

Bacterial genomes is extracted test kit, ultra-thin recovery test kit and nitrite ion Wei Tiangen company product;

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

Embodiment 1: the DNA sequence dna of methicillin-resistant staphylococcus aureus Hla (H35L delta110-150) is synthetic by Shanghai JaRa Bioisystech Co., Ltd.

Embodiment 2: the H part that is used for building HF2 with PCR method amplification.

1. the connection peptides SEQ ID NO:3 that considers H section, designs following primer and clones H gene from synthetic DNA sequence dna:

SEQ ID NO:7gc ggatccat ggcagattct gatattaata ttaa (underscore shows restriction enzyme site BamHI)

SEQ ID NO:8aactaagctg ccaccgccac catttgtcat ttc

2.PCR clone gene

PCR system:

Template (100ng/ μ l)	0.5μl
		SEQ ID NO:7(50μM)	1μl
SEQ ID NO:8(50μM)	1μl
		Primerstar Taq enzyme	0.5μl
dNTP	4μl
		5X Buffer	10μl
Sterilizing distilled water	33μl
		Cumulative volume	50μl

98 ℃ of sex change 10s of pcr amplification reaction condition, 60 ℃ of annealing 10s, 72 ℃ are extended 1min30s, 30 circulations, 72 ℃ are extended 6min completely.Use after completion of the reaction 1% sepharose detection pcr amplification result.

Use gel to reclaim test kit and reclaim H gene product.

Embodiment 3: the clone of methicillin-resistant staphylococcus aureus fibronectin binding protein A active fragments FNBA2 active fragments (F2)

1. first according to MRSA-252FnBA albumen full-length gene order, applying biological information software carries out structural analysis, determines the FnBA1 goal gene fragment that needs amplification.

2. according to analytical results, adopt PCR method from MRSA-252 genome amplification FnBA1 goal gene fragment, amplification step is as follows:

1) design PCR primer is as follows, is respectively SEQ ID NO:9-10

SEQ ID NO:9ggtggcggtg gcagcttagt tttatatagt aataaagc

SEQ ID NO:10ttttcctttt gcggccgcct aacctttgtt tgttgattct tctc(underscore shows restriction enzyme site NotI)

2) in-80 ℃ of freezers, taking out the methicillin-resistant staphylococcus aureus MRSA-252 bacterial strain of preserving coats on MRSA-252 special solid substratum, in 37 ℃ of overnight incubation, picking list colony inoculation is cultivated 8 hours in the special liquid bulk substratum of MRSA-252 again, with reference to bacterial genomes extraction agent box extracting MRSA genome.

3) take MRSA-252 complete genome DNA as template pcr amplification FNBA2 gene fragment

PCR system:

Template (239.5ng/ μ l)	2.5μl
		SEQ ID NO:9(50μM)	1μl
SEQ ID NO:10(50μM)	1μl
		Primerstar Taq enzyme	0.5μl
dNTP	4μl
		5X Buffer	10μl
Sterilizing distilled water	31μl
		Cumulative volume	50μl

98 ℃ of denaturation 5min of pcr amplification reaction condition, 98 ℃ of sex change 10s, 60 ℃ of annealing 10s, 72 ℃ are extended 1min30s, 30 circulations, 72 ℃ are extended 6min completely.Use after completion of the reaction 1% sepharose detection pcr amplification result.

4) use gel to reclaim test kit and reclaim FNBA2PCR product.

The clone of embodiment 4:HF2 gene

PCR system:

Template H+F2	2μl
		SEQ ID NO:7(50μM)	1μl
SEQ ID NO:10(50μM)	1μl
		Primerstar Taq enzyme	0.5μl
dNTP	4μl
		5X Buffer	10μl
Sterilizing distilled water	31.5μl
		Cumulative volume	50μl

98 ℃ of sex change 10s of pcr amplification reaction condition, 60 ℃ of annealing 10s, 72 ℃ are extended 2min30s, 30 circulations, 72 ℃ are extended 6min completely.Use after completion of the reaction 1% sepharose detection pcr amplification result.

Use gel to reclaim test kit and reclaim HF2 product, result is as accompanying drawing 1.

Embodiment 5: the structure of restructuring HF2 gene clone

1.BamH I and Not I enzyme are cut pGEX-6P-2 plasmid and FnBA1PCR product

Endonuclease reaction system:

BamH I	3μl
		Not I	3μl
10×K Buffer	3μl
		0.1%BSA	6μl
Product	45μl

Cumulative volume

60μl

37 ℃ of enzymes are cut 2h.

2. the PCR product that uses ultra-thin recovery test kit to reclaim pGEX-6P-2 plasmid and cut through BamH I and Not I enzyme.

3. connect and transform

By ultraviolet spectrophotometer, measure HF2 enzyme and cut back to close product nucleic acid concentration: 15ng/ μ l, pGEX-6P-2 enzyme cuts back to close product nucleic acid concentration: 60ng/ μ l.

Ligation system:

Solution I	5μl
		HF2 enzyme cuts back to close product	4μl
PGEX-6P-2 enzyme cuts back to close product	2μl
		Cumulative volume	10μl

Mix, 16 ℃ connect 1h.

From-80 ℃ of refrigerators, get 3 pipe intestinal bacteria XL1blue competent cells, the first pipe adds pGEX-6P-2 plasmid, makes positive control; The second pipe adds DNA to connect product; 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 l LB, mix, be placed in 37 ℃ 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 ℃ of incubators and cultivates 24h.

4.pGEX-6p-2/HF2 the screening of positive recombinant plasmid, evaluation

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 ℃ 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:

BamH I	0.5μl
		Not I	0.5μl
10×K Buffer	0.5μl
		0.1%BSA	1μl
Recombinant plasmid	7.5μl
		Cumulative volume	10μl

37 ℃ of enzymes are cut 2h;

4. 1% agarose gel electrophoresis detects double digestion result, and result is as Fig. 2, and visible swimming lane 2 samples are the pGEX-6p-2-HF2 recombinant plasmid successfully constructing;

5. pGEX-6p-2-HF2 recombinant plasmid is sent to the order-checking of Dalian TaKaRa company, and sequencing result is CQS5997, is shown in Fig. 4 with expected sequence SEQ ID NO:5 comparison result, and the sequence of the goal gene fragment of visible recombinant plasmid is correct.

Embodiment 6: the evaluation of recombination fusion protein HF2 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-HF2/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 standby), 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, be placed in again 30 ℃ of 3h of shaking table abduction delivering, 25 ℃ of 5h, 16 ℃ 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 ℃ 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 ℃, 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 14000rpm.

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 standby 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-HF2/XL-1blue is soluble proteins and no significant difference at 16 ℃, 25 ℃, 30 ℃.

The preparation of embodiment 7:HF2 antigen

1. amplification culture is obtained albumen

Go bail for and exist in 4 ℃ of refrigerators standby pGEX-6P-2-HF2/XL-1blue bacterium liquid 400 μ L to join 20mL once to activate containing in the TB substratum of Amp resistance, after 37 ℃ 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 ℃ 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 ℃ 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 HF2FnBA1 target protein

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 HF2 molecular weight of albumen that enzyme scales off is between 95-130kDa, be consistent with expection molecular weight of albumen size, see Fig. 3.

3.BCA method is measured protein content, and maximum concentration is 1.5mg/mL.

Embodiment 8: infect the foundation with staphylococcus aureus strains (international standard strain MRSA-252) standard quantitative curve

Inoculation is placed in to 37 ℃ 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 ℃ of constant-temperature table concussions and cultivates the centrifugal 10min collection of 6000rpm thalline after 6 hours, with physiological saline, washs 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 ℃ 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 9: the structure of pyemia animal model

1. inoculation being placed in to 37 ℃ 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 ℃ 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 carry out systemic infection by the BALB/C mice (100 μ l/ are only) that each group bacterium liquid is 18 ~ 20g by 6 ~ 8 weeks age of tail vein injection, body weight, physiological saline control group is set simultaneously, observe and within 7 days, also add up the mortality ratio of respectively organizing mouse;

2, infecting rear 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 20 μ L blood samples with after 10 times of 180 μ L heparin dilutions for bacterial count, get 50 μ L and be applied to flat board, be placed in 37 ℃, counting clone number after 24h; After taking blood sample, mouse is put to death and put into after 75% alcohol soaking disinfection, its four limbs are fixed in taking-up, 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 ℃, 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 when 48h, reach peak value, maximum field planting amount reaches 8.0 * 10 ⁹cFU/ml, when 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 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 10: the detection of immune animal and antibody

1. immune animal

1) first immunisation, with PBS dilution HF2 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;

2. after immunity for the third time the 7th and 14 days, gather the blood of BALB/C mice, with ELISA, detect after mouse immune IgG, IgG1, IgG2 _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, is adjusted to 9.6 with PH meter by 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 with PH meter, pH is adjusted to 7.4;

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

5. nitrite ion (TMB) ，Wei Tiangen 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 HF2 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 ℃ spend the night after with washings washing 3 times, emptyly with preservative film, wrap after dry, be placed in 4 ℃ of refrigerators standby;

3. sealing: enzyme plate adds confining liquid 100 μ L/ holes, is placed in 37 ℃ 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 ℃ 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 ℃ 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 HF2 proteantigen immune mouse reaches 1:512000; After immunity, the antibody positive rate of the 7th day reaches 90%, illustrates that the HF2 recombinant protein that the present invention builds can make generation antibody in immune mouse body.

Embodiment 11: by immune mouse, determine the malicious protection of attacking of HF2 recombinant protein immune animal

With the immunization protocol of embodiment 6, after immune mouse, at the 14th day, adopt lethal dose, tail vein injection MRSA-252 viable bacteria to attack poison experiment for the third time, every BALB/C mice injection bacterium liquid measure is 1.25 * 10 ⁹cFU, observes 10 days, and statistics is respectively organized the survival rate of mouse.The results are shown in table 2.

The poison of attacking after table 2HF2 recombinant protein immune mouse is protected effect

Table 2 shows: the average immune protective rate of negative control group and blank group is respectively 15% and 20%, and recombination fusion protein HF2 adds Al (OH) ₃the average immune protective rate of adjuvant group is 80%.Therefore; HF2 recombinant protein of the present invention has good immunogenicity; and can MRSA-252 be infected and be played a protective role, can induce body to produce immunne response, can be aided with aluminium adjuvant and prepare subunit vaccine for preventing the infection of streptococcus aureus.

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, such as coated reagent, detection antibody, developer, terminator etc. prepared related kit, for example whether detection kit, infect golden yellow staphylococcus, determine prognosis etc. for diagnosis.

Those skilled in the art can be by HF2 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 (15)

1. a recombinant protein hF2it is characterized in that: this albumen is combined albumen (FNBA) fragment by streptococcus aureus alpha hemolysin (Hla) and is formed with fibronectin, wherein, Hla and FNBA merge by connection peptides, and the aminoacid sequence of this recombinant protein is as shown in SEQ ID NO:2.

2. recombinant protein according to claim 1 hF2, it is characterized in that, described connection peptides is Linker, this connection peptides is-(Linker) n-, wherein Linker is GGGGS or GGSGG or YAPVDV, n is 1-4.

3. recombinant protein according to claim 2 hF2, it is characterized in that, described connection peptides Linker is GGGGS, n is 1.

4. recombinant protein according to claim 1 hF2, it is characterized in that, described Hla is the sudden change Hla (H35L, delta110-150) of 110-150 disappearance.

5. the recombinant protein of stating according to claim 4 hF2it is characterized in that: described Hla is the mutant Hla (H35L of 110-150 disappearance, delta110-150) streptococcus aureus alpha hemolysin holotoxin Hla, its DNA sequence dna is as shown in SEQ ID NO:3, and aminoacid sequence is as shown in SEQ ID NO:4.

6. recombinant protein according to claim 1 hF2, it is characterized in that: described FNBA be streptococcus aureus fibronectin in conjunction with protein film outskirt, its DNA sequence dna is as shown in SEQ ID NO:5, aminoacid sequence is as shown in SEQ ID NO:6.

7. the arbitrary described recombinant protein of claim 1-6 hF2preparation method, it is characterized in that, comprise the following steps:

1) full gene is synthetic to obtain the DNA sequence dna of coding Hla (H35L delta110-150);

2) forward and the reverse primer as shown in SEQ ID NO:7 and SEQ ID NO:8 with nucleotide sequence, amplifies Hla;

3) with nucleotide sequence if DNA sequence dna is forward and the reverse primer as shown in SEQ ID NO:9 and SEQ ID NO:10, from MRSA genome amplification, go out FNBA;

4) with Hla and FNBA, do template, forward and reverse primer with nucleotide sequence as shown in SEQ ID NO:7 and SEQ ID NO:10 amplify recombination fusion protein HF2 fragment;

5) by obtained HF2 gene fragment clone to expression vector, be then converted into Host Strains, Host Strains after Induction Transformation is expressed HF2 recombinant protein;

6) purification of recombinant proteins.

8. an expression vector, is characterized in that: comprise recombinant protein described in coding claim 1 hF2nucleotide sequence.

9. expression vector according to claim 8, is characterized in that, described expression vector is pGex serial carrier, pET serial carrier.

10. expression vector according to claim 9, is characterized in that, described expression vector is pGex-6P-2.

The Host Strains of expression vector as described in 11. 1 kinds of expression are as arbitrary in claim 8-10.

12. Host Strains according to claim 11, is characterized in that, described Host Strains is E.colistrain XL1 blue, BL21 series or HMS174 series.

13. Host Strains according to claim 12, is characterized in that, described Host Strains is E.colistrain XL1 blue.

14. recombinant proteins claimed in claim 1 hF2application in preparing the subunit vaccine of methicillin-resistant staphylococcus aureus resistance.

15. recombinant proteins claimed in claim 1 hF2application in preparing methicillin-resistant staphylococcus aureus detection kit.

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