CN103694323B

CN103694323B - MntC recombinant protein of staphylococcus aureus and preparation method and application thereof

Info

Publication number: CN103694323B
Application number: CN201310665113.9A
Authority: CN
Inventors: 曾浩; 邹全明; 樊绍文; 卢陆; 杨茜; 敬海明
Original assignee: Third Military Medical University TMMU; Chengdu Olymvax Biopharmaceuticals Inc
Current assignee: Third Military Medical University TMMU; Chengdu Olymvax Biopharmaceuticals Inc
Priority date: 2013-12-09
Filing date: 2013-12-09
Publication date: 2017-01-18
Anticipated expiration: 2033-12-09
Also published as: CN103694323A

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

Staphylococcus aureus mntc recombinant protein and its preparation method and application

Technical field

The invention belongs to biological technical field, it is related to a kind of staphylococcus aureus (sa) mntc recombinant protein.

Background technology

Staphylococcus aureus (staphylococcus aureus, sa), hereinafter referred to as S. aureus L-forms, there is " thermophilic meat bacterium " Another name.As the representative of gram-positive bacteria, it is a kind of important pathogenic bacteria causing hospital infection and Nosocomial Infections.Infection with Acute, suppurative be characterized, local infection can cause the pyogenic infection of skin and soft tissue etc., does not recover for a long time；General infection May result in the severe infections such as osteomyelitis, septic arthritis, endocarditis, pneumonia, septicopyemia and complication, the death rate is up to 20%.Meanwhile, the exotoxin of S. aureus L-forms also can cause food poisoning, scalded skin syndrome and TSS etc. Whole body lethal infection.Therefore, strengthen the immune protection research to S. aureus L-forms infection, develop safe and effective new S. aureus L-forms Recombinant vaccine by effective control S. aureus L-forms drug resistance is spread and clinical S. aureus L-forms extensively infection have important strategy and Realistic meaning.

The living necessities many kinds of metal ions of sa, they participate in various physiological processes, such as metabolism, dna synthesis and poison Supervision of the power factor etc..The picked-up of these ions is relevant with specific albumen, using these specific protein immune animals thus producing Raw specific antibody be can yet be regarded as a kind of selection of effective vaccine.Iron ion determine albumen isdb just by merck company as candidate's epidemic disease Seedling carried out clinical testing.Manganese ion is also that sa growth institute is required, and it can protect sa to kill from host's neutrophil cell, Important function has been played in the immune evasion of sa.Sa passed through to raise manganese ion transport protein c(mntc in infection early stage) and obtain Obtaining manganese ion is to escape the key mechanism being killed.However, the picked-up of this element excessively can lead to toxicity, therefore, the picked-up of manganese is Highly adjusted.Manganese ion metabolism GAP-associated protein GAP is expressed than isdb earlier when sa infects, and these albumen comprise an atp and close Become albumen mnta, an a complete cell membrane transporter albumen mntb and manganese ion transport protein mntc.Wherein mntc is one Plant highly conserved cell surface protein.Recently, mntc is defined in the expression of sa surface of cell membrane, then in multiple In vivo infections Occur in model.Thus using mntc as antigen-immunized animal, induce body to produce the antibody being directed to this albumen, should be able to be in early stage Block it and adjust the function of manganese metabolism thus playing the effect resisting sa infection.

Content of the invention

The purpose of the present invention aims to provide a kind of staphylococcus aureus manganese ion transport protein mntc of restructuring, is used Make antigen, prepare the biological products for detecting, preventing or treat sa infection.

In the first aspect of the invention, provide a kind of mntc recombinant protein of staphylococcus aureus, described mntc weight The amino acid sequence of histone is as shown in seq id no.1.

In the second aspect of the invention, provide the nucleotide sequence of the mntc recombinant protein of the coding present invention, and bag Carrier containing described nucleotide sequence and host.

In the third aspect of the invention, provide a kind of method of the mntc recombinant protein preparing the present invention.

In the fourth aspect of the invention, provide a kind of fermentation mntc host and the method preparing mntc recombinant protein.

In the fifth aspect of the invention, provide a kind of method of the mntc recombinant protein purifying the present invention.

In the sixth aspect of the invention, provide the application as antigen for the mntc recombinant protein of the present invention, and described Mntc recombinant protein is in preparation for detecting, preventing or treating the application in the biological products that sa infects.

In the seventh aspect of the invention, provide the polyclonal antibody being produced by the mntc recombinant protein immunity of the present invention with And described polyclonal antibody in preparation for detecting, preventing or treat the application in the biological products that sa infects.

In the eighth aspect of the invention, provide the composition of mntc recombinant protein comprising the present invention or kit.

The present invention adopts the recombinant expressed described fusion protein of technique for gene engineering, and not only expression is high, be easy to isolate and purify, And immunogenicity is strong, safety non-toxic.Prove through animal experiment, the mntc recombinant protein of the present invention can produce effective stimulus body The higher HI of life and good immanoprotection action, can be used in preparing detection, prevention or treatment sa infection Biological products.

Brief description

The pcr amplification of Fig. 1, mntc genetic fragment: m:dna molecular weight standard；1st, 2: genes of interest fragment mntc (855bp).

Fig. 2, the digestion qualification result of expression vector pgex-6p-2-mntc: m: nucleic acid (dna) molecular weight standard；1、2、3、 4: recombinant expression plasmid pgex-6p-2-mntc1,2,3,4, wherein No. 3, No. 4 is to recombinate successful plasmid, detached after digestion Fragment 4800bp and 855bp.

Fig. 3, mntc affinity chromatography result: the fusion protein digestion result containing gst label: m: Protein Marker；1: Before digestion, the fusion protein containing gst label；2: after digestion, in the destination protein (password optimization engineering bacteria) of supernatant acquisition；3: After digestion, non-specific binding is (close with the enzyme specifically binding in gel beads and gst label in the destination protein in gel beads Code optimization engineering bacteria)；4: after digestion, in the destination protein (password is not optimised engineering bacteria) of supernatant acquisition；5: after digestion, non-specific Property combine that (password is not optimised engineering in the destination protein in gel beads and enzyme in gel beads for the specific binding and gst label Bacterium).

Growth curve in four kinds of culture mediums for Fig. 4, mntc engineering bacteria.

Fig. 5, mntc expression in m9 and tb culture medium；1: protein molecular weight standard；2:m9；3:tb.

Fig. 6, the different impact to mntc protein expression for the iptg concentration；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 kind impact to protein expression for the daughter bacteria different vaccination amount；1: protein molecular weight standard；2:5%；3:10%；4: 15%.

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

Figure 10, po₂During variable concentrations, the expression of mntc albumen；1: protein molecular weight standard；2:25%；3:45%； 4:65%.

Figure 11, the impact to mntc protein expression for the glycerine of variable concentrations；1: protein molecular weight standard；2:5ml/l；3: 10ml/l；4:15ml/l.

Figure 12, the situation that destination protein is expressed in different inducing temperatures and time；1: protein molecular weight standard；2: lure Lead 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- Ago-Gel 4b purifies mntc result；M: protein molecular weight standard；1: be incorporated on filler Gst-mntc fusion protein；2: the albumen on filler after digestion wash-out；3: the destination protein mntc after digestion wash-out.

Figure 16, mmc chromatographic purifying mntc protein chromatography figure.

Figure 17, mmc chromatographic purifying mntc protein electrophoresis figure.M: protein molecular weight standard；1: sample；2: flow through；3: wash De- peak.

Figure 18, phenyl hp chromatographic purifying mntc protein chromatography figure.

Figure 19, phenyl hp chromatographic purifying mntc protein electrophoresis figure.M: protein molecular weight standard；1: flow through；2-4: wash De- peak collecting pipe 1-3.

Mmc chromatographic purifying mntc protein chromatography figure under Figure 20, high conductance.

Mmc chromatographic purifying mntc protein electrophoresis figure under Figure 21, high conductance；M. protein molecular weight standard；1: sample introduction sample； 2: flow through sample 3:mmc elution samples.

Mmc chromatographic purifying mntc protein chromatography figure under Figure 22, low conductance.

Mmc chromatographic purifying mntc protein electrophoresis figure under Figure 23, low conductance；M. protein molecular weight standard；1: sample introduction sample； 2: flow through sample；3:mmc elution samples.

First amplification of Figure 24, mntc albumen purifies mmc chromatogram.

First amplification of Figure 25, mntc albumen purifies q hp chromatogram.

Figure 26, first mmc and q hp chromatographic purifying protein electrophoresis figure；M: protein molecular weight standard；1: sample introduction sample； 2:mmc elution samples；3:g25 elution samples；4:q hp flows through sample.

Figure 27, mntc albumen second batch amplifies purifying mmc chromatogram.

Figure 28, mntc albumen second batch amplifies purifying q hp chromatogram.

Figure 29, second batch mmc and q hp chromatographic purifying protein electrophoresis figure；M: protein molecular weight standard；1: sample introduction sample； 2:q hp flows through sample；3:g25 elution samples；4:mmc elution samples.

The 3rd batch, Figure 30, mntc albumen amplifies purifying mmc chromatogram.

The 3rd batch, Figure 31, mntc albumen amplifies purifying q hp chromatogram.

Figure 32, the 3rd batch of mmc and q hp chromatographic purifying protein electrophoresis figure；M: protein molecular weight standard；1: sample introduction sample； 2:mmc elution samples；3:g25 elution samples；4:q hp flows through sample.

The hplc testing result of Figure 33, mntc final sample；13.409 points of main peak retention time, main peak ratio 99.5%.

Figure 34, mntc stoste and bacterin preparation centrifugation supernatant 10%sds-page；1-5: bacterin preparation centrifugation supernatant is (in conjunction with 1 ～5 hours, sample per hour)；6:mntc stoste.

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

Specific embodiment

Present invention will be illustrated below, but the scope of the present invention will be not limited to detailed description below and enforcement Example.

The present invention is based on prior art, provides a kind of staphylococcus aureus manganese ion transport protein mntc of restructuring, its Can be applicable to prepare the detection kit of the subunit vaccine of infection of staphylococcus aureus and correlation, described mntc albumen Amino acid sequence is as shown in seq id no.1.The optimization based on wild mntc albumen for the 6-290 amino acids of this albumen, the 1-5 amino acids (gplgs, seq id no.6) are the amino acid of the modification from expression vector, the coding of this 5 amino acid Sequence is preferably gggcccctgggatcc(seq id no.7).

On the other hand, the present invention provides the nucleotide sequence encoding described mntc albumen.In known gal4 amino acid In the case of sequence, those skilled in the art can design the suitable nucleosides encoding described amino acid sequence completely as needed Acid sequence, and so that it is expressed.In a preferred embodiment, described nucleotide sequence is as shown in seq id no.2.

On the other hand, the present invention provides a kind of recombinant expression carrier for expressing mntc recombinant protein, described expression Carrier comprises nucleotide sequence and the carrier sequence encoding described mntc recombinant protein.Preferably, described expression vector is Pgex or pet serial carrier；It is highly preferred that described expression vector is pgex-6p-2 carrier.The collection of illustrative plates of pgex serial carrier is as schemed Shown in 35.

Present invention preferably employs pgex-6p-2 carrier its to be mainly characterized by carrier being connected to a molecular weight be 26kda Glutathione-s- transferase (gst), just containing a gst label in expressed fusion protein, this label is protein purification Mark.Compared with other fusion vectors, pgex serial carrier has that purification condition is gentle, step simple, do not need denaturant Addition so that albumen after purification can keep its space conformation and immunogenicity to greatest extent.

On the other hand, the present invention also provides a kind of host of the mntc recombinant protein of the expression present invention, including the present invention Recombinant expression carrier.Described host can be any expression cell well known by persons skilled in the art it is preferable that described host It is Escherichia coli xl1-blue.

On the other hand, the present invention also provides a kind of method preparing mntc recombinant protein, and methods described can be chemistry Synthetic method or nucleotides representation.Those skilled in the art are completely permissible according to the sequence of mntc recombinant protein and common sense in the field Know how to prepare the mntc recombinant protein of the present invention.

In a preferred embodiment, the method preparing mntc recombinant protein of the present invention comprises the following steps:

1) coded sequence according to wild mntc albumen (seq id no.5) design forward primer and reverse primer；Preferably Ground, as shown in seq id no.3, described reverse primer is as shown in seq id no.4 for described forward primer；

2) use forward primer and the reverse primer of step 1) design, amplify the gene of coding mntc albumen by pcr Fragment；The codon of mntc albumen is optimized, specifically, the 9th by mntc albumen coded sequence (seq id no.5) Codon (coding leu) is optimized for ctg, the 13rd, 14 bit codons (coding thr) be optimized for acc, the 27th, 65,282 passwords Son (coding gly) is 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, then converts to Host Strains；

4) the Host Strains expression mntc recombinant protein after Induction Transformation.

Preferably, described expression vector is pgex-6p-2, and described Host Strains are Escherichia coli xl1-blue.

On the other hand, the present invention also provides a kind of zymotechnique of mntc host.

Described technique includes a certain amount of kind of daughter bacteria is inoculated in the fermentation medium containing glycerine and certain dissolved oxygen amount, Certain time is induced to express recombinant protein through derivant.

Preferably, described technique is related to culture medium, plants daughter bacteria inoculum concentration, the amounts of glycerol in culture medium, dissolved oxygen amount, derivant Species, inducer concentrations, inducing temperature, induction time several respects factor.Wherein, described culture medium can be animal derived tb, Animal derived m9, plant-derived tb, plant-derived m9, preferably animal derived tb；The inoculum concentration of described kind of daughter bacteria is 5%～ 15%, preferably 10%；Described amounts of glycerol is 5-15ml/l culture medium, preferably 10ml/l culture medium；Described dissolved oxygen amount is 25%～ 65%, preferably 45%；Described derivant can be lactose or iptg, preferably iptg；The concentration of described derivant is 0.1～ 1mm, preferably 0.2mm；Described inducing temperature is 16～37 DEG C, preferably 30 DEG C；Described induction time is 1～6 hour, preferably For 5 hours.

In a kind of specific embodiment of described zymotechnique, basal medium selects animal derived tb culture medium, its Middle amounts of glycerol is 10ml/l；When fermentation starts, the inoculum concentration ratio planting daughter bacteria is 10%；Oxyty keeps during the fermentation 45%；During induction, temperature be adjusted to 30 DEG C, iptg concentration be 0.2mm, induction 5h.

On the other hand, the present invention also provides a kind of technique purifying mntc recombinant protein after fermentation mntc host, bag Include the Glutathione Sepharose 4b affinity chromatography carrying out successively, cation-exchange chromatography, desalination, de- endotoxin.Preferred In embodiment, before carrying out affinity chromatography, need to break bacterium to genetic engineering bacterium, albumen is discharged from thalline.Excellent Selection of land, Glutathione Sepharose 4b affinity chromatography combines pp enzyme digestion；Described cation-exchange chromatography is mmc chromatography；Institute State desalination and use g25 post；Described de- endotoxin uses q hp column chromatography.

Preferably, the buffer solution that described mmc chromatography uses includes: buffer solution a:10mm his+0.01% PLURONICS F87, ph6.0；Buffer solution 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 conductance is less than 10ms/cm.

On the other hand, invention also provides mntc recombinant protein in preparation for detecting, preventing or treating the life of sa infection Application in Tetramune.Preferably, described biological products are vaccine.Those skilled in the art can milli according to prior art content Undoubtedly free burial ground for the destitute knows how to apply this mntc recombinant protein.

On the other hand, the present invention also provides the antibody being produced by the mntc recombinant protein immunity of the present invention, described antibody For polyclonal antibody, can be used for detection, the prevention and treatment of the disease related to sa, or be used for preparing biological system accordingly Product.

On the other hand, the present invention also provides a kind of composition comprising mntc albumen of the present invention or kit.Such Composition can be reagent, medicine (as vaccine), for preventing, detecting or treat sa infection.Such kit can be inspection Any form kit known in the art such as test agent box or therapeutic reagent box.

The expression of genetic engineering recombinant protein of the present invention and fundamental characteristics include:

1) mntc expression of recombinant proteins plasmid abduction delivering in prokaryotic expression system Escherichia coli；

2), when selecting pgex-6p-2 carrier, mntc recombinant protein is with fusion protein form expression；Expressed fusion protein In contain a gst label, this label just becomes the mark of protein purification so that purification condition is gentle, step simple, do not need The addition of denaturant, thus albumen after purification can keep its space conformation and immunogenicity to greatest extent；It is purified Mntc recombinant protein purity is more than 95%；

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

The subunit vaccine of the present invention can carry out immunity inoculation by subcutaneous (muscle) injecting pathway, and excitating organism produces high Titre igg antibody and cellullar immunologic response.And animal experiment proves that, described genetic engineering recombinant subunit vaccine has well Anti- sa infection immune protective effect.It is further combined vaccine and the research of many subunits fusion bacterins lays the first stone, with When for vaccine, therapeutic antibodies and diagnostic kit development and application have great importance.

Bacterial strain used in this part is as follows with various reagents:

1. bacterial strain

Staphylococcus aureus atcc international standard strain mrsa-252 is provided by U.S. atcc；

Bacterial strain xl1-blue Escherichia coli are Agilent company of the U.S. product.

2. plasmid

Plasmid pgex-6p-2 is U.S.'s ge healthcare Products.

3. reagent

Primestar hs dna polymerase, dna molecular weight standard, restriction enzyme bamh i and not i, albumen divide Sub- amount standard, dna ligase are Dalian takara Products；

Plasmid extraction kit and gel reclaims kit are U.S.'s omega Products；

Mh culture medium: purchased from Beijing extensive and profound in meaning star biotechnology Co., Ltd (powdered beef 2.0g, soluble starch 1.5g, acid hydrolyzed casein 17.5g), add water to 1l, ph value 7.4 ± 0.2；

Mh flat board: mh culture medium adds agarose extremely final concentration of 1.5g/100ml；

Pbs(potassium dihydrogen phosphate (kh₂po₄) 0.2g(domestic pure analysis pure), disodium hydrogen phosphate (na₂hpo₄·12h₂o)2.9g (domestic pure analysis pure), sodium chloride (nacl) 8.0g(domestic pure analysis pure), potassium chloride (kcl) 0.2g, add water to 1000ml, Ph7.4)；

20mm pb buffer solution: potassium dihydrogen phosphate (kh₂po₄) 0.2g, disodium hydrogen phosphate (na₂hpo₄·12h₂O) 2.9g, chlorine Change potassium (kcl) 0.2g, add water to 1000ml, ph7.0；

Ampicillin, kanamycins (North China pharmacy)；

5 × protein sample-loading buffer (250mm tris-hcl(ph6.8), 10%(w/v) sds, 0.5%(w/v) bromine phenol Indigo plant, 50%(v/v) glycerine, 5%(w/v) beta -mercaptoethanol)；

Ge healthcare company of the Glutathione Sepharose 4b(U.S.)；

Aluminium phosphate adjuvant: general chemical company of the U.S. (20mg/ml)；

Vaccine diluent (histidine (merck company of the U.S., pharmaceutical grade) 10mm, nacl0.9%(Sichuan Cologne, injection Physiological saline), PLURONICS F87 (merck company of the U.S., pharmaceutical grade) 0.01%, ph6.0), apyrogeneity；

Remaining reagent such as agar powder, Tween-20 is domestic market and buys.

Embodiment 1: the structure of the codon optimization of coding mntc and expression vector and expression engineering bacteria

According to Escherichia coli password Preference, mrsa-252 genome (gi:49240382) is encoded with the gene of mntc Sar0641 carries out password optimization, specific as follows:

9th bit codon (coding leu) of wild type mntc albumen coded sequence (seq id no.5) is optimized for ctg, 13rd, 14 bit codons (coding thr) are optimized for acc, the 27th, 65,282 bit codons (coding gly) be optimized for ggt, the 214th Individual codon (coding arg) is optimized for cgt, the nucleotide sequence (seq id no.2) after being optimized, the ammonia of this sequential coding Base acid sequence is as shown in the 6th～290 of seq id no.1.

The sequence having optimized is transferred to Shanghai JaRa Bioisystech Co., Ltd to carry out full genome synthesis, with bamhi and Noti connects into pgex-6p-2 for head and the tail restriction enzyme site, constitutes pgex-6p-2-mntc (r) expression plasmid, converts xl1-blue Bacterial strain.

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

1st, mrsa-252 bacterial strain is taken to coat mh agar plate (mueller-hinton agar, the extensive and profound in meaning star in Beijing biology skill Art Co., Ltd, lot:02-051) recovery, 37 DEG C of aerobic overnight incubation.Picking single bacterium colony inoculates 1000ml mh (mueller-hinton, Beijing extensive and profound in meaning star biotechnology Co., Ltd, lot:02-052) fluid nutrient medium, 37 DEG C aerobic 210rpm shaken cultivation 7h, extracts mrsa genome.

2nd, the genome being extracted with step 1 is template, expands mntc genes of interest fragment, and amplification step is as follows:

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

Forward primer: pmntcbamhi1(seq id no.3:

5'-ctgggatccagcagtgataagtcaaatgcaaac-3'；

bamhi

Reverse primer: pmntcnoti2(seq id no.4):

5'-atgcggccgccttattatttcatgcttccgtg-3'；

notⅰ

2) pcr system:

3) 98 DEG C of denaturation 10s of pcr amplification reaction condition, 62 DEG C of annealing 30s, 72 DEG C of extension 1min30s, 30 circulations, 72 DEG C fully extended 7min.Detect pcr amplification using 1.5% Ago-Gel after completion of the reaction, pcr amplification is shown in In Fig. 1.

4) gel reclaims kit is used to reclaim mntc pcr product.

3rd, the identification of pcr product and clone, step is as follows:

1) bamh i and not i digestion pgex-6p-2 plasmid and mntc pcr product, endonuclease reaction system:

37 DEG C of digestion 2h.

2) reclaim pgex-6p-2 plasmid and the pcr product through bamh i and not i digestion.

3) connect and convert:

Coupled reaction system:

Measuring mntc digestion recovery product nucleic acid concentration by ultraviolet specrophotometer is 43ng/ μ l, pgex-6p-2 digestion Recovery product nucleic acid concentration is 55ng/ μ l, and according to the general ratio of carrier and exogenous sequences molal quantity for 1:2～10, design connects below Connect reaction system:

Mix, 16 DEG C of connection 2h.

4) 3 pipe Escherichia coli xl1-blue competent cells are taken, first pipe adds pgex-6p-2 plasmid, makees positive control； Second pipe adds dna connection product；3rd pipe is not added with exogenous dna, makees negative control.Ice bath 30min, thermal shock in 42 DEG C of metal baths 90s, rapid ice bath 2min.Add 600 μ l lb blank cultures, mix, be placed in 200rpm shaking 1h in 37 DEG C of shaking tables.

Each pipe is centrifuged 3min with 5000rpm room temperature, discards 300 μ l supernatants, more resuspended thalline, takes 200 μ l to coat dense eventually Spend the lb flat board for 100 μ g/ml amp.Flat board is inverted in culture 24h in 37 DEG C of incubators.

5) screening of pgex-6p-2-mntc (wt) positive recombinant plasmid, identification:

1. negative control plates do not have bacterium colony to occur；Positive control flat board covers with bacterium colony, illustrates that competent cell just makes Really, credible result.Separate good bacterium colony on picking connection product conversion flat board, be inoculated in final concentration of 100 μ g/ml amp's In lb culture medium, 37 DEG C of shaken cultivation are overnight；

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

3. plasmid dna carries out bamh i and not i double digestion；

Double digestion reaction system:

37 DEG C of digestion 2h；

4. 1.5% agarose gel electrophoresis detection double digestion result, result is as shown in Figure 2 it is seen that swimming lane 3,4 samples are Pgex-6p-2-mntc (wt) recombinant plasmid successfully constructing；

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

Embodiment 3:mntc abduction delivering in prokaryotic expression system-Escherichia coli

1. destination protein abduction delivering

1) the correct pgex-6p-2-mntc of identification (wt)/xl1-blue bacterium solution and full genome synthesis pgex-6p-2- are taken The each 100 μ l of mntc (r)/xl1-blue bacterium solution add and contain in the lb culture medium of 100 μ g/ml amp to 10ml, and 80rpm37 DEG C overnight Culture, takes bacterium solution 2ml of incubated overnight to add respectively and contains in the lb culture medium of 100 μ g/ml amp to 100ml；To od600 it is During 0.8-1.0, add iptg to make its final concentration of 200 μm, then be placed in 30 DEG C of 3h of shaking table abduction delivering, 16 DEG C overnight induce table Reach.

2) take out the bacterium solution after abduction delivering, 5min, supernatant discarded are centrifuged with 10000rpm, add 5ml lysis buffer (pbs) mix, ultrasonic (300 watts of power) cracking 10min (work 6 seconds is rested 9 seconds), then 4 DEG C of 14000rpm are centrifuged 15min, point From supernatant precipitation.

2. process supernatant:

Supernatant combines: takes Glutathione Sepharose 4b80 μ l, washed with pbs after 3 times, add ready supernatant, Room temperature combines 1h.It is centrifuged after 3min with 14000rpm at 4 DEG C, washed 2 times using pbs-0.25% polysorbas20, pbs washing three Secondary.Take precipitation 16 μ l, add 4 μ l5 × protein sample-loading buffer, boil 5min, 14000rpm is centrifuged 3min.

Carry out digestion wash-out with pp enzyme (prescission protease, ge company of the U.S.): the use of pp enzyme is by manufacturer's recommended Method carry out.Pp enzyme by being used carries gst label, is beneficial to removal pp enzyme.After the completion of digestion, supernatant is collected by centrifugation And precipitation.Respectively take supernatant to precipitate 16 μ l, add 4 μ l5 × protein sample-loading buffer, boil 5min, 14000rpm is centrifuged 3min.Carry out 10%sds-page, result is as shown in Figure 3.

As seen from the figure, after password optimizes, engineering bacterium expression amount ratio is significantly increased before being not optimised.Hereinafter experiment will make Carried out with the engineering bacteria after password optimization.

Embodiment 4:mntc engineering bacterium fermentation

1st, the determination of fermentation condition:

1) impact to engineering bacteria growing and destination protein expression for the culture medium:

The impact to mntc protein expression for four kinds of culture mediums is tested in shaking flask by such as aforementioned cultural method:

Plant-derived improvement tb(potassium dihydrogen 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 dihydrogen phosphate 2.3g, disodium hydrogen phosphate 13g, glycerine 5ml, yeast extract 24g, animal sources tryptone 12g, magnesium sulfate 1g, add water to 1l)；

Plant-derived m9-caa(disodium hydrogen phosphate 15.6g, potassium dihydrogen phosphate 4.3g, ammonium chloride 1g, magnesium sulfate 1g, chlorination Sodium 0.67g, glucose 5g, soy peptone 3.6g, plant source dusty yeast 4g, acid hydrolyzed casein 6g, add water to 1l)；

Animal derived m9-caa(disodium hydrogen phosphate 15.6g, potassium dihydrogen phosphate 4.3g, ammonium chloride 1g, magnesium sulfate 1g, chlorination Sodium 0.67g, glucose 5g, animal tryptone 3.6g, animal sources dusty yeast 4g, acid hydrolyzed casein 6g, add water to 1l).

Pgex-6p-2-mntc (r)/xl-1blue is taken to be inoculated in amp⁺In lb flat board, 37 DEG C of incubation 16h-20h, picking list Bacterium colony is in 10mlamp⁺In lb culture medium, it is placed in 37 DEG C in shaking table, 200rpm shakes od₆₀₀When about 2 about, by 1:100 respectively Be inoculated in tetra- kinds of culture mediums of 100ml, 37 DEG C, 200rpm shake 14 hours, every 2 hours sampling survey od₆₀₀, its result is shown in Fig. 4.

As seen from the figure: plant-derived culture medium is all worse than animal derived, and engineering bacteria is all just to grow during about 8h to slow down, and In animal derived culture medium with regard to growing way very well, it is constantly in logarithmic phase, and tb is better than m9, thus (dynamic from animal sources culture medium Abbreviation tb, m9 after property improvement tb and m9-caa of thing source).

By fresh mntc engineering bacteria bacterium solution (od₆₀₀About 2) it is inoculated in respectively in 100mltb and m9 culture medium by 1:100, 37 DEG C, 200rpm shakes to od₆₀₀0.8 about, add the iptg of 1mm, 25 DEG C of induction 12h.Take 100ml bacterium solution to be centrifuged, abandon supernatant, Weigh tb:2.4g, m9:1.5g.Broken with 1g:10ml plus pbs ultrasonic (300 watts of power) cracking 10min (work 6 seconds is rested 9 seconds) Bacterium, in conjunction with gst4b(process detailed in Example 5), carry out 10%sds-page, result is as shown in Figure 5.

As shown in Figure 5: unit destination protein expression tb is better than m9, and unit bacterium weight in wet base tb is more than m9, therefore mntc fermentation Select tb culture medium with basal medium.

2) impact that iptg concentration is expressed to destination protein:

The impact to destination protein expression for the iptg of the different final concentrations of investigation.The final concentration of iptg be respectively 0.1mm, 0.2mm, 0.5mm, 1mm(culture medium), it is compared and selects optium concentration.By fresh mntc engineering bacteria bacterium solution (od₆₀₀Greatly About 2) it is inoculated in respectively in four 100ml tb by 1:100,37 DEG C, 200rpm shakes to od₆₀₀0.8 about, being separately added into iptg makes Its concentration corresponds to above-mentioned four kinds of concentration (one bottle of concentration), 25 DEG C of induction 12h respectively.Four bottles of bacterium solution are centrifuged respectively, abandon Clearly, pbs, ultrasonic (300 watts of power) cracking 10min (work 6 seconds is rested 9 seconds) is added to break bacterium, in conjunction with gst4b with 1g:10ml Afterwards, carry out 10%sds-page, result is as shown in Figure 6.

As shown in Figure 6: during iptg final concentration of 0.2mm, protein expression is essentially identical with protein expression when 0.5mm, 1mm, Significantly better than protein expression during 0.1mm, so selecting 0.2mm to be that iptg final concentration is used in fermentation.

3) impact that the different vaccination amount of kind daughter bacteria is expressed to growth and the destination protein of engineering bacteria:

Research 5%, 10%, 15% 3 kind different plant the impact to fermentation for the daughter bacteria inoculum concentrations.By growth curve of bacteria, unit Expressing quantity is determining optimum inoculation amount.As kind of a daughter bacteria od₆₀₀When 2 about, pour into and in fermentation tank, start timing, every 1 hour Od is surveyed in sampling₆₀₀, terminate before induction, draw engineering bacteria early growth phase curve (Fig. 7), can determine whether that bacteria growth is fast Slowly.After induction samples after terminating, process according to method before, carry out 10%sds-page, judge unit destination protein expression (Fig. 8).As shown in Figure 7,5% inoculum concentration bacterial growth is the slowest and highest od₆₀₀Much lower compared with other two inoculum concentration, 10% and 15% Inoculum concentration is more or less the same.As shown in Figure 8, different vaccination amount unit destination protein expression.Expression most preferably 5% inoculum concentration, its Secondary is 10%, but is more or less the same, and 15% is worst.

In sum: 5% inoculum concentration expression is best, but the speed of growth is slow, and final bacterium yield is few；15% inoculum concentration growth speed Degree is fast, but expression is worst；The expression of 10% inoculum concentration is more or less the same than 5%, and the speed of growth is also more or less the same than 15%, institute To select 10% inoculum concentration as fermentation inoculum concentration.

4) impact to engineering bacteria growing and destination protein expression for the oxygen concentration:

This engineering bacteria is facultative aerobe, and the height of oxygen concentration is very big on bacterial growth impact, so during the fermentation Just seem particular importance to the control of dissolved oxygen, now investigate respectively dissolved oxygen bacterium when 25%, 45%, 65% growing state (Fig. 9) and Destination protein expression (Figure 10).

Understand from bacterial growth situation: 45% dissolved oxygen condition, bacterium grows best；Come from unit-protein expression See, under 45% dissolved oxygen condition, expression is also best, so oxyty selects 45%.

5) determination of glycerine consumption:

Induce in different bacterium amount, the expression of mntc can be affected, and in culture medium when fermenting, the amount of glycerine can direct shadow Ring bacterium amount number.When in tank, glycerine consumption is over, bacterium stops growing, and ph value and oxygen dissolving value rise rapidly at short notice, Iptg now should be added start to induce at once.So how many direct decision induction starting times of glycerine.By study 5ml/l, 10ml/l, To destination protein, the impact expressed and final weight in wet yield to determine optimal induction starting time to the amounts of glycerol of 15ml/l.Result such as Figure 11 Shown.

Knowable to figure: during 5ml/l glycerol concentration, expression highest；Secondly 10ml/l, but is more or less the same；15ml/l is just poor Many.But during 5ml/l glycerol concentration, last thalline weight in wet base is much lower；10ml/l is more or less the same with 15ml/l, so Determine that amounts of glycerol adds 10ml/l eventually.

6) impact that different inducing temperatures and time express to destination protein:

Investigate 30 DEG C, 25 DEG C, the 16 DEG C three different impacts to protein expression for the inducing temperature, and when reaching maximum expression Time.After induction, every 2h sampling, induces 10h, processes sample, carry out 10%sds-page, its result is as shown in figure 12.

From electrophoretogram: 30 DEG C of unit abduction delivering amounts are highests, and the time used by maximum expression that reaches is the shortest , the differential expression between 4h-6h is little.And 25 DEG C, 16 DEG C of unit expressions be just not so good as 30 DEG C, and expression time is long.Therefore lure Lead temperature and time and select 30 DEG C, 5h.

According to the studies above, the preferred zymotechnique finally determining the mntc engineering bacteria of the present invention is:

(1) basal medium selects animal derived tb culture medium, and wherein amounts of glycerol is 10ml/l；

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

(3) oxyty remains at 45% about during the fermentation；

(4) induce when, temperature be adjusted to 30 DEG C, iptg concentration be 0.2mm, induction 5h.

2nd, engineering bacterium fermentation technique is amplified

According to above-mentioned optimal conditions, fermentation-scale is enlarged (25l), and obtains mntc engineering bacteria growth curve (figure 13) and unit-protein expression electrophoretogram (Figure 14).

As shown in Figure 13, after technique is amplified, whole growth curve standard of comparison, early stage bacterial growth is very fast, and the later stage induces When curve more steady, finally obtain strain density (od₆₀₀) reach 38, unit bacterium weight in wet base 47g/l.

As shown in Figure 14, express from unit destination protein: expression prolongation in time is significantly increased, 5h expression Highest.

In sum: mntc zymotechnique amplifies successfully, complies fully with expected results.The zymotechnique of the present invention is applicable In the application of large-scale industrial production.

The purifying process of embodiment 5:mntc recombinant protein

1st, break bacterium and prepare supernatant:

The thalline 200-500g that embodiment 1 is built with 20mmol/l pb, ph7.0 buffer solution, by weight: volume ratio 1: 10 ratios mix and suspend, 4 DEG C of precoolings.

High-pressure homogenization: using distilled water flushing high pressure homogenizer (high pressure homogenizer apv-1000, Denmark an invernsys Group) pipeline, low-temperature circulating system open be cooled in advance 1-4 DEG C standby.The suspension bacteria liquid of precooling is added high pressure homogenizer, pressure Power maintains 60-80mpa and breaks bacterium 3-5 time, takes pbs smear to carry out violet staining, under each visual field under oil mirror, unbroken bacterium is little It is considered as brokenly bacterium (broken bacterium rate is more than 90%) completely in 2.

High speed centrifugation: the liquid after broken bacterium loads centrifugal barrel (beckman, the U.S.), and 4 DEG C, 10,000-15,000g are centrifuged 15-30min, collects supernatant standby.

2nd, Glutathione Sepharose 4b affinitive layer purification

Break to every liter of mntc engineering bacteria and in bacterium solution, add 200ml gst filler, 20～25 DEG C combine more than 4h, cohesive process Adopt the combination to promote mntc albumen and gst filler of the method for vertical rotary or stirring.Gst by above-mentioned combination mntc albumen Filler adopts pbs to wash 5 volumes to remove the foreign protein not being combined with gst filler.Then fill out to filler by every 100ml gst Material adds the pp enzyme of 20ml, suction filtration collect filtrate after digestion 6 hours under the conditions of 20～25 DEG C, obtains digestion and removes gst label Mntc albumen afterwards, and carry out sds-page analysis.Result is as shown in figure 15.

3rd, cation-exchange chromatography

1) selection of chromatographic stuffing

Relatively adopt the purification effect to mntc albumen for the mmc and phenyl hp, the sample of use is the mntc of above-mentioned acquisition Thick purification of samples.

Instrument system: akta-expolrer100/avant25 liquid chromatographic system (ge company)；

Chromatographic stuffing: mmc, phenyl hp(is ge company)；

Post specification: (φ) 2.6cm × (h) 20cm, (φ) 1.6cm × (h) 10cm；

Packed column volume: 54ml, 5ml；

Mmc buffer solution: buffer solution a:10mm his+0.01% PLURONICS F87, ph6.0；Buffer solution b:10mmhis+ 0.01% PLURONICS F87+1m nacl, ph6.0；

Phenyl hp buffer solution: buffer solution a:10mm his+0.01% PLURONICS F87+1.5m (nh₄)₂so₄,ph6.0； Buffer solution b:10mm his+0.01% PLURONICS F87, ph6.0；

Loading sample: after taking gst affinity chromatography sample adjust respectively standby to ph consistent with mmc and phenyl hp buffer solution a With.

(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 is as shown in Figures 18 and 19.

In each elution program, the buffer solution of remaining share is corresponding buffer solution a.

Collect: each for destination protein elution samples are carried out sds-page purity analysis, evaluates purification effect.

From Figure 16-19 as can be seen that under the conditions of different chromatographies, mmc is stronger to the binding ability of mntc than phenyl hp, Penetrate almost without mntc destination protein in peak, eluting peak mntc purity can reach 95% about, destination protein yield is high, has Chromatographic purifying effect well.

Accordingly, it is determined that selecting mmc to be used as the filler of first step chromatographic purifying.

2) optimization of chromatographic purifying technique

Establish be used mmc as first step filler on the basis of, condition optimizing is carried out to this chromatographic stuffing, main optimization Condition be loading sample conductance, evaluation index is the purity of mntc and yield.

Instrument system: akta-explorer100 liquid chromatographic system (ge healthcare)；

Chromatographic stuffing: mmc；

Post specification: (φ) 2.6cm × (h) 20cm；

Packed column volume: 54ml；

Buffer solution:

1. buffer solution a:10mm his+0.01% PLURONICS F87+0.15m nacl, ph6.0；

Buffer solution b:10mm his+0.01% PLURONICS F87+1m nacl, ph6.0；

2. buffer solution a:10mm his+0.01% PLURONICS F87, ph6.0；

Buffer solution b:10mm his+0.01% PLURONICS F87+1m nacl, ph6.0；

Loading sample: take the thick purification of samples of mntc to adjust to consistent with two kinds of buffer solution a respectively.

Flow velocity: 20ml/min

Elution program: 0-100%b, 10 column volumes (cv).

Each for destination protein elution samples are collected, and carries out sds-page purity analysis, evaluate purification effect.

From Figure 20-23 as can be seen that under the conditions of low loading sample conductance, destination protein no flows through, and the purpose purifying Purity of protein is high, accordingly, it is determined that selecting to be less than 10ms/cm using loading sample condition.

4th, desalination

Desalination g25 post is balanced using vaccine diluent, upper step is purified the sample obtaining buffer solution is replaced by desalting column.

This process concretely comprises the following steps: the sample that upper step is obtained, and with vaccine diluent dissolving, balances tomographic system (akta Explorer100, U.S. ge healthcare) and chromatographic column xk50-60(U.S. ge healthcare) (600ml Sephadex g 25), with flow velocity 20ml/min desalination.

5th, second step purifies de- endotoxin

Chromatographic stuffing: q hp；

Post specification: (φ) 2.6cm × (h) 20cm；

Packed column volume: 50ml；

Buffer solution: buffer solution a: vaccine diluent, endotoxin-free；Buffer solution b:1m naoh；

Loading sample: sample after g25 desalination；

With buffer solution b (1mol/l naoh) incumbent firms 5 column volumes of sterilization, after placing half an hour, diluted using vaccine Liquid balance system is 6.0 to ph, then loading.Flow velocity: 8ml/min.It is destination protein peak that collection penetrates peak.

6th, technique is amplified

Carry out technique amplification according to loading determined above and elution mode: the mmc chromatographic column scale being adopted is amplified For (φ) 2.6cm × (h) 20cm, packed column volume cv=70ml, q hp chromatographic column scale is enlarged into (φ) 2.6cm × (h) 20cm, Packed column volume cv=50ml.Repeat three batches experiments, 10%sds-page analyzes mntc effect after purification and yield, evaluates this work Stability after skill amplification and repeatability.

From Figure 24-32 as can be seen that after the mmc chromatography technique of mntc albumen amplifies, chromatographic purifying pattern and small trials The no significant change of chromatographic purifying chromatogram, purified after mntc purity reach more than 97%.

7th, hplc purity detecting

Agilent company of the hplc instrument agilent1260(U.S.), analytical column zorbax sb-300- Agilent company of the c34.6x150mm3.5micron(U.S.).

Mobile phase: a:0.1% trifluoroacetic acid (tedia, the U.S.), water (18.2m ω)；B:0.1% trifluoroacetic acid (tedia, beautiful State), acetonitrile (tedia, the U.S.).

60 DEG C of column temperature, 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 as shown in Figure 33 and Biao 1,13.409 points of mntc main peak retention time as seen from the figure, main peak area ratio 99.5%.

The hplc testing 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

8th, the mntc recombinant protein that sequence verification obtains.

Entrust the n end of the mntc recombinant protein to acquisition for the Research Centre for Proteome Analysis(Shanghai), c end to be sequenced, Carry out molecular weight determination and amino acid composition analysis, result is completely the same with the mntc recombinant protein of design.

Embodiment 6:mntc protein endotoxins assay

1st, the sample apirogen water (Zhanjiang Bo Kang Marine Bio Co., Ltd.) that embodiment 5 obtains is diluted to 50 μ g/ Ml is as testing sample.The highest model that can be detected according to endotoxin detection kit (Zhanjiang Bo Kang Marine Bio Co., Ltd.) Enclose 0.25eu/ml, testing sample is diluted.Suppose that testing sample endotoxin content is 5eu/ml, then use apirogen water Dilution testing sample is to 2.5 μ g/ml (dilution 20 times) further；

2nd, prepare endotoxin standard positive control according to kit (Zhanjiang Bo Kang Marine Bio Co., Ltd.) specification Solution, testing sample working solution, testing sample detection solution；

3rd, the preparation of TAL: according to the quantity of testing sample and reference substance, take TAL, cotton ball soaked in alcohol is sterilized bottleneck, Open after drying, every addition inspection water 0.1ml, gently shake up standby；

4th, it is loaded: be separately added into testing sample in the TAL preparing and detect that solution, the endotoxin standard positive are right According to liquid, the inspection each 0.1ml of water, jog mixes, and sealed membrane seals, 37 DEG C of water-baths 60 ± 2 minutes, and period forbids to move；Check It is negative control with water；

5th, detect: take out sample, gently 180 ° of vertical rotary, observe bottom of bottle, liquid solidification is not flowed for the positive, and flowing is not It is solidified as feminine gender；

6th, measurement result: negative, less than 5eu/ml.

The preparation of embodiment 7:mntc vaccine

Aluminum phosphate is general chemical company of U.S. imported with original packaging product (20mg/ml)

1st, prepare staphylococcus aureus restructuring mntc vaccine

1) measure Aluminium phosphate adjuvant 80 μ l, add it to prepare in bottle；Measure vaccine diluent 220 μ l, cumulative volume 300 μ l, fully mixes；

2) with vaccine diluent, mntc recombinant protein is diluted to 30 μ g/300 μ l, fully mixes；

3) assist agent solution after isopyknic dilution is added to sub-bottling with the protein solution after dilution, temperature range 4 Under the conditions of DEG C -32 DEG C, vertical it is suspended or horizontal stirring and adsorbing obtained final product after 1 hour.

2nd, antigen protein Aluminium phosphate adjuvant adsorption uniformity and completeness in 10%sds-page identification mntc vaccine

1) sample 1ml from mntc vaccine, 4 DEG C, 6000rpm is centrifuged 5 minutes, carefully draws supernatant, and samples 40 μ from supernatant l；

2) fill into isopyknic dissociation solution (1m na with supernatant₂co₃), under room temperature condition, vertical suspension 1 hour, sample 40 μ l；

3) method pressing above-mentioned 1 prepares the protein solution not containing Aluminium phosphate adjuvant, and volume shared by aluminum phosphate is diluted with vaccine Liquid is supplied, and samples 40 μ l after fully mixing；

4) samples taken is added 10 μ l5 × albumen sample-loading buffer, 100 DEG C are heated 5 minutes, cooling simultaneously brief centrifugation Afterwards, 10 μ l loadings are taken；

5) 10%sds-page electrophoresis, voltage elder generation 80v electrophoresis 20 minutes, then it is adjusted to 180v, electrophoresis 40 minutes, then by glue Take out, be placed in coomassie brilliant blue staining liquid vibration dyeing, then after being placed in vibration decolouring in destainer, observe under imaging system As a result, result is shown in Figure 34 it is seen that Aluminium phosphate adjuvant can fully adsorb recombinant protein.

Embodiment 8:mntc vaccine immunity animal and the detection of antibody

1st, immune animal

1) animal used as test: 66 week old balb/c mouse (Beijing China Fukang), body weight is about 16g

2) immune programme for children: the bacterin preparation according to embodiment 7 preparation is with 30 μ g/600 μ l in three times in quadriceps muscle of thigh intramuscular injection Immunity in (0,14,21 days).

2nd, the 7th day after third time immunity, the serum of collection balb/c mouse, with igg response after elisa detection mouse immune Level.

3、elisa

1) prepare liquid

1. it is coated the preparation of liquid: weigh nahco₃1.6g, na₂co₃2.9g, is dissolved in 1l ddh₂O, ph is adjusted to 9.6；

2. the preparation of confining liquid: 1g cow's serum (sigma, the U.S.), it is dissolved in 100ml antibody diluent (1:100)；

3. the preparation of antibody diluent: nacl8g, kh are weighed on electronic balance₂po₄0.2g, na₂hpo₄· 12h₂O2.9g, kcl0.2g, polysorbas20 0.5ml, adjust ph to 7.4, plus distilled water is settled to 1000ml；

4. the preparation of cleaning solution: weigh 2.42g tris and be dissolved in 1l ddh₂O, adds 500 μ l polysorbas20s, then ph is adjusted To 7.4；

5. nitrite ion (tmb), is Tiangeng Products；

6. terminate liquid (2m h₂so₄) preparation: the 22.2ml concentrated sulfuric acid is poured into 177.8ml ddh₂In o.

2) antibody titer that elisa detection mntc recombinant protein immune mouse produces:

1. it is coated: mntc recombinant protein is diluted to 1 μ g/ml with being coated liquid, is coated 96 orifice plates (corning, the U.S.), 200 μ l/ holes, 4 DEG C are overnight washed with cleaning solution 3 times afterwards, wrapped with preservative film, be placed in standby in 4 DEG C of refrigerators after sky is dry；Setting Pbs control wells；

2. close: add confining liquid, 100 μ l/ holes to ELISA Plate, be placed in 2 hours in 37 DEG C of incubators, wash 3 times；

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

4. take the ELISA Plate closed, sequentially add the serum of dilution, 100 μ l/ holes, be placed in 37 DEG C of incubator 30min, wash Wash 3 times, empty dry；

5. the sheep anti-Mouse igg antibody adding hrp mark is preserved liquid, dilute 1:5000, make antibody working solution；

6. add the antibody working solution of dilution, 100 μ l/ holes, be placed in 37 DEG C of incubator 1h, wash three times, empty dry；

7. add substrate nitrite ion (tmb) 100 μ l/ hole, room temperature lucifuge reacts 5min；

8. add terminate liquid (2mol/l h₂so₄), it is immediately placed on ELIASA to measure od value at 450nm wavelength；

9. result judges: a_Sample/a_{Negative value}>=2.1 is positive (negative control dilutes for 1:1000 times of serum before mouse immune).

Result: the antibody titer that detection mntc recombinant protein antigen immune mouse produces reaches 1:512000, and this is described The mntc * subunit recombinant protein of bright structure can make to produce antibody in immune mouse body.

Embodiment 9: attack malicious protected effect by what mntc vaccine immune mouse determined immune animal

With the immunization protocol of embodiment 8, after third time immune mouse, adopted lethal dose, tail vein injection at the 14th day Mrsa-252 viable bacteria carries out challenge viral dosage, and every balb/c injected in mice bacterium solution amount is 1.25 × 10⁹Cfu, observes 10 days, statistics The survival rate of each group mouse.3 wheel animal protection tests (10/wheel) result is shown in table 2.

Table 2:mntc recombinant protein immune mouse is protected to the poison of attacking of animal

Group	Mouse (only)	Immune component	Survival number after 10 days	3 take turns average protective rate (%)
					Experimental group	30	mntc+alpo₄Adjuvant	12	40
Negative control group	30	Vaccine diluent	2	6.7

Table 2 shows: the 3 average immune protective rates of wheel of negative control group are respectively 6.7%, mntc and add alpo₄The 3 of adjuvant group Taking turns average immune protective rate is 40%.Therefore it was demonstrated that the mntc vaccine of the present invention has good immunogenicity, machine can be induced Body produces protective immune response, and can play immanoprotection action to sa infection, can be aided with aluminium adjuvant Prepare restructuring Subunit vaccine is used for preventing the infection of staphylococcus aureus.

By above example, those skilled in the art can apparently be applied using ordinary skill knowledge Recombinant protein prepared by the present invention and other related reagents, for example, be coated reagent, detection antibody, developer, terminator, adjuvant Deng the related recombinant subunit vaccine of preparation, therapeutic antibodies and detection kit, for immune protection and the golden yellow grape of diagnosis The application of coccus infection.

Claims

1. the nucleotide sequence of the mntc recombinant protein of coding staphylococcus aureus, described nucleotide sequence such as seq id Shown in no.2；

The amino acid sequence of described mntc recombinant protein is as shown in seq id no.1.

2. a kind of carrier including nucleotide sequence as claimed in claim 1 or host, described carrier is expression vector pgex- 6p-2, described host is Escherichia coli xl1-blue.

3. a kind of method preparing mnt c recombinant protein as described in claim 1, methods described includes step: 1) basis The coded sequence design of the wild mntc albumen as shown in seq id no.5 is positive, reverse primer amplification coding mntc albumen Genetic fragment；2) codon of mntc albumen is optimized, specifically, the mntc albumen as shown in seq id no.5 is compiled The 9th bit codon encoding leu in code sequence is optimized for ctg, is separately encoded the 13rd of thr the, 14 bit codons and is optimized for acc, Be separately encoded gly the 27th, 65,282 bit codons be optimized for ggt, the 214th codon optimization of coding arg is cgt；3) By step 2) gene fragment clone that obtains makes it express to expression vector, conversion Host Strains.

4. method according to claim 3, wherein, described forward primer as shown in seq id no.3, described reverse primer As shown in seq id no.4.

5. a kind of fermentation method to express the mntc recombinant protein described in claim 1 for the host as claimed in claim 2, Including the animal derived tb that the kind daughter bacteria of 10% inoculum concentration is inoculated in the glycerine containing 10ml/l culture medium and 45% dissolved oxygen amount In fermentation medium, at 30 DEG C, the iptg through 0.2mm induces 5 hours and ferments.

6. a kind of ferment host as claimed in claim 2 after purify mntc recombinant protein as described in claim 1 Method, including carrying out affinity chromatography, cation-exchange chromatography, desalination, de- endotoxin successively；Before carrying out affinity chromatography, need Bacterium is broken to host, albumen is discharged from thalline；Described affinity chromatography is that gst Ago-Gel 4b chromatography combines Prescission protease digestion；Described cation-exchange chromatography is mmc chromatography, the buffer solution bag that described mmc chromatography uses Include: buffer solution a:10mm his+0.01% PLURONICS F87, ph 6.0；Buffer solution b:10mm his+0.01% poloxamer 188+1m nacl,ph6.0；Loading flow velocity is 12ml/min, and elution flow rate is 12ml/min, the wash-out of described elution buffer Program is with 0-100% elution buffer b, and 7 column volumes carry out gradient elution, and loading sample conductance is less than 10ms/cm；Described Desalination uses g25 post；Described de- endotoxin uses q hp post.

7. nucleotides sequence according to claim 1 is listed in the application in the preparation of preparation prevention sa infection.

8. application according to claim 7, wherein, described preparation is vaccine.

9. include kit or the combination of carrier described in nucleotide sequence or the claim 2 described in claim 1 or host Thing.