CN103694323A

CN103694323A - MntC recombinant protein of staphylococcus aureus and preparation method and application thereof

Info

Publication number: CN103694323A
Application number: CN201310665113.9A
Authority: CN
Inventors: 曾浩; 邹全明; 樊绍文; 卢陆; 杨茜; 敬海明
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: 2013-12-09
Filing date: 2013-12-09
Publication date: 2014-04-02
Anticipated expiration: 2033-12-09
Also published as: CN103694323B

Abstract

The invention belongs to the field of biotechnology, and relates to a MntC recombinant protein of staphylococcus aureus (SA), a carrier comprising the recombinant protein, a host, a composition or a kit, application, preparation, fermentation and purification method of the protein. The MntC recombinant protein prepared by the method has strong immunogenicity, is safe and non-toxic, and is proved by animal tests to be able to effectively stimulate an organism to generate high efficient humoral immune response and good immune protection.

Description

Streptococcus aureus MntC recombinant protein and its preparation method and application

Technical field

The invention belongs to biological technical field, relate to a kind of streptococcus aureus (SA) MntC recombinant protein.

Background technology

Streptococcus aureus (Staphylococcus aureus, SA), hereinafter to be referred as golden Portugal bacterium, has the another name of " having a liking for meat bacterium ".As the representative of gram-positive microorganism, it is a kind of important pathogenic bacteria that causes that hospital infection and community infect.It is acute, suppurative that infection be take is feature, and local infection can cause the pyogenic infection of skin and soft tissue etc., does not prolongedly heal; Systemic infection can cause severe infections and the complication such as osteomyelitis, septic arthritis, endocarditis, pneumonia, pyemia, and mortality ratio is up to 20%.Meanwhile, the extracellular toxin of golden Portugal bacterium also can cause the whole body lethal infections such as food poisoning, scalded skin syndrome and toxic shock syndrome.Therefore, strengthen the immune protection research that Dui Jin Portugal bacterium infects, develop safe and effective novel golden Portugal bacterium recombinant vaccine and extensively infect and there is important strategy and realistic meaning the golden Portugal of effective control bacterium resistance being spread with clinical golden Portugal bacterium.

The living necessities many kinds of metal ions of SA, they participate in various physiological processes, as metabolism, DNA is synthetic and the supervision of virulence factor etc.The picked-up of these ions is relevant with specific albumen, thereby utilizes these specific protein immune animals to produce can yet be regarded as a kind of selection of effective vaccine of specific antibodies.Iron ion determines that protein I sdBJiu Bei Merck company carried out clinical trial as candidate vaccine.Mn ion is also that SA growth institute is essential, and it can be protected SA to avoid host's neutrophilic granulocyte and kill and wound, and in the immune evasion of SA, has brought into play vital role.SA infect early stage by raising mn ion translocator C(MntC) and to obtain mn ion be the key mechanism that escape is killed and wounded.Yet this element picked-up too much can cause toxicity, therefore, the picked-up of manganese is by altitude mixture control.Mn ion metabolism associated protein is than the more Zao expression of IsdB when SA infects, and these albumen comprise an ATP synthetic proteins MntA, a complete cell membrane transporter albumen MntB and a mn ion translocator MntC.Wherein MntC is a kind of cell surface protein of high conservative.Recently, MntC is defined in SA surface of cell membrane and expresses, and then in multiple In vivo infection model, occurs.Thereby using MntC as antigen-immunized animal, induction body produces the antibody for this albumen, thereby should be able to block in early days its function that regulates manganese metabolism, play the effect that SA infects of resisting.

Summary of the invention

Object of the present invention aims to provide a kind of streptococcus aureus mn ion translocator MntC of restructuring, used as antigen, for the preparation of the biological products that detect, prevent or treatment SA infects.

Of the present invention, aspect first, provide the MntC recombinant protein of a kind of streptococcus aureus, the aminoacid sequence of described MntC recombinant protein is as shown in SEQ ID NO.1.

Aspect second of the present invention, the nucleotide sequence of coding MntC recombinant protein of the present invention is provided, and the carrier that comprises described nucleotide sequence and host.

Aspect the 3rd of the present invention, provide a kind of method of preparing MntC recombinant protein of the present invention.

Aspect the 4th of the present invention, a kind of MntC of fermentation host is provided and prepares the method for MntC recombinant protein.

Aspect the 5th of the present invention, provide the method for a kind of purifying MntC recombinant protein of the present invention.

Aspect the 6th of the present invention, the application of MntC recombinant protein of the present invention as antigen is provided, and the application of described MntC recombinant protein in the biological products for the preparation of detecting, prevent or treat SA to infect.

Aspect the 7th of the present invention, provide the polyclonal antibody and the application of described polyclonal antibody in the biological products for the preparation of detecting, prevent or treat SA to infect that by MntC recombinant protein immunity of the present invention, are produced.

Aspect the 8th of the present invention, provide the composition or the test kit that comprise MntC recombinant protein of the present invention.

The present invention adopts the recombinant expressed described fusion rotein of genetic engineering technique, not only expression amount high, be convenient to separation and purification, and immunogenicity is strong, safety non-toxic.Through animal experiment, prove, MntC recombinant protein of the present invention can produce higher humoral immunoresponse(HI) and good immanoprotection action by effective stimulus body, can be used in the biological products that preparation detects, prevents or treatment SA infects.

Accompanying drawing explanation

The pcr amplification result of Fig. 1, MntC gene fragment: M:DNA molecular weight standard; 1,2: goal gene fragment MntC (855bp).

The enzyme of Fig. 2, expression vector pGEX-6p-2-MntC is cut qualification result: M: nucleic acid (DNA) molecular weight standard; 1,2,3,4: recombinant expression plasmid pGEX-6p-2-MntC1,2,3,4 is for wherein No. 3, No. 4 the successful plasmid of restructuring, and enzyme is cut fragment 4800bp and the 855bp of rear separation.

Fig. 3, MntC affinity chromatography result: the fusion protease containing GST label is cut result: M: molecular weight of albumen standard; 1: before enzyme is cut, the fusion rotein that contains GST label; 2: after enzyme is cut, the target protein obtaining at supernatant (password optimizing project bacterium); 3: after enzyme is cut, target protein and specific binding enzyme and the GST label (password optimizing project bacterium) on gel beads of non-specific binding in gel beads; 4: after enzyme is cut, the target protein obtaining at supernatant (password is optimizing project bacterium not); 5: after enzyme is cut, target protein and specific binding enzyme and the GST label (password not optimizing project bacterium) on gel beads of non-specific binding in gel beads.

Fig. 4, the growth curve of MntC engineering bacteria in four kinds of substratum.

Fig. 5, the MntC expression in M9 and TB substratum; 1: protein molecular weight standard; 2:M9; 3:TB.

Fig. 6, the impact of different IP TG concentration on MntC protein expression; 1: protein molecular weight standard; 2:0.1mM; 3:0.2mM; 4:0.5mM; 5:1mM.

Fig. 7, different vaccination amount growth curve of bacteria figure before induction.

Fig. 8, the impact of kind daughter bacteria different vaccination amount on protein expression; 1: protein molecular weight standard; 2:5%; 3:10%; 4:15%.

MntC engineering bacteria growth curve under Fig. 9, different oxygen concentrations.

Figure 10, PO ₂during different concns, the expression of MntC albumen; 1: protein molecular weight standard; 2:25%; 3:45%; 4:65%.

The impact of the glycerine of Figure 11, different concns on MntC protein expression; 1: protein molecular weight standard; 2:5ml/L; 3:10ml/L; 4:15ml/L.

Figure 12, the situation that target protein is expressed when different inducing temperatures and time; 1: protein molecular weight standard; 2: induction 0h; 3: induction 2h; 4: induction 4h; 5: induction 6h; 6: induction 8h; 7: induction 10h.

The growth curve of Figure 13,25L fermentation MntC engineering bacteria.

During Figure 14,25L fermentation MntC engineering bacteria, MntC protein expression situation; 1: protein molecular weight standard; 2: induction 0h; 3: induction 1h; 4: induction 2h; 5: induction 3h; 6: induction 4h; 7: induction 5h.

Figure 15, GST-sepharose 4B purifying MntC result; M: protein molecular weight standard; 1: be incorporated into the GST-MntC fusion rotein on filler; 2: enzyme is cut the albumen on filler after wash-out; 3: enzyme is cut the target protein MntC after wash-out.

Figure 16, MMC chromatography purification MntC albumen color atlas.

Figure 17, MMC chromatography purification MntC protein electrophoresis figure.M: protein molecular weight standard; 1: sample; 2: stream is worn; 3: elution peak.

Figure 18, Phenyl HP chromatography purification MntC albumen color atlas.

Figure 19, Phenyl HP chromatography purification MntC protein electrophoresis figure.M: protein molecular weight standard; 1: stream is worn; 2-4: elution peak collection tube 1-3.

Figure 20, high electricity are led lower MMC chromatography purification MntC albumen color atlas.

Figure 21, high electricity are led lower MMC chromatography purification MntC protein electrophoresis figure; M. protein molecular weight standard; 1: sample introduction sample; 2: stream is worn sample 3:MMC elution samples.

Figure 22, low electricity are led lower MMC chromatography purification MntC albumen color atlas.

Figure 23, low electricity are led lower MMC chromatography purification MntC protein electrophoresis figure; M. protein molecular weight standard; 1: sample introduction sample; 2: stream is worn sample; 3:MMC elution samples.

Figure 24, first amplification purifying of MntC albumen MMC color atlas.

Figure 25, first amplification purifying of MntC albumen Q HP color atlas.

Figure 26, first MMC and Q HP chromatography purification protein electrophoresis figure; M: protein molecular weight standard; 1: sample introduction sample; 2:MMC elution samples; 3:G25 elution samples; 4:Q HP stream is worn sample.

Figure 27, MntC albumen second batch amplify purifying MMC color atlas.

Figure 28, MntC albumen second batch amplify purifying Q HP color atlas.

Figure 29, second batch MMC and Q HP chromatography purification protein electrophoresis figure; M: protein molecular weight standard; 1: sample introduction sample; 2:Q HP stream is worn sample; 3:G25 elution samples; 4:MMC elution samples.

Figure 30, the 3rd batch, MntC albumen amplify purifying MMC color atlas.

Figure 31, the 3rd batch, MntC albumen amplify purifying Q HP color atlas.

Figure 32, the 3rd crowd of MMC and Q HP chromatography purification protein electrophoresis figure; M: protein molecular weight standard; 1: sample introduction sample; 2:MMC elution samples; 3:G25 elution samples; 4:Q HP stream is worn sample.

The HPLC detected result of Figure 33, MntC final sample; Main peak retention time 13.409 minutes, main peak ratio 99.5%.

The centrifugal supernatant 10%SDS-PAGE of Figure 34, MntC stoste and vaccine preparation; 1-5: the centrifugal supernatant of vaccine preparation (in conjunction with 1～5 hour, sampling per hour); 6:MntC stoste.

The collection of illustrative plates of Figure 35, pGEX serial carrier.

Embodiment

To illustrate content of the present invention below, but scope of the present invention is not limited to following embodiment and embodiment.

The present invention is based on prior art, a kind of streptococcus aureus mn ion translocator MntC of restructuring is provided, it can be applicable to prepare the subunit vaccine of infection of staphylococcus aureus and relevant detection kit, and the aminoacid sequence of described MntC albumen is as shown in SEQ ID NO.1.The optimization of the 6-290 amino acids of this albumen based on wild MntC albumen, 1-5 amino acids (GPLGS, SEQ ID NO.6) be the amino acid from the modification of expression vector, these 5 amino acid whose encoding sequences are preferably gggcccctgggatcc(SEQ ID NO.7).

On the other hand, the invention provides the nucleotide sequence of the described MntC albumen of coding.The in the situation that of known protein amino acid sequence, those skilled in the art can design the nucleotide sequence of suitable encode such amino acid sequences completely as required, and make its expression.A kind of preferred embodiment in, described nucleotide sequence is as shown in SEQ ID NO.2.

On the other hand, the invention provides a kind of for expressing the recombinant expression vector of MntC recombinant protein, nucleotide sequence and carrier sequence that described expression vector comprises the described MntC recombinant protein of encoding.Preferably, described expression vector is pGEX or pET serial carrier; More preferably, described expression vector is pGEX-6p-2 carrier.The collection of illustrative plates of pGEX serial carrier as shown in figure 35.

Its principal feature of pGEX-6p-2 carrier that the present invention preferably adopts is on carrier, to be connected to the glutathione-S-transferase that a molecular weight is 26kDa (GST), just contains a GST label in expressed fusion rotein, and this label is the mark of protein purification.Compare with other fusion vector, 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.

On the other hand, the present invention also provides a kind of host who expresses MntC recombinant protein of the present invention, comprises recombinant expression vector of the present invention.Described host can be any express cell well known by persons skilled in the art, and preferably, described host is intestinal bacteria XL1-Blue.

On the other hand, the present invention also provides a kind of method of the MntC of preparation recombinant protein, and described method can be chemical synthesis or Nucleotide representation.Those skilled in the art can know how to prepare MntC recombinant protein of the present invention completely according to the sequence of MntC recombinant protein and this area general knowledge.

A kind of preferred embodiment in, the method for the MntC of preparation recombinant protein of the present invention comprises the following steps:

1) according to the encoding sequence of wild MntC albumen (SEQ ID NO.5) design forward primer and reverse primer; Preferably, described forward primer is as shown in SEQ ID NO.3, and described reverse primer is as shown in SEQ ID NO.4;

2) use forward primer and the reverse primer of step 1) design, by pcr amplification go out the to encode gene fragment of MntC albumen; Codon to MntC albumen is optimized, be specially, the 9th bit codon of MntC albumen coded sequence (SEQ ID NO.5) (coding Leu) is optimized for to CTG, 13rd, 14 bit codons (coding Thr) are optimized for ACC, 27th, 65,282 bit codons (coding Gly) are optimized for GGT, and the 214th codon (coding Arg) is optimized for CGT;

3) by step 2) gene fragment clone that obtains is to expression vector, is then converted into Host Strains;

4) Host Strains after Induction Transformation is expressed MntC recombinant protein.

Preferably, described expression vector is pGEX-6p-2, and described Host Strains is intestinal bacteria XL1-Blue.

On the other hand, the present invention also provides a kind of MntC host's zymotechnique.

Described technique comprises a certain amount of kind of daughter bacteria is inoculated in the fermention medium that contains glycerine and certain dissolved oxygen amount, through inductor induction certain hour and express recombinant protein.

Preferably, described technique relates to substratum, plants amounts of glycerol, dissolved oxygen amount, inductor kind, inductor concentration, inducing temperature, induction time several respects factor in daughter bacteria inoculum size, substratum.Wherein, described substratum can be animal derived TB, animal derived M9, plant-derived TB, plant-derived M9, is preferably animal derived TB; The inoculum size of described kind of daughter bacteria is 5%～15%, is preferably 10%; Described amounts of glycerol is 5-15ml/L substratum, is preferably 10ml/L substratum; Described dissolved oxygen amount is 25%～65%, is preferably 45%; Described inductor can be lactose or IPTG, is preferably IPTG; The concentration of described inductor is 0.1～1mM, is preferably 0.2mM; Described inducing temperature is 16～37 ℃, is preferably 30 ℃; Described induction time is 1～6 hour, is preferably 5 hours.

In a kind of embodiment of described zymotechnique, basic medium is selected animal derived TB substratum, and wherein amounts of glycerol is 10ml/L; When fermentation starts, the inoculum size ratio of planting daughter bacteria is 10%; Oxyty remains on 45% during the fermentation; During induction, it is 0.2mM, induction 5h that temperature is adjusted to 30 ℃, IPTG concentration.

On the other hand, the present invention also provide a kind of after fermentation MntC host the technique of purifying MntC recombinant protein, comprise the glutathione agarose gel 4B affinity chromatography, cation-exchange chromatography, desalination, the de-intracellular toxin that carry out successively.In preferred embodiments, before carrying out affinity chromatography, need to be to the broken bacterium of genetic engineering bacterium, so that albumen is discharged from thalline.Preferably, glutathione agarose gel 4B affinity chromatography is cut in conjunction with PP enzyme enzyme; Described cation-exchange chromatography is MMC chromatography; G25 post is used in described desalination; Described de-intracellular toxin is used Q HP column chromatography.

Preferably, the damping fluid that described MMC chromatography is used comprises: buffer A: 10mM His+0.01% PLURONICS F87, pH6.0; Buffer B: 10mM His+0.01% PLURONICS F87+1M NaCl, pH6.0; MMC: loading flow velocity is 12ml/min; Elution flow rate is 12ml/min; Elution program: 0-100%B, 5-10 column volume carries out gradient elution, preferably 7 column volumes (CV); Loading sample electricity is led and is less than 10ms/cm.

On the other hand, invention also provides the application of MntC recombinant protein in the biological products for the preparation of detecting, prevent or treat SA to infect.Preferably, described biological products are vaccine.Those skilled in the art according to prior art content can be beyond all doubt know how to apply this MntC recombinant protein.

On the other hand, the present invention also provides the antibody being produced by MntC recombinant protein of the present invention immunity, and described antibody is polyclonal antibody, can be for detection, prevention and the treatment of the disease relevant to SA, or for the preparation of corresponding biological products.

On the other hand, the present invention also provides a kind of composition that comprises MntC albumen of the present invention or test kit.Such composition can be reagent, medicine (as vaccine), for prevention, detection or treatment SA, infects.Such test kit can be any form test kits known in the art such as detection kit or treatment test kit.

Expression and the fundamental characteristics of genetically engineered recombinant protein of the present invention comprise:

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

2), while selecting pGEX-6p-2 carrier, MntC recombinant protein is with fusion protein form expression; In expressed fusion rotein, 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 MntC recombinant protein purity is out greater than 95%;

3) MntC recombinant protein all can induce body to produce specific protection antibody.

Subunit vaccine of the present invention can carry out immunization by subcutaneous (muscle) injecting pathway, and excitating organism produces high titre IgG antibody and cellullar immunologic response.And confirm through experimentation on animals, described genetically engineered recombinant subunit vaccine has the immune protective effect that good anti-SA infects.For further combined vaccine He Duo subunit fusion bacterin research lays the first stone, simultaneously for development and the application of vaccine, therapeutic antibodies and diagnostic kit has great importance.

The bacterial strain and all ingredients that in this part, use are as follows:

1. bacterial strain

Streptococcus aureus ATCC international standard strain MRSA-252 is provided by U.S. ATCC;

Strain X L1-blue intestinal bacteria are U.S. Agilent company product.

2. plasmid

Plasmid pGEX-6p-2 is U.S. GE Healthcare company product.

3. reagent

PrimeSTAR HS archaeal dna polymerase, DNA molecular amount standard, restriction enzyme BamH I and Not I, molecular weight of albumen standard, DNA ligase are Dalian TakaRa company product;

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

MH substratum: purchased from Beijing extensive and profound in meaning star biotechnology limited liability company (beef powder 2.0g, Zulkovsky starch 1.5g, acid hydrolyzed casein 17.5g), add water to 1L, pH value 7.4 ± 0.2;

MH is dull and stereotyped: it is 1.5g/100mL that MH substratum adds agarose to final concentration;

PBS(potassium primary phosphate (KH ₂pO ₄) the domestic analytical pure of 0.2g(), Sodium phosphate dibasic (Na ₂hPO ₄12H ₂o) the domestic analytical pure of 2.9g(), the domestic analytical pure of sodium-chlor (NaCl) 8.0g(), Repone K (KCl) 0.2g, adds water to 1000mL, pH7.4);

20mM PB damping fluid: potassium primary phosphate (KH ₂pO ₄) 0.2g, Sodium phosphate dibasic (Na ₂hPO ₄12H ₂o) 2.9g, Repone K (KCl) 0.2g, adds water to 1000mL, pH7.0;

Ampicillin Trihydrate, kantlex (North China pharmacy);

5 * protein sample-loading buffer (250mM Tris-HCl(pH6.8), 10%(W/V) SDS, 0.5%(W/V) tetrabromophenol sulfonphthalein, 50%(V/V) glycerine, 5%(W/V) beta-mercaptoethanol);

Glutathione agarose gel 4B(U.S. GE Healthcare company);

Aluminum phosphate adjuvant: U.S. GENERAL CHEMICAL company (20mg/ml);

Vaccine diluent (Histidine (U.S. Merck company, pharmaceutical grade) 10mM, Cologne, NaCl0.9%(Sichuan, injection physiological saline), PLURONICS F87 (U.S. Merck company, pharmaceutical grade) 0.01%, pH6.0), pyrogen-free;

All the other reagent such as agar powder, tween 20 are domestic market and buy.

Embodiment 1: the coding codon optimized and expression vector of MntC and the structure of expression engineering bacteria

According to intestinal bacteria password Preference, the gene SAR0641 of MRSA-252 genome (GI:49240382) coding MntC is carried out to password optimization, specific as follows:

The 9th bit codon of wild-type MntC albumen coded sequence (SEQ ID NO.5) (coding Leu) is optimized for to CTG, 13rd, 14 bit codons (coding Thr) are optimized for ACC, 27th, 65,282 bit codons (coding Gly) are optimized for GGT, the 214th codon (coding Arg) is optimized for CGT, nucleotide sequence after being optimized (SEQ ID NO.2), the aminoacid sequence of this sequence encoding is as shown in 6th～290 of SEQ ID NO.1.

Transfer to Shanghai JaRa Bioisystech Co., Ltd to carry out full gene the sequence of having optimized and synthesize, take BamHI and NotI as head and the tail restriction enzyme site connects into pGEX-6P-2, form pGEX-6p-2-MntC (r) expression plasmid, transform XL1-Blue bacterial strain.

Embodiment 2: the structure of wild MntC expression vector and expression bacterium

1, get MRSA-252 bacterial strain and coat MH agar plate (Mueller-Hinton Agar, Beijing extensive and profound in meaning star biotechnology limited liability company, LOT:02-051) recovery, 37 ℃ of aerobic overnight incubation.The single colony inoculation 1000ml of picking MH(Mueller-Hinton, Beijing extensive and profound in meaning star biotechnology limited liability company, LOT:02-052) liquid nutrient medium, 37 ℃ of aerobic 210rpm shaking culture 7h, extracting MRSA genome.

2, with the genome that step 1 is extracted, be template, amplification MntC goal gene fragment, amplification step is as follows:

1) design PCR primer is as follows, is respectively (underscore shows restriction enzyme site base sequence)

Forward primer: PMNTCBAMHI1(SEQ ID NO.3:

5'-CTG GGATCCAGCAGTGATAAGTCAAATGCAAAC-3'；

BamHI

Reverse primer: PMNTCNOTI2(SEQ ID NO.4):

5'-AT GCGGCCGCCTTATTATTTCATGCTTCCGTG-3'；

NotⅠ

2) PCR system:

3) 98 ℃ of sex change 10s of pcr amplification reaction condition, 62 ℃ of annealing 30s, 72 ℃ are extended 1min30s, 30 circulations, 72 ℃ are extended 7min completely.Use after completion of the reaction 1.5% sepharose detection pcr amplification result, pcr amplification the results are shown in Fig. 1.

4) use gel to reclaim test kit and reclaim MntC PCR product.

3, the evaluation of PCR product and clone, step is as follows:

1) BamH I and Not I enzyme are cut pGEX-6P-2 plasmid and MntC PCR product, endonuclease reaction system:

37 ℃ of enzymes are cut 2h.

2) reclaim pGEX-6P-2 plasmid and the PCR product of cutting through BamH I and Not I enzyme.

3) connect and transform:

Ligation system:

By ultraviolet spectrophotometer, measuring MntC enzyme, to cut back to close product nucleic acid concentration be 43ng/ μ l, and it is 55ng/ μ l that pGEX-6P-2 enzyme cuts back to close product nucleic acid concentration, general than being 1:2～10 according to carrier and external source fragment mole number, designs following ligation system:

Mix, 16 ℃ connect 2h.

4) get 3 pipe intestinal bacteria XL1-Blue 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 30min, 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 200rpm 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 the LB flat board that final concentration is 100 μ g/ml Amp.Flat-plate inverted is placed in 37 ℃ of incubators and cultivates 24h.

5) screening, the evaluation of the positive recombinant plasmid of pGEX-6p-2-MntC (wt):

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 connects product and transforms the dull and stereotyped upper good bacterium colony of separating, and being inoculated in final concentration is in the LB substratum of 100 μ g/ml Amp, 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:

37 ℃ of enzymes are cut 2h;

4. 1.5% agarose gel electrophoresis detects double digestion result, and as shown in Figure 2, visible swimming lane 3,4 samples are pGEX-6p-2-MntC (wt) recombinant plasmid successfully constructing to result;

5. pGEX-6p-2-MntC (wt) recombinant plasmid is sent to the handsome company in Shanghai sequence verification, retains the recombinant plasmid of correct sequence.

Embodiment 3:MntC abduction delivering in prokaryotic expression system-intestinal bacteria

1. target protein abduction delivering

1) get and identify that correct pGEX-6P-2-MntC (wt)/XL1-blue bacterium liquid and synthetic each the 100 μ L of pGEX-6P-2-MntC (r)/XL1-blue bacterium liquid of full gene are added to 10mL containing in the LB substratum of 100 μ g/ml Amp, 80rpm37 ℃ of incubated overnight, the bacterium liquid 2mL that gets respectively incubated overnight is added to 100mL containing in the LB substratum of 100 μ g/ml Amp; While being 0.8-1.0 to OD600, adding IPTG to make its final concentration is 200 μ M, then is placed in 30 ℃ of 3h of shaking table abduction delivering, 16 ℃ of abduction deliverings that spend the night.

2) take out the bacterium liquid after abduction delivering, with the centrifugal 5min of 10000rpm, supernatant discarded, add 5mL lysis buffer (PBS) to mix, ultrasonic (300 watts of power) cracking 10min (work 6 seconds, have a rest 9 seconds), 4 ℃ of centrifugal 15min of 14000rpm again, cleer and peaceful precipitation in separation.

2. process supernatant:

Supernatant combination: get glutathione agarose gel 4B80 μ l, with after PBS washing 3 times, add ready supernatant, 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 washing three times.Get precipitation 16 μ l, add 4 μ l5 * protein sample-loading buffers, boil 5min, the centrifugal 3min of 14000rpm.

With PP enzyme (Prescission proteolytic enzyme, U.S. GE company), carry out enzyme and cut wash-out: the use of PP enzyme is undertaken by the method for manufacturer's recommended.Because used PP enzyme is with GST label, be beneficial to remove PP enzyme.After enzyme cuts into, cleer and peaceful precipitation in centrifugal collection.Respectively get cleer and peaceful precipitation 16 μ l, add 4 μ l5 * protein sample-loading buffers, boil 5min, the centrifugal 3min of 14000rpm.Carry out 10%SDS-PAGE, result as shown in Figure 3.

As seen from the figure, after password is optimized, engineering bacterium expression amount is significantly increased before not optimizing.Below test the engineering bacteria accessing to your password after optimizing is carried out.

Embodiment 4:MntC engineering bacterium fermentation

1, determining of fermentation condition:

1) impact that substratum is expressed engineering bacteria growth and target protein:

By testing the impact of four kinds of substratum on MntC protein expression as aforementioned cultural method in shaking flask:

Plant-derived improvement TB(potassium primary phosphate 2.3g, disodium hydrogen phosphate 13g, glycerine 5ml, yeast extract 24g, soy peptone 12g, magnesium sulfate 1g, add water to 1L);

Animal derived improvement TB(potassium primary phosphate 2.3g, disodium hydrogen phosphate 13g, glycerine 5ml, yeast extract 24g, animal source Tryptones 12g, magnesium sulfate 1g, add water to 1L);

Plant-derived M9-CAA(Sodium phosphate dibasic 15.6g, potassium primary phosphate 4.3g, ammonium chloride 1g, magnesium sulfate 1g, sodium-chlor 0.67g, glucose 5g, soy peptone 3.6g, plant-sourced yeast powder 4g, acid hydrolyzed casein 6g, add water to 1L);

Animal derived M9-CAA(Sodium phosphate dibasic 15.6g, potassium primary phosphate 4.3g, ammonium chloride 1g, magnesium sulfate 1g, sodium-chlor 0.67g, glucose 5g, animal Tryptones 3.6g, animal source yeast powder 4g, acid hydrolyzed casein 6g, add water to 1L).

Get pGEX-6P-2-MntC (r)/XL-1blue and be inoculated in Amp ⁺in LB flat board, hatch 16h-20h for 37 ℃, picking list bacterium colony is in 10mlAmp ⁺in LB substratum, be placed in 37 ℃ of shaking tables, 200rpm shakes OD ₆₀₀during about 2 left and right, by 1:100, be inoculated in respectively in tetra-kinds of substratum of 100ml, 37 ℃, 200rpm shake 14 hours, and OD is surveyed in sampling in every 2 hours ₆₀₀, it the results are shown in Figure 4.

As seen from the figure: plant-derived substratum is all worse than animal derived, when engineering bacteria is all about 8h, just growth slows down, and it is fine with regard to growing way in animal derived substratum, always in logarithmic phase, and TB is better than M9, therefore select animal source substratum (being called for short TB, M9 after animal derived improvement TB and M9-CAA).

By fresh MntC engineering bacteria bacterium liquid (OD ₆₀₀about 2) by 1:100, be inoculated in respectively in 100mlTB and M9 substratum, 37 ℃, 200rpm shake to OD ₆₀₀0.8 left and right, adds the IPTG of 1mM, 25 ℃ of induction 12h.Get 100ml bacterium liquid centrifugal, abandon supernatant, the TB:2.4g that weighs, M9:1.5g.With 1g:10ml, add the broken bacterium of PBS ultrasonic (300 watts of power) cracking 10min (work 6 seconds, have a rest 9 seconds), in conjunction with GST4B(process, refer to embodiment 5), carry out 10%SDS-PAGE, result is as shown in Figure 5.

As shown in Figure 5: the target protein expression amount TB of unit is better than M9, and the bacterium weight in wet base TB of unit is greater than M9, therefore MntC fermentation is selected TB substratum with basic medium.

2) impact that IPTG concentration is expressed target protein:

The impact of the IPTG that investigates different final concentrations on target protein expression level.The final concentration of IPTG is respectively 0.1mM, 0.2mM, 0.5mM, 1mM(substratum), it is compared and selects optimum concn.By fresh MntC engineering bacteria bacterium liquid (OD ₆₀₀about 2) by 1:100, be inoculated in respectively in four 100ml TB, 37 ℃, 200rpm shake to OD ₆₀₀0.8 left and right, adds respectively IPTG to make the respectively corresponding above-mentioned four kinds of concentration (one bottle of concentration) of its concentration, 25 ℃ of induction 12h.Four bottles of bacterium liquid are centrifugal respectively, abandon supernatant, with 1g:10ml, add PBS, ultrasonic (300 watts of power) cracking 10min (work 6 seconds, have a rest 9 seconds) breaks bacterium, after GST4B, carries out 10%SDS-PAGE, and result is as shown in Figure 6.

As shown in Figure 6: protein expression when IPTG final concentration is 0.2mM when protein expression and 0.5mM, 1mM is basic identical, the protein expression during significantly better than 0.1mM, so select 0.2mM for fermentation IPTG final concentration.

3) impact of the different vaccination amount of kind daughter bacteria on the growth of engineering bacteria and target protein expression:

Research 5%, 10%, 15% 3 kind of different daughter bacteria inoculum size of planting are on the impact of fermenting.By growth curve of bacteria, unit expressing quantity, determine optimum inoculation amount.As kind of a daughter bacteria OD ₆₀₀when 2 left and right, to pour in fermentor tank and start timing, OD is surveyed in sampling in every 1 hour ₆₀₀, until finish before induction, draw engineering bacteria early growth phase curve (Fig. 7), can judge bacteria growth speed.Induction finishes after rear sampling, according to method processing before, carries out 10%SDS-PAGE, judgement unit target protein expression amount (Fig. 8).As shown in Figure 7, the slowest and the highest OD of 5% inoculum size bacterial growth ₆₀₀more another two kinds of inoculum sizes are much lower, and 10% and 15% inoculum size is more or less the same.As shown in Figure 8, different vaccination amount unit target protein expression amount.Expressing best is 5% inoculum size, is secondly 10%, but is more or less the same, and 15% is the poorest.

In sum: 5% inoculum size is expressed best, but the speed of growth is slow, final bacterium yield is few; 15% inoculum size fast growth, but expression is the poorest; The expression amount of 10% inoculum size is more or less the same than 5%, and the speed of growth is also more or less the same than 15%, so select 10% inoculum size as fermentation inoculum size.

4) impact of oxygen concn on engineering bacteria growth and target protein expression:

This engineering bacteria is facultative aerobe, the height of oxygen concn is very large on bacterial growth impact, so during the fermentation to the control of the dissolved oxygen particularly important that just seems, existing growing state (Fig. 9) and the target protein expression amount (Figure 10) of investigating respectively dissolved oxygen bacterium 25%, 45%, 65% time.

Known from bacterial growth situation: 45% dissolved oxygen condition, bacterium looks best; From unit expressing quantity, under 45% dissolved oxygen condition, it is also best expressing, so oxyty selects 45%.

5) determining of glycerine consumption:

When different bacterium amount, induce, can affect the expression amount of MntC, and in when fermentation substratum the amount of glycerine can directly affect bacterium amount number.In tank, glycerine consumption is over, and bacterium stops growing, and pH value and oxygen dissolving value rise rapidly at short notice, now should add IPTG at once and start induction.So how many direct decision induction starting times of glycerine.The impact of target protein being expressed by the amounts of glycerol of research 5ml/L, 10ml/L, 15ml/L and final weight in wet base yield are determined best induction starting time.Result as shown in figure 11.

From scheming: during 5ml/L glycerol concentration, expression amount is the highest; Secondly 10ml/L, but is more or less the same; 15ml/L is just much poor.But during 5ml/L glycerol concentration, last thalline weight in wet base is much lower; 10ml/L and 15ml/L are more or less the same, so finally determine that amounts of glycerol adds 10ml/L.

6) different inducing temperatures and the time impact on target protein expression:

Investigate the impact of 30 ℃, 25 ℃, 16 ℃ three different inducing temperatures on protein expression, and reach the time of using when maximum is expressed.Every 2h sampling after induction, induction 10h, processing sample, carries out 10%SDS-PAGE, and its result is as shown in figure 12.

From electrophorogram: 30℃ unit's abduction delivering amount is the highest, and reach that maximum to express the time used be the shortest, the differential expression between 4h-6h is little.And 25 ℃, 16℃ unit's expression amount are just not as 30 ℃, and expression time is long.Therefore inducing temperature and time are selected 30 ℃, 5h.

According to above-mentioned research, finally determined that the preferred zymotechnique of MntC engineering bacteria of the present invention is:

(1) basic medium is selected animal derived TB substratum, and wherein amounts of glycerol is 10ml/L;

(2), when fermentation starts, the inoculum size ratio of planting daughter bacteria is 10%;

(3) oxyty remains at 45% left and right during the fermentation;

(4) when induction, it is 0.2mM, induction 5h that temperature is adjusted to 30 ℃, IPTG concentration.

2, engineering bacterium fermentation technique is amplified

According to above-mentioned optimal conditions, fermentation scale is expanded to (25L), and obtain MntC engineering bacteria growth curve (Figure 13) and unit protein expression electrophorogram (Figure 14).

As shown in Figure 13, after technique is amplified, whole growth curve standard of comparison, early stage, bacterial growth was very fast, and when the later stage induces, curve is more steady, finally obtains nectar degree (OD ₆₀₀) reach 38, the bacterium weight in wet base 47g/L of unit.

As shown in Figure 14, from unit target protein, express: expression amount prolongation is in time significantly increased, and 5h expression amount is the highest.

In sum: MntC zymotechnique amplifies successfully, meet expected results completely.Zymotechnique of the present invention is applicable to industrial production application on a large scale.

The purifying process of embodiment 5:MntC recombinant protein

1, broken bacterium is prepared supernatant:

The thalline 200-500g that embodiment 1 is built is with 20mmol/L PB, pH7.0 damping fluid, by weight: volume ratio 1:10 ratio mixes suspension, 4 ℃ of precoolings.

High-pressure homogenization: use distilled water flushing high pressure homogenizer (high pressure homogenizer APV-1000, Denmark An Invernsys Group) pipeline, cold cycle system open be chilled in advance 1-4 ℃ standby.The suspension bacteria liquid of precooling is added to high pressure homogenizer, and pressure maintains the broken bacterium of 60-80Mpa 3-5 time, gets PBS smear and carries out violet staining, and under oily mirror, under each visual field, not broken bacterium is less than 2 and is considered as brokenly bacterium (broken bacterium rate is greater than 90%) completely.

High speed centrifugation: the liquid after broken bacterium packs centrifugal barrel (Beckman, the U.S.) into, 4 ℃, 10,000-15, the centrifugal 15-30min of 000g, collects supernatant standby.

2, glutathione agarose gel 4B affinitive layer purification

In the broken bacterium liquid of every liter of MntC engineering bacteria, add 200ml GST filler, 20～25 ℃ in conjunction with more than 4h, and cohesive process adopts the method for vertical rotary or stirring to promote the combination of MntC albumen and GST filler.Adopt 5 volumes of PBS washing to remove the foreign protein of not being combined with GST filler the GST filler of above-mentioned combination MntC albumen.Then to filler, by every 100ml GST filler, add the PP enzyme of 20ml, suction filtration collect filtrate after enzyme is cut 6 hours under 20～25 ℃ of conditions, obtains the MntC albumen after enzyme excision GST label, and carries out SDS-PAGE analysis.Result as shown in figure 15.

3, cation-exchange chromatography

1) selection of chromatographic stuffing

Relatively adopt MMC and the purification effect of Phenyl HP to MntC albumen, the thick purification of samples of MntC that the sample of use is above-mentioned acquisition.

Instrument system: AKTA-expolrer100/Avant25 liquid chromatographic system (GE company);

Chromatographic stuffing: MMC, Phenyl HP(are GE company);

Post specification: (Φ) 10cm of 20cm, (Φ) 1.6cm of 2.6cm * (H) * (H);

Dress column volume: 54ml, 5ml;

MMC damping fluid: buffer A: 10mM His+0.01% PLURONICS F87, pH6.0; Buffer B: 10mMHis+0.01% PLURONICS F87+1M NaCl, pH6.0;

Phenyl HP damping fluid: buffer A: 10mM His+0.01% PLURONICS F87+1.5M (NH ₄) ₂sO ₄, pH6.0; Buffer B: 10mM His+0.01% PLURONICS F87, pH6.0;

Loading sample: after getting GST affinity chromatography, sample is adjusted to respectively standby with MMC and the consistent pH of Phenyl HP buffer A.

(1) MMC: loading flow velocity: 12ml/min, elution flow rate: 12ml/min;

Elution program: 0-100%B, 7 column volumes (CV), result is as shown in FIG. 16 and 17.

(2) Phenyl HP: loading flow velocity: 8ml/min, elution flow rate: 8ml/min;

Elution program: 0-100%B, 10 column volumes (CV).Result as shown in Figures 18 and 19.

In each elution program, the damping fluid of all the other shares is corresponding buffer A.

Collect: each elution samples of target protein is carried out to SDS-PAGE purity check, evaluate purification effect.

From Figure 16-19, can find out, under different chromatography conditions, MMC is stronger to the binding ability of MntC than Phenyl HP, penetrate in peak almost without MntC target protein, elution peak MntC purity can reach 95% left and right, and target protein yield is high, has good chromatography purification effect.

Therefore, determine that choice for use MMC is as the filler of the first step chromatography purification.

2) optimization of chromatography purification technique

Establishing on the basis of use MMC as the first step filler, this chromatographic stuffing is carried out to condition optimizing, the main condition of optimizing is that loading sample electricity is led, the purity that evaluation index is MntC and yield.

Instrument system: AKTA-explorer100 liquid chromatographic system (GE Healthcare);

Chromatographic stuffing: MMC;

Post specification: (Φ) 20cm of 2.6cm * (H);

Dress column volume: 54ml;

Damping fluid:

1. buffer A: 10mM His+0.01% PLURONICS F87+0.15M NaCl, pH6.0;

Buffer B: 10mM His+0.01% PLURONICS F87+1M NaCl, pH6.0;

2. buffer A: 10mM His+0.01% PLURONICS F87, pH6.0;

Buffer B: 10mM His+0.01% PLURONICS F87+1M NaCl, pH6.0;

Loading sample: get respectively the thick purification of samples of MntC and be adjusted to consistent with two kinds of buffer A.

Flow velocity: 20ml/min

Elution program: 0-100%B, 10 column volumes (CV).

Each elution samples of target protein is collected, and carry out SDS-PAGE purity check, evaluate purification effect.

From Figure 20-23, can find out, under low loading sample electricity sliver part, target protein is worn without stream, and the target protein purity of purifying is high, therefore, determines that choice for use loading sample condition should be lower than 10ms/cm.

4, desalination

Adopt vaccine diluent balance desalination G25 post, the sample that upper step purifying is obtained is replaced damping fluid by desalting column.

These process concrete steps are: the sample that upper step is obtained, by vaccine diluent, dissolve, balance layer analysis system (AKTA Explorer100, U.S. GE Healthcare) and chromatography column XK50-60(U.S. GE Healthcare) (600mL Sephadex G 25), with flow velocity 20mL/min desalination.

5, the de-intracellular toxin of second step purifying

Chromatographic stuffing: Q HP;

Post specification: (Φ) 20cm of 2.6cm * (H);

Dress column volume: 50ml;

Damping fluid: buffer A: vaccine diluent, without intracellular toxin; Buffer B: 1M NaOH;

Loading sample: sample after G25 desalination;

With 5 column volumes of buffer B (1mol/L NaOH) cleaning and sterilizing in place, place after half an hour, using vaccine diluent equilibrium system is 6.0 to pH, loading then.Flow velocity: 8ml/min.It is target protein peak that collection penetrates peak.

6, technique is amplified

According to above definite loading and elution mode, carry out technique amplification: the MMC chromatography column scale adopting is enlarged into the 20cm of (Φ) 2.6cm * (H), dress column volume CV=70ml, Q HP chromatography column scale is enlarged into the 20cm of (Φ) 2.6cm * (H), dress column volume CV=50ml.Repeat three batches of experiments, 10%SDS-PAGE analyzes effect and the yield after MntC purifying, evaluates stability and repeatability after this technique is amplified.

From Figure 24-32, can find out, after the MMC chromatography technique of MntC albumen is amplified, chromatography purification pattern and test chromatography purification color atlas without considerable change in a small amount, the MntC purity after purified reaches more than 97%.

7, HPLC purity detecting

HPLC instrument Agilent1260(U.S. Agilent company), analytical column ZorBax SB-300-C34.6x150mm3.5micron(U.S. Agilent company).

Moving phase: A:0.1% trifluoroacetic acid (Tedia, the U.S.), water (18.2M Ω); B:0.1% trifluoroacetic acid (Tedia, the U.S.), acetonitrile (Tedia, the U.S.).

60 ℃ of column temperatures, flow velocity 0.5mL/min, loading 10 μ l.

Detection method: 0-30min:90%A, 10%B; 30-35min:100%B; 35-40min:90%A, 10%B; 40-45min:90%A, 10%B.

Result is as shown in Figure 33 and table 1, and MntC main peak retention time is 13.409 minutes as seen from the figure, main peak area ratio 99.5%.

The HPLC detected result of table 1:MntC sample

Peak #	Retention time (min)	Type	Peak width (min)	Peak area (mAU*s)	Peak area %
						1	11.158	?	0.0000	0.00000	0.0000
2	13.409	BV	0.1356	3606.82300	99.5314
						3	13.918	VB	0.1400	16.98190	0.4686
4	32.252	?	0.0000	0.00000	0.0000

8, the MntC recombinant protein that sequence verification obtains.

Entrust Research Centre for Proteome Analysis(Shanghai) to check order, carry out molecular weight determination and amino acid composition analysis the N end of the MntC recombinant protein obtaining, C end, the MntC recombinant protein of result and design is in full accord.

Embodiment 6:MntC albumen endotoxin assay

1, the apirogen water for sample (Zhanjiang Bo Kang marine organisms company limited) embodiment 5 being obtained is diluted to 50 μ g/mL as testing sample.The highest scope 0.25EU/mL that can detect according to intracellular toxin detection kit (Zhanjiang Bo Kang marine organisms company limited), dilutes testing sample.Suppose that testing sample endotoxin content is 5EU/mL, by apirogen water, further dilute testing sample to 2.5 μ g/mL (diluting 20 times);

2, according to test kit (Zhanjiang Bo Kang marine organisms company limited) specification sheets preparation endotoxin standard positive control solution, testing sample working fluid, testing sample, detect solution;

3, the preparation of tachypleus amebocyte lysate: according to the quantity of testing sample and reference substance, get tachypleus amebocyte lysate, cotton ball soaked in alcohol sterilization bottleneck, dries rear unlatching, every adds inspection water 0.1ml, shakes up gently standby;

4, application of sample: to adding respectively in the tachypleus amebocyte lysate preparing testing sample to detect solution, endotoxin standard positive control solution, check each 0.1ml of water, jog mixes, sealed membrane sealing, 37 ℃ of water-baths 60 ± 2 minutes, during forbid mobile; Check the negative contrast of water;

5, detect: take out sample, vertical rotary is 180 ° gently, observe the bottle end, liquid solidifies not mobile positive, flows and is not solidified as feminine gender;

6, measurement result: feminine gender, is less than 5EU/ml.

The preparation of embodiment 7:MntC vaccine

Aluminum phosphate is U.S. GENERAL CHEMICAL company imported with original packaging product (20mg/ml)

1, preparation streptococcus aureus restructuring MntC vaccine

1) measure aluminum phosphate adjuvant 80 μ L, joined in preparation bottle; Measure vaccine diluent 220 μ L, cumulative volume 300 μ L, fully mix;

2) by vaccine diluent, MntC recombinant protein is diluted to 30 μ g/300 μ L, fully mixes;

3) protein solution after assist agent solution after isopyknic dilution and dilution is added in sub-bottling, under 4 ℃ of-32 ℃ of conditions of temperature range, vertical suspendible or horizontal whip attachment are after 1 hour and get final product.

2, antigen protein aluminum phosphate adjuvant absorption homogeneity and thoroughness in 10%SDS-PAGE identification of M ntC vaccine

1) from MntC vaccine sampling 1ml, 4 ℃, centrifugal 5 minutes of 6000rpm, carefully draws supernatant, and samples 40 μ l from supernatant;

2) fill into and the isopyknic dissociation solution of supernatant (1M Na ₂cO ₃), under room temperature condition, vertical suspendible 1 hour, sampling 40 μ l;

3) by above-mentioned 1 method, prepare the not protein solution of phosphoric acid aluminium adjuvant, the shared volume of aluminum phosphate is supplied with vaccine diluent, fully mixes rear sampling 40 μ l;

4) institute's sample thief is added to 10 μ l5 * albumen sample-loading buffers, 100 ℃ heating 5 minutes, cooling and instantaneous centrifugal after, get 10 μ l loadings;

5) 10%SDS-PAGE electrophoresis, the first 80v electrophoresis of voltage 20 minutes, be adjusted to again 180v, electrophoresis 40 minutes, then takes out glue, is placed in coomassie brilliant blue staining liquid vibration dyeing, be placed in again after destainer vibration decolouring, observations under imaging system, the results are shown in Figure 34, and visible aluminum phosphate adjuvant can fully adsorb recombinant protein.

The detection of embodiment 8:MntC vaccine immunity animal and antibody

1, immune animal

1) laboratory animal: 6 BALB/c mouse in 6 week age (Beijing China Fukang), body weight is about 16g

2) immune programme for children: divide three times in quadriceps muscle of thigh intramuscular injection (0,14,21 day) immunity with 30 μ g/600 μ l according to the vaccine preparation of embodiment 7 preparations.

2, immunity is for the third time latter the 7th day, gathers the serum of BALB/C mice, with IgG after ELISA detection mouse immune, replys level.

3、ELISA

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 by pH;

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

3. the preparation of antibody diluent: take NaCl8g on electronic balance, KH ₂pO ₄0.2g, Na ₂hPO ₄12H ₂o2.9g, KCl0.2g, polysorbas20 0.5mL, regulates pH to 7.4, and adding distil water is settled to 1000mL;

4. the preparation of washings: take 2.42g Tris and be dissolved in 1L ddH ₂o, then add 500 μ L polysorbas20s, then 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 MntC recombinant protein immune mouse produces:

1. coated: MntC recombinant protein is diluted to 1 μ g/mL with coating buffer, coated 96 orifice plates (Corning, the U.S.), 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; PBS control wells is set;

2. sealing: add confining liquid to enzyme plate, 100 μ L/ holes, are placed in 37 ℃ of incubators 2 hours, wash 3 times;

3. serum is carried out to doubling dilution according to 1:100,1:500,1:1000,1:2000,1:4000,1:8000;

4. get the enzyme plate having sealed, add successively the serum of dilution, 100 μ L/ holes, are placed in 37 ℃ of incubator 30min, wash 3 times, empty dry;

5. the goat anti-mouse igg antibody that adds HRP mark is preserved to liquid, dilution 1:5000, makes antibody working fluid;

6. the antibody working fluid that adds dilution, 100 μ L/ holes, are placed in 37 ℃ of incubator 1h, wash three times, empty dry;

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

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

9. result judgement: A _sample/ A _{negative value}>=2.1 positive (negative control is that before mouse immune, serum 1:1000 doubly dilutes).

Result: the antibody titer that detects the generation of MntC recombinant protein antigen immune mouse reaches 1:512000, illustrates that the MntC subunit recombinant protein that the present invention builds can make generation antibody in immune mouse body.

Embodiment 9: the poison of attacking of determining immune animal by MntC vaccine immune mouse is protected effect

With the immunization protocol of embodiment 8, 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.3 take turns animal protection test (10/wheel) the results are shown in table 2.

Table 2:MntC recombinant protein immune mouse is to the malicious protection of attacking of animal

Group	Mouse (only)	Immune component	The number of surviving after 10 days	3 take turns average protection ratio (%)
					Experimental group	30	MntC+AlPO ₄Adjuvant	12	40
Negative control group	30	Vaccine diluent	2	6.7

Table 2 shows: 3 of negative control group is taken turns average immune protective rate and is respectively 6.7%, MntC and adds AlPO ₄it is 40% that 3 of adjuvant group is taken turns average immune protective rate.Therefore; confirm that MntC vaccine of the present invention has good immunogenicity; can induce body to produce protective immune response, and can infect and play immanoprotection action SA, can be aided with aluminium adjuvant and prepare recombinant 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, preparation relevant recombinant subunit vaccine, therapeutic antibodies and detection kit such as coated reagent, detection antibody, developer, terminator, adjuvant, for the application of immune protection and diagnosis infection of staphylococcus aureus.

Claims

1. a MntC recombinant protein for streptococcus aureus, the aminoacid sequence of described albumen is as shown in SEQ ID NO.1.

Coding MntC recombinant protein as claimed in claim 1 nucleotide sequence, preferably, described nucleotide sequence is as shown in SEQ ID NO.2.

3. carrier or a host who comprises nucleotide sequence as claimed in claim 2; Preferably, described carrier is pGEX or pET series expression vector, and further preferably, described carrier is expression vector pGEX-6P-2; Preferably, described host is intestinal bacteria XL1-Blue.

4. a method of preparing Mnt C recombinant protein as claimed in claim 1, described method is chemical synthesis or Nucleotide representation; Preferably, described method comprises step: 1) according to the gene fragment of the encoding sequence of wild MntC albumen (SEQ ID NO.5) design forward, reverse primer amplification coding MntC albumen; Preferably, described forward primer is as shown in SEQ ID NO.3, and described reverse primer is as shown in SEQ ID NO.4; 2) codon of MntC albumen is optimized, be specially, the 9th bit codon of MntC albumen coded sequence (SEQ ID NO.5) (coding Leu) is optimized for to CTG, 13rd, 14 bit codons (coding Thr) are optimized for ACC, 27th, 65,282 bit codons (coding Gly) are optimized for GGT, and the 214th codon (coding Arg) is optimized for CGT; 3) by step 2) gene fragment clone that obtains to expression vector, transform Host Strains and make its expression.

5. fermentation host as claimed in claim 3, to express a method for the MntC recombinant protein of claim 1, comprises a certain amount of kind of daughter bacteria is inoculated in the fermention medium that contains glycerine and certain dissolved oxygen amount, through inductor induction certain hour, ferments; Preferably, described fermention medium is selected from animal derived TB, animal derived M9, plant-derived TB or plant-derived M9; Preferably, the inoculum size of described kind of daughter bacteria is 5%～15%, more preferably 10%; Preferably, in described fermention medium, amounts of glycerol is 5-15ml/L substratum, more preferably 10ml/L substratum; Preferably, in described fermention medium, dissolved oxygen amount is 25%～65%, more preferably 45%; Preferably, described fermentation inducement agent is lactose or IPTG; Preferably, the concentration of described inductor is 0.1～1mM, more preferably 0.2mM; Preferably, described inducing temperature is 16～37 ℃, enters one and is preferably 30 ℃; Preferably, induction time is 1～6 hour, more preferably 5 hours.

6. a method for purifying MntC recombinant protein as claimed in claim 1 after fermentation host as claimed in claim 3, comprises and carries out successively affinity chromatography, cation-exchange chromatography, desalination, de-intracellular toxin; Preferably, before carrying out affinity chromatography, need to be to the broken bacterium of host, so that albumen is discharged from thalline; Preferably, described affinity chromatography is that GST affinity chromatography is cut in conjunction with Prescission proteolytic enzyme enzyme, and further preferably, described GST affinity chromatography is GST sepharose 4B chromatography; Preferably, described cation-exchange chromatography is MMC chromatography, and further preferably, the damping fluid that described MMC chromatography is used comprises: buffer A: 10-20mM His, 0.01% PLURONICS F87, pH6.0; Buffer B: 10-20mM His, 0.01% PLURONICS F87,1M NaCl, pH6.0; Preferably, the elution program of described elution buffer is with 0-100% elution buffer B, and 5-10 column volume carries out gradient elution; Preferably, G25 post is used in described desalination; Preferably, described de-intracellular toxin is used Q HP post.

7. the application in the preparation that MntC recombinant protein according to claim 1 detects, prevents in preparation or treatment SA infects; Preferably, described preparation is vaccine.

8. the test kit or the composition that comprise albumen as claimed in claim 1 or nucleotide sequence claimed in claim 2 or carrier claimed in claim 3 or host.

9. the antibody being produced by MntC recombinant protein immunity claimed in claim 1.

10. the application of antibody according to claim 9 in the preparation for the preparation of detecting, prevent or treat SA to infect.