CN103820414B

CN103820414B - The purification process of Streptococcus mutans recombinant subunit recombinant vaccine candidate antigens Glu

Info

Publication number: CN103820414B
Application number: CN201410082929.3A
Authority: CN
Inventors: 张卫军; 刘开云; 付强; 邹全明; 郭刚; 张怡; 孙红武; 付启欢; 樊绍文; 卢陆; 高莺
Original assignee: Third Military Medical University TMMU; Chengdu Olymvax Biopharmaceuticals Inc
Current assignee: Third Military Medical University TMMU; Chengdu Olymvax Biopharmaceuticals Inc
Priority date: 2014-03-07
Filing date: 2014-03-07
Publication date: 2016-08-17
Anticipated expiration: 2034-03-07
Also published as: CN103820414A

Abstract

The present invention provides a kind of Streptococcus mutans recombinant subunit genetic engineering to be selected the purification process of vaccine antigen Glu, comprise step: collect the coli somatic of the Streptococcus mutans recombinant subunit recombinant vaccine Glu albumen of fermentation, high-pressure homogenization is used to break bacterium, collect supernatant, centrifugal supernatant, collects precipitation；Inclusion body washs；Inclusion body cracks: resuspended with urea-containing PBS, centrifugal, abandons precipitation, preserves supernatant；Preliminary renaturation: take urea-containing PB buffer, solubilization of inclusion bodies liquid is dropwise slowly added in the buffer of above-mentioned stirring, centrifugal, abandon precipitation, column purification on supernatant；Affinitive layer purification: be purified destination protein under urea-containing PB buffer conditions, after destination protein is combined with filler, carries out enzyme action eluting with the PB buffer of containing sodium fluoride, collects eluent and is the Glu albumen of chromatography purification.The antigen purity using the method purification of the present invention is high, and technique is simple and direct.

Description

The purification process of Streptococcus mutans recombinant subunit recombinant vaccine candidate antigens Glu

Technical field

The invention belongs to biotechnological pharmaceutics field, relate to a kind of restructuring Streptococcus mutans dental caries vaccine candidate and resist Purification process in former Glu preparation.

Background technology

Dental caries is under the many factors effect based on antibacterial, chronic, the Progressive symmetric erythrokeratodermia that hard tooth tissue occurs A kind of infectious disease destroyed.Being characterized in that sickness rate is high, distribution is wide.Average caries incidence can be 50% Left and right, is the main commonly encountered diseases in oral cavity, is also one of most common disease of the mankind, and World Health Organization (WHO) will It is listed as the big keypoint control disease of the mankind three with cancerous protuberance and cardiovascular disease.

Research at present thinks that antibacterial is the pathogenetic essential conditions of dental caries, it is considered that cariogenic bacteria has two types, One is that acid-producing bacteria belongs to, and the most predominantly Streptococcus mutans, actinomyces and lactobacillus can make carbon hydrate Thing decomposes product acid, causes tooth inanimate matter demineralization；Another kind is gram-positive cocci, can destruction of organic material, Tooth can be made to form cavity through long term.The main cariogenic bacteria generally acknowledged at present is Streptococcus mutans.

Since the eighties in 20th century, people begin to prepare with the Effective Antigens composition of Streptococcus mutans Subunit anti-caries vaccine.Though traditional inactivation killed vaccine and attenuated live vaccine can suppress Streptococcus mutans to facing Adhesion, but the antibody of full cell induction and heart nephridial tissue have cross reaction, attenuated live vaccine to have attenuation not Abundant or virulence is replied then can cause and the injury of body is all dfficult to apply to clinic.The most multiple with Anti-caries vaccine based on S.Mutans antigen can significantly reduce animal dental caries incidence rate, and antigen mainly has: table The antigens such as face proteantigen I/II (Ag I/II), glucosyltransferase (GTF) and glucan-binding protein (GBP) Molecule (Hajishengallis G.Infect Immun, 1998,66 (4): 1740-1743;Katz J.Infect Immun,1993,61(5):1964-1971;Childers NK.Oral Microbiol Immunol, 2006,21(5):309-313;Childers NK.J Dent Res,2002,81(1):48-52.;Peacock ZS.Oral Microbiol Immunol,2005,20(1):60-64;Smith D J.Infect Immun, 2005,73 (5): 2797-2804).

Glucosyltransferase (Glucosyltransferase, GTF) be Streptococcus mutans synthesis important cariogenic because of Son, the catalysing sucrose synthesis multiple extracellular polysaccharide including glucosan.GTF structure includes catalytic domain Two the function sections in (Catalytie, Cat) and glucan binding domian district (Glucan binding, Glu).Catalysis activity District has the activity of catalysing sucrose hydrolysis, it is possible to catalysing sucrose produces glucosan；Glucan binding domian district gathers with Portugal Sugar combines, and this combination is specificity, irreversible, and adhesion is very strong, has thus mediated deformation chain Interaction between coccus, between thalline and glucosan, thalline and facing, so that Streptococcus mutans glues Attached, be gathered in the surface of tooth and produce acid, acid causes dental surface demineralization, ultimately results in the generation of dental caries, It is the pathogenetic principal elements of dental caries.Glu has good immunogenicity and immune protective, and it can efficiently be induced Anti-GTF antibody, thus suppress the ability of GTF synthesis glucosan, reduce bacterial plaque and formed.(Xu QA, Vaccine. 2007Jan26;25(7):1191-5；Jespersgaard C,Infect Immun.1999Feb;67(2):810-6； Taubman MA,Infect Immun.2001Jul;69 (7): 4210-6.) therefore, suppression GTF catalyzes and synthesizes Portugal The generation of polysaccharide likely anticaries.

Summary of the invention

The purpose of the present invention, is to provide a kind of Streptococcus mutans recombinant subunit genetic engineering and is selected vaccine antigen The purification process of Glu.Use its aminoacid sequence of antigen Glu(such as SEQ ID of the method purification of the present invention Shown in NO:1, its nucleotide sequence is as shown in SEQ ID NO:2), its purity >=90%, complete whole Without additionally replacing buffer again after purge process, be a kind of technique target protein simple and direct, obtained purity height, The preferable purification process of the response rate.

A kind of Streptococcus mutans recombinant subunit recombinant vaccine candidate antigens Glu's that the present invention provides is pure Change method, it mainly comprises step:

1) thalline, broken bacterium are collected: collect the Streptococcus mutans recombinant subunit recombinant vaccine Glu of fermentation The coli somatic of albumen, uses high-pressure homogenization to break bacterium, collects supernatant, centrifugal supernatant, collects precipitation；

2) inclusion body washing: the precipitation that step 1) is collected, resuspended with PBS, wash repeatedly, It is then centrifuged for, collects precipitation；

3) inclusion body cracking: resuspended with urea-containing PBS, centrifugal, abandon precipitation, preserve supernatant；

4) preliminary renaturation: take urea-containing PB buffer, solubilization of inclusion bodies liquid is dropwise slowly added into State in the buffer of stirring, centrifugal, abandon precipitation, column purification on supernatant；

5) affinitive layer purification: select protein purification system, to mesh under urea-containing PB buffer conditions Albumen be purified, after destination protein is combined with filler, carries out enzyme action with the PB buffer of containing sodium fluoride and wash De-, collect eluent and be the Glu albumen of chromatography purification.

The purification process of Streptococcus mutans recombinant subunit recombinant vaccine candidate antigens Glu, wraps further Containing following steps:

6) dialysis concentrates: be placed in bag filter by the Glu albumen of purification, and sealing is suspended in equipped with containing carbamide PB buffer in, stirring dialysis, take out bag filter high molecular polymer be sprinkling upon outside bag filter, treat volume After concentration, take out.

The purification process of Streptococcus mutans recombinant subunit recombinant vaccine candidate antigens Glu, further also Comprise step:

7) cosolvent is added: preserve after adding L-arginine, glycerol, trehalose or PEG.

Preferably, step 1) particularly as follows: will fermentation Streptococcus mutans recombinant subunit recombinant vaccine The 10-20mmol/L PBS of the coli somatic pH7.0-7.5 of Glu albumen is mixed in 1:9 ratio Even suspension, uses high-pressure homogenization to break bacterium after pre-cooling, the sample after crushing is centrifuged 30 minutes through 750 × g, collects Supernatant, supernatant is centrifuged 30 minutes with 13000 × g again, collects precipitation.

Preferably, step 2) particularly as follows: centrifugation is contained 1%TRITON X-100 with 9 times of volumes PBS resuspended, fully mix, by agitator gentle agitation 30 minutes, then with 10000 × g from The heart 30 minutes, abandons supernatant, and precipitation repeats and washed once；Centrifugation is contained 1mol/L with 9 times of volumes The PBS of carbamide is resuspended, fully after mixing, abandons supernatant, and precipitation repeats and washed once, after being centrifuged Collect precipitation.

Preferably, step 3) is particularly as follows: every gram of inclusion body 19ml PBS weight containing 8M carbamide Outstanding, 4 DEG C of gentle agitation 12h, 13000G is centrifuged 30 minutes, abandons precipitation, supernatant 4 DEG C preservation.

Preferably, step 4) particularly as follows: take certain volume containing 2M carbamide pH7.0100mmol/L PB Buffer, be placed in agitator stirring, solubilization of inclusion bodies liquid be dropwise slowly added into the slow of above-mentioned stirring Rushing in liquid, 13000g is centrifuged 30 minutes, abandons precipitation, column purification on supernatant.

Preferably, step 5) particularly as follows: select Profinity eXactTM protein purification system be purified, Use and under the pH7.0100mmol/L PB buffer conditions containing 2mol/L carbamide, target protein be purified, After destination protein is combined with filler, carry out enzyme action eluting with the PB buffer containing 100mmol/L sodium fluoride, Collect eluent and be the Glu albumen of chromatography purification.

Preferably, step 6) particularly as follows: the Glu albumen of purification is placed in the bag filter of MW10000, Sealing.Being suspended in equipped with in the 100mmol/L PB buffer containing 1mol carbamide pH7.0,4 DEG C of stirrings are thoroughly Analyse 10 hours；Changing buffer is the most urea-containing 100mmol/L PB buffer, 4 DEG C of stirring dialysis 10 Hour；Take out bag filter high molecular polymer or sucrose is sprinkling upon outside bag filter, treat 20 times of volumes of volume concentration After, take out.

Preferably, high molecular polymer is Polyethylene Glycol or polyvinylpyrrolidone.

Described antigen through the following steps that preparation:

1) design forward primer and reverse primer is expanded by PCR or full genome synthesis is to obtain coding deformation chain The nucleotide sequence of coccus recombinant subunit recombinant vaccine candidate antigens Glu protein active fragment；

2) nucleotide sequence that step 1) is obtained is cloned into expression vector establishment recombinant vector, then should Recombinant vector converts to Host Strains；

3) Host Strains after Induction Transformation expresses recombiant protein.

The advantage of said method of the present invention is:

In step 1), the 60-80MPa high-pressure homogenization in production or pilot scale purification is used to break bacterium technology, broken Bacterium rate is more than 96%, after taking out broken bacterium fragment by differential centrifugation, then carries out high speed centrifugation acquisition inclusion body egg In vain.

In step 4): the preliminary renaturation of described albumen, the carbamide in solubilization of inclusion bodies liquid is passed through dilution refolding Mode be slowly dropped to about 2mol/L from the concentration of 8mol/L, in the process, due to the fall of urea concentration As little as 2M, inclusion body protein obtains the effect of preliminary renaturation, and the final concentration of albumen controls at 0.05-0.1mg/ml Left and right, it is to avoid because protein concentration is too high, intermolecular force strengthens, and causes polymerization between protein molecule to tend to increase Greatly, form oligomer and polymer produces precipitation.

In step 5): described affinitive layer purification, the filler used is that Profinity eXact merges mark Label system, is integrated purification and removal label by sodium fluoride, simplifies purge process.

In step 6): the method that described dialysis concentrates can use Bag filter method or hollow fiber column method to carry out, full The requirement that foot technique is amplified.

Use purification process of the present invention mainly have inclusion body washing and cracking, dilution refolding, affinity chromatograph, Dialysis concentrates.Make 12%SDS-PAGE by the Glu of above-mentioned PROCESS FOR TREATMENT different batches, present single Target protein band, molecular weight is about 43kD, and purity is more than 90%.Isoelectric point, IP is at about pH9.1.Pure Glu Yu LTB adjuvant common Nasal immunization Waster rat after change, finds that Glu is with immunological adjuvant group antigen IgG level in specificity saliva sIgA and serum is significantly higher than negative control group (PBS group) (P < 0.01), Prove that the Glu using purification process of the present invention to obtain can produce higher immunne response by effective stimulus body.

For above and other objects of the present invention, feature and advantage can be become apparent, cited below particularly preferably Embodiment, and coordinate accompanying drawing, it is described in detail below.

Accompanying drawing explanation

Fig. 1 is Glu gene PCR amplification figure, in figure, swimming lane 1: nucleic acid (DNA) molecular weight standard (sky root Marker III)；Swimming lane 2-3: for Glu PCR primer；

Fig. 2 is BamH I and the enzyme action qualification result of Not I double digestion qualification recombiant plasmid, in figure, and swimming lane 1: nucleic acid (DNA) molecular weight standard (sky root Marker III)；Swimming lane 2-3: recombinant expression plasmid pPAL7-Glu Qualification result after double digestion, fragment 5900bp separated after enzyme action and 1140bp；

Fig. 3 is restructuring Glu engineering bacteria IPTG abduction delivering figure, wherein swimming lane 1 recombination engineering (pPAL7-Glu/XL1Blue) before induction；Swimming lane 2 be protein molecular weight standard (Fermentas, #SM0671)；Swimming lane 3-7 is respectively recombination engineering induction 1-5h；

Fig. 4 is Glu albumen affinity chromatograph and sodium fluoride enzyme action SDS-PAGE result figure, in figure, swimming lane 1: Restructuring Glu inclusion body protein sample；Swimming lane 2: upper prop flows through sample；Swimming lane 3: protein molecular weight standard (Fermentas, #SM0671)；Swimming lane 4-9: elution samples after sodium fluoride enzyme action；Swimming lane 10: phosphoric acid is clear Elution samples after washing；

Fig. 5 is Glu albumen affinitive layer purification tomographic map；Wherein peak 1 is for flowing through peak, and for the purpose of peak 2, albumen is washed De-peak, peak 3 cleans peak for phosphoric acid.

Detailed description of the invention

Below in conjunction with embodiment, the present invention is described in detail:

Embodiment 1The preparation of antigen Glu

(1) clone of Streptococcus mutans Glu gene

1. (Chinese people solve Streptococcus mutans UA159 (deriving from American Type Culture Collection center, ATCC) Fang Jun Third Military Medical University clinical microbiology and immunology teaching and research room preserve)

2. the Streptococcus mutans UA159 bacterial strain taking out preservation in liquid nitrogen container is coated on BHI solid medium, In 37 DEG C, overnight incubation.Genome extraction agent box extracting Streptococcus mutans genome.

3. use PCR method to expand the encoding gene of Glu respectively from Streptococcus mutans genome.

1) design of primers synthesis is following (underscore shows restriction enzyme site base sequence)

The gene order announced according to GenBank and design of primers principle, design corresponding primer, introduces enzyme Cut site.

2) the PCR amplification of genes of interest:

With Streptococcus mutans genomic DNA as template, P1 and P2 is primer amplification Glu gene, uses such as Lower PCR system and program:

Template DNA 1 μ l；10 × PCR buffer (magnesium chloride containing) 5 μ l；DNTPs(10mmol/L) 4 μ l； The each 1 μ l of upstream and downstream primer (0.025mmol/L)；Taq archaeal dna polymerase (5u/ μ l) 0.5 μ l, adds Deionized water is to final volume 50 μ l.

Reaction system is mixed, after centrifugal treating, adds 30 μ l paraffin oil.94 DEG C of denaturations 5 minutes, 94 DEG C Degeneration 30 seconds, 60 DEG C of annealing 1min seconds, 72 DEG C extend the 1min second, and 35 circulations, 72 DEG C fully extended 10 minutes.Take 1 μ l product, 1.2% agarose gel electrophoresis detection PCR effect after completion of the reaction.

3) (reclaiming test kit purchased from sky, Beijing is Time Inc., uses by test kit in the recovery of PCR primer Description operates)

(1) agarose gel of 1.0% is recorded；

(2) being added by pcr amplification product in electrophoresis loading hole, indicator migrates to stop electricity during appropriate location Swimming；

(3) under Burdick lamp, separate the gel containing purpose fragment, move into 1.5ml EP pipe；

(4) adding DNA and combine buffer, 65 DEG C of water-baths make gel dissolve completely and keep pH value of solution to exist Between 5.0～6.0；

(5) sol solutions moving into separation pipe, 12000g is centrifuged 1 minute, discards the liquid in collecting pipe；

(6) adding 500 μ l lavation buffer solutions, 12000g is centrifuged 1 minute, discards the liquid in collecting pipe；

(7) step (6) is repeated；

(8) 12000g is centrifuged 1 minute, separates another clean 1.5ml EP of pipe dislocation pipe, adds the TE of 20 μ l Buffer, 65 DEG C

Hatching 10 minutes, 12000g is centrifuged 1 minute, takes 2 μ l electrophoresis, and effect is reclaimed in UVP UV scanning inspection Really.PCR amplification Glu genetic results is the most as shown in Figure 1.

The clone of 4.PCR product

1) coupled reaction

Reclaiming production concentration according to PCR with pMD18T carrier by exogenous sequences with carrier mole ratio is respectively The principle of 1: 2～10, design coupled reaction system is as follows:

Purpose fragment connects: table 1 below

Table 1

Reclaim fragment (200ng/ μ l)	1μl
		pMD18(50ng/μl)	1μl
ddH₂O	3μl
		Connect solution	5μl
Cumulative volume	10μl

16 DEG C connect 3 hours.

2) connect product to convert and the screening of recon, qualification

(1) Amp containing X-Gal, IPTG⁺The preparation of LB flat board

The front fusing of LB solid medium, treats that temperature is down to about 50 DEG C and is added Amp to final concentration of 100mg/L, mixing hypsokinesis is to flat board, natural coagulation.Use within first 2～3 hours, take Amp⁺LB flat board, adds Enter 40 μ l X-Gal (20mg/ml), 5 μ l IPTG (200mg/ml), uniform by L rod coating, it is placed in 37 DEG C and incubates Case is standby.

(2) connect product to convert

Take three pipe each addition 100 μ l DH5 α competence bacterias the most respectively (limited purchased from sky, Beijing root biochemical technology Company) bacterium solution EP pipe, first pipe add coupled reaction product, second pipe add comparison inserting paragraph (control Insert) DNA connects product, and as positive control, the 3rd pipe is not added with foreign DNA, as negative control, Ice-water bath 60 minutes.42 DEG C of water-bath heat shocks 90 seconds, place rapidly ice-water bath 1～2 minutes.Often manage and add 700 μ l LB culture fluid, 37 DEG C of shaking tables are cultivated 1 hour.Each pipe is centrifuged 1 minute with 800g, inhales and abandons 400 μ l Mixing precipitation after supernatant, respectively takes 50 μ l and is coated with Amp⁺LB flat board, 37 DEG C of incubator overnight incubation.

(3) screening of purpose recon and qualification

1. picking connect product convert separate good indigo plant on flat board, white macula bacterium colony inoculates Amp respectively⁺LB Flat board, cultivates 12～16 hours for 37 DEG C.Transferred species is in Amp⁺In LB culture fluid, 37 DEG C of shaking table overnight incubation.

2. the blue white macula plasmid of plasmid DNA extracting (using Omega company plasmid extraction test kit) extracting respectively

Taking bacterium solution to be sub-packed in 1.5ml centrifuge tube, 12000g is centrifuged 3 minutes, leaves and takes precipitation.

Often pipe adds 100 μ l solution I suspensions, mixing of fully vibrating.

Add 100 μ l solution II, softly mix, ice-water bath 5 minutes.

Adding 250 μ l solution III, mixing of gently shaking, room temperature is placed 10 minutes.

4 DEG C, 12000g be centrifuged 10 minutes, move to supernatant separate in pipe.

12000g is centrifuged 1 minute, topples over the waste liquid in collecting pipe.

Add 500 μ l lavation buffer solutions in separating in pipe, ibid centrifugal and discard the waste liquid in collecting pipe.

Repeat previous step.

12000g is centrifuged 1 minute, makes ethanol volatilize completely.

Separation pipe is placed in another clean EP pipe and adds a certain amount of TE buffer, 65 DEG C of water-baths 5 points Clock, 12000g is centrifuged 1 minute.

Taking a certain amount of eluent and carry out electrophoresis, remaining is placed in-20 DEG C and saves backup.

3. enzyme action is identified: respectively locus coeruleus plasmid DNA and white macula plasmid DNA are carried out double digestion.(limit Property restriction endonuclease purchased from Dalian TaKaRa company)

Table 2

Locus coeruleus plasmid DNA	1μl
		BamHI₂	1μl
10 × buffer (K)	1μl
		ddH₂O	7μl
Cumulative volume	10μl

Table 3

Recombinant plasmid dna	5μl
		NdeI or NcoI	0.5μl
XhoI	0.5μl
		10 × buffer (K)	1μl
ddH₂O	3μl
		Cumulative volume	10μl

After mixing, 37 DEG C of water-baths 4 hours.

The sequence analysis of 5.PCR product

TA is cloned positive transformants bacterial strain and delivers to Huada Gene Research Center, Beijing, extract matter according to a conventional method Grain, uses double deoxidation chain termination method, Insert Fragment is carried out sequencing.

(2) recombinant gene expression plasmid and the structure of efficient expression engineering and screening

1. the structure of recombiant plasmid

By the pMD18T-Glu plasmid containing genes of interest and empty carrier pPAL7 plasmid double digestion, digestion products Reclaim after purification through 1.0% sepharose electrophoresis, purpose fragment glue, connect with ligase, convert escherichia coli DH5 α, extracts plasmid, double digestion, and 1.0% agarose gel electrophoresis is identified.

Relevant operation specifically comprises the following steps that

1.1 plasmid DNA extractings (use Omega company plasmid extraction test kit, by test kit operation instruction Book operates)

(1) bacterium colony transferred species single on picking flat board is in in the LB culture fluid of corresponding antibiotic, 37 DEG C of shaking tables Overnight incubation；

(2) taking bacterium solution to be sub-packed in 1.5mL centrifuge tube, 12000g is centrifuged 3 minutes, leaves and takes precipitation；

(3) often pipe adds 100 μ L solution I suspensions, mixing of fully vibrating；

(4) add 100 μ L solution II, softly mix, ice-water bath 5 minutes；

(5) adding 250 μ L solution III, mixing of gently shaking, room temperature is placed 10 minutes；

(6) 4 DEG C, 12000g be centrifuged 10 minutes, move to supernatant separate in pipe；

(7) 12000g is centrifuged 1 minute, topples over the waste liquid in collecting pipe；

(8) 500 μ L lavation buffer solutions are added in separating in pipe, ibid centrifugal and discard the waste liquid in collecting pipe, Repeated washing is once；

(9) 12000g is centrifuged 1 minute, makes ethanol volatilize completely；

(10) separation pipe is placed in another clean EP pipe and adds a certain amount of TE buffer, 65 DEG C of water-baths 5 minutes, 12000g was centrifuged 1 minute；

(11) taking a certain amount of eluent and carry out electrophoresis, remaining is placed in-20 DEG C and saves backup.

1.2 agarose gel electrophoresiies:

1.0% agarose gel, 1 × TAE buffer, 120-150mA, electrophoresis 20-40 minute.

50 × TAE stores formula of liquid: 2.0mol/L Tris alkali, 1.0mol/L NaAc, 0.1mol/L Na₂EDTA；PH8.3 is regulated with glacial acetic acid.

The endonuclease reaction of 1.3 plasmid DNA:

1 μ g plasmid DNA

1 μ l 10 × K buffer (see the raw work Products description in Shanghai)

1 μ l restricted enzyme BamH I/Not I(10u/ μ l)

Mend to 10 μ l with distilled water

Mix rear 37 DEG C of incubations 1-2 hour.

The target DNA recovery purification of 1.4 sepharose electrophoresis glue:

The target DNA electrophoresis band observed under uviol lamp and cut on agarose gel, moves into 1.5mL EP Pipe.

The DNA adding Omega company glue recovery test kit combines buffer, and 65 DEG C of water-baths make gel the most molten Change and keep pH value of solution between 5.0～6.0.Being moved into by sol solutions and separate pipe, 12000g is centrifuged 1 minute, Discard the liquid in collecting pipe.

Adding supporting lavation buffer solution, 12000g is centrifuged 1 minute, discards the liquid in collecting pipe.Repeat Wash 1 time.

12000g is centrifuged 1 minute, separates another clean 1.5mL EP of pipe dislocation pipe, adds certain volume TE buffer, hatches 10 minutes for 65 DEG C, and 12000g is centrifuged 1 minute, takes a certain amount of electrophoresis, and UVP is purple Purification effect is reclaimed in the detection of outer scanner.

1.5 coupled reactions (use Shanghai Sheng Gong company DNA ligation kit)

By UV spectrophotometer measuring target DNA fragment and the concentration of carrier segments, according to exogenous sequences With the principle that carrier mole ratio is generally 1:2～10, design coupled reaction system is as follows:

Target DNA 1 μ l；Plasmid vector 1～2 μ l；Connect solution 5 μ l；ddH₂O2～3 μ l, always Volume 10 μ l.22 DEG C connect 12-16 hour.

The preparation (CaCl2 method) of 1.6 competence bacterias

(1) aseptic inoculation ring dips-70 DEG C of frozen antibacterial conservation liquid, trilinear method streak inoculation in LB flat board, Cultivate 12～16h for 37 DEG C.

(2) the single colony inoculation of picking is in 2mL LB culture fluid, and 37 DEG C of shaking tables cultivate 12～16h.

(3) by the BL21 of incubated overnight in 1% ratio transferred species to LB culture fluid, 37 DEG C of shaking tables are cultivated extremely OD₆₀₀During to 0.2～0.4,8000g is centrifuged 5 minutes and collects antibacterial.

(4) the 0.1mol/L CaCl of 1ml pre-cooling is added₂Resuspended precipitation, ice-water bath 3 hours.4℃8000g Centrifugal 5 minutes, abandon supernatant.Add the 0.1mol/L CaCl of 100 μ l pre-coolings₂Suspend precipitation, ice-water bath 1 Hour, standby.

1.7 connect product converts

(1) take competence bacterium solution 100 μ l, add coupled reaction product；Ice-water bath 60 minutes, 42 DEG C of water-bath heat Shock 100s, rapid placement ice-water bath 1～2 minutes.

(2) adding 100 μ l LB culture fluid, 37 DEG C of shaking tables are cultivated 1 hour.

(3) it is centrifuged 10 minutes with 8000g, inhales and abandon mixing precipitation after 100 μ l supernatants, respectively take 50 μ l coatings flat Plate, 37 DEG C of incubator overnight incubation.

The Screening and Identification of 1.8 positive engineering bacterias

(1) picking converts the cultivation of flat board list bacterium colony

(2) extracting plasmid (method is the same)

(3) double digestion identifies (method is the same)

2. the efficiently induction of expressed fusion protein engineering bacteria and protein expression

The recombinant bacterium identified of learning from else's experience is inoculated in the 10ml LB culture fluid containing Amp, and 37 DEG C of shaking tables are carried out once Activate 10 hours.By the recombination engineering that once activates in 1% ratio transferred species in the 1L LB containing Amp In culture fluid, 37 DEG C of shaking tables carry out re-activation 5 hours.Bacterium after re-activation is connect by the ratio in 10% Planting in 10L fermentation tank, culture medium is TB, ferments, and cultivates to bacteria log trophophase with IPTG for 37 DEG C Abduction delivering 4 hours, the expression of SDS-PAGE detection fusion albumen.

Embodiment 2Height crushes bacterium, is centrifuged

1. by the engineered E. coli of the expression Streptococcus mutans recombinant subunit engineered protein Glu of structure Bacterium, by fermentation, target protein expression rate is 26%, and centrifugal collection thalline is standby.

2. thalline 200-500g is with PBS (10-20mM, pH7.0-7.5) buffer, by weight: volume ratio 1: 10 ratio mixings suspend, 4 DEG C of pre-coolings.

3. high pressure homogenizer: use distilled water flushing high pressure homogenizer pipeline, cold cycle system opens pre-cooling Standby to 1-4 DEG C.

4. the suspension bacteria liquid of pre-cooling adds high pressure homogenizer, and pressure maintains 60-80Mpa and breaks bacterium 3-5 time, takes Broken bacterium solution smear row violet staining, under oil mirror under each visual field unbroken bacterium to be considered as brokenly bacterium less than 1-2 complete Entirely.

5. differential centrifugation: the liquid after broken bacterium loads centrifugal barrel, and 4 DEG C, 750g is centrifuged 30min, collects supernatant.

6. high speed centrifugation: centrifugal supernatant loads centrifugal barrel, and 4 DEG C, 13000g is centrifuged 30min, collects precipitation.

Embodiment 3Inclusion body washing, cracking

1. the pH7.0-7.520mM PB that centrifugation contains 1% triton x-100 with 9 times of volumes is delayed Rush liquid resuspended, and after fully mixing with high speed disperser, by agitator gentle agitation 30min, then with 10000 × g Centrifugal 30min, collects precipitation, is repeated once；

2. the pH7.0-7.520mM PB buffer with 9 times of volumes, centrifugation being contained 2M carbamide is resuspended, And after fully mixing with high speed disperser, by agitator gentle agitation 15min, then be centrifuged with 10000 × g 30min, collects precipitation, is repeated once；

3. weigh precipitation weight in wet base, by weight: volume ratio 1:10 ratio adds the PB containing 8M carbamide (10-20mM, pH7.0-7.5) buffer, 4 DEG C of stirring and dissolving overnight, 13000g high speed centrifugation 30 minutes, Collect supernatant.

Embodiment 4The preliminary renaturation of albumen

1. configuration PB (10-20mM, the pH7.0-7.5) buffer containing 2M carbamide is as basis renaturation buffering Liquid, is loaded in beaker, is placed on the magnetic stirring apparatus of 4 DEG C of environment and is stirred vigorously；

2. the inclusion body protein solution 1ml dissolved in Example 3 is dropwise slowly added into above-mentioned being stirred vigorously Buffer in, make protein solution moment dilution refolding, the final concentration of protein after dilution controls About 0.05-0.1mg/ml；

3., by the protein sample 13000g high speed centrifugation 30 minutes of dilution refolding, supernatant is preliminary renaturation Protein sample.

Embodiment 5 affinitive layer purification

1. select Profinity eXact purification media filling chromatographic column, use containing 2M carbamide PB (100mM, pH7.0-7.5) buffer balance layer analysis system and chromatographic column, treat conductance and UV Exponential Stability Rear loading collection flow through；

2. after end of the sample, use PB (300mM, the pH7.0-7.5) buffer containing 2M carbamide to wash post, to go Except the foreign protein with chromatographic column non-specific binding, till UV280 no longer declines；

3. by the PB (100mM, pH7.0-7.5) containing 2M carbamide and 100mM NaF of a column volume Buffer adds in chromatographic column, and retains 30-60min, be allowed to fully to induce affinity tag and destination protein it Between enzyme action；

4. use PB (100mM, pH7.0-7.5) the buffer solution elution chromatographic column containing 2M carbamide, restructuring Glu albumen i.e. elutes from chromatographic column, collects the elution samples after cutting, and this sample is final mesh Albumen；

5. finally peel off Profinity with PB (100mM, the pH7.0-7.5) buffer containing 100mM phosphoric acid Affinity tag on eXact medium thus regenerate chromatographic column, chromatographic column is with containing 0.02% sodium azide PB (100mM, pH7.0-7.5) buffer preserving.

Embodiment 6Dialysis concentrates

1. purification Glu sample embodiment 5 collected is placed in the bag filter of MW10000, sealing two ends After be suspended in the carbamide PB containing 1mol (100mM, pH7.0-7.5) dialysis buffer liquid, 4 DEG C stirring dialysis 10 Hour；

Being changed to by bag filter in the most urea-containing PB (100mM, pH7.0-7.5) buffer, 4 DEG C are stirred the most again Mix dialysis 10 hours；

3. take out bag filter, use high molecular polymer such as Polyethylene Glycol (carbowax), polyvinylpyrrolidone etc. Or the adsorbent such as sucrose is sprinkling upon outside bag filter, being placed at 4 DEG C, the solvent in bag filter oozes out and is i.e. adsorbed agent and inhales Attached, after 20 times of volumes of volume concentration, take out.

4. the method for hollow fiber column be may be used without for the dialysis of large volume purification of samples and concentration process to enter OK.

Embodiment 7Add cosolvent

Purification of samples after embodiment 6 being concentrated adds L-arginine (glycerol, the sea of the allowed content of pharmacopeia Algae sugar or PEG etc.) after cosolvent is sufficiently stirred for, i.e. the antigen of purification, 4 DEG C preserve and carry out SDS-PAGE Electroresis appraisal.

Embodiment 8Rat immunity is tested

1. prepared by immunogen

Take restructuring Glu albumen and LTB respectively, use PBS(phosphate buffer, pH7.0) be diluted to required Concentration is standby, and its final concentration is respectively as follows:

Table 4

Glu(mg/ml)	LTB(mg/ml)	Mass ratio
			10	1	10:1
5	1	5:1
			1	1	1:1

2. immune rat

Take 3 week old female SPF Waster rat 120, be randomly divided into 5 groups, 30/group.Specifically It is divided into and tests I group: Glu200 μ g+LTB20 μ g；Experiment II group: Glu100 μ g+LTB20 μ g；Experiment III group: Glu20 μ g+LTB20 μ g；Adjuvant group: LTB20 μ g；PBS group: PBS.Use Nasal immunization, 20 μ l/ Mus.Immunity 1 time weekly, totally 3 times.

3. antibody test

(1) saliva antigenic specificity sIgA detection

After last 2 weeks, lumbar injection 50 μ l(5 μ g) pilocarpine nitrate, collect secretion after 3～5 minutes Saliva.Taking rat saliva about 0.3ml, 14000g is centrifuged 10min, takes supernatant.Use antibody diluent 1:5 Dilution saliva sample, is coated (100ng/ hole) ELISA Plate by 100 μ l/ hole addition restructuring Glu albumen, carries out ELISA detects (the goat anti rat IgA of HRP labelling, BETHYL, Code:A110-102P).

Through statistical analysis (t inspection), vaccine immunity group compares with adjuvant group and PBS group, vaccine immunity group Saliva antigenic specificity sIgA level significantly raises (P < 0.01).

(2) serum antigen specific IgG detection

After last 2 weeks, tail venous blood sampling, separate serum.Serum to be checked is diluted with antibody diluent 1:200 times, Carry out ELISA detection (the goat anti rat IgG of HRP labelling, ZSGB-BIO, Code:ZB-2307).

Through statistical analysis (t inspection), vaccine immunity group compares with adjuvant group and PBS group, vaccine immunity group Serum antigen specific IgG level significantly raises (P < 0.01).

Although the present invention discloses as above with preferred embodiment, so it is not limited to the present invention, Ren Hesuo Belong to those skilled in the art, without departing from the spirit and scope of the invention, when making a little change With improvement, therefore the protection domain of the present invention is when being as the criterion depending on as defined in claim.

Claims

1. the purification process of a Streptococcus mutans recombinant subunit recombinant vaccine candidate antigens Glu, it is characterised in that the aminoacid sequence of this antigen is as shown in SEQ ID NO:1, and its purification process comprises step:

1) thalline, broken bacterium are collected: collect the coli somatic of the Streptococcus mutans recombinant subunit recombinant vaccine Glu albumen of fermentation, high-pressure homogenization is used to break bacterium, collect supernatant, centrifugal supernatant, collect precipitation, particularly as follows: the coli somatic of the Streptococcus mutans recombinant subunit recombinant vaccine Glu albumen of fermentation is suspended in the mixing of 1:9 ratio with the 10-20mmol/L PBS buffer of pH 7.0-7.5,4 DEG C of pre-coolings, 60-80MPa high-pressure homogenization breaks bacterium, sample after Po Sui is centrifuged 30 minutes through 750 × g, collecting supernatant, supernatant is again with 13000 × g Centrifugal 30 minutes, collect precipitation；

2) inclusion body washing: the precipitation that step 1) is collected, resuspended with PBS, washing repeatedly, is then centrifuged for, and collects precipitation, particularly as follows: the PBS that with 9 times of volumes, centrifugation is contained 1%TRITON X-100 is resuspended, fully mixing, by agitator gentle agitation 30 minutes, then is centrifuged 30 minutes with 10000 × g, abandoning supernatant, precipitation repeats and washed once；The PBS that with 9 times of volumes, centrifugation is contained 1mol/L carbamide is resuspended, fully after mixing, abandons supernatant, and precipitation repeats and washed once, and collected after centrifugation precipitates；

4) preliminary renaturation: take urea-containing PB buffer, solubilization of inclusion bodies liquid is dropwise slowly added in the buffer of above-mentioned stirring, centrifugal, abandon precipitation, column purification on supernatant；Particularly as follows: take the buffer containing 2M carbamide pH 7.0 100mmol/L PB of certain volume, it is placed in agitator to be stirred vigorously, solubilization of inclusion bodies liquid is dropwise slowly added in the buffer of above-mentioned stirring, make protein solution moment dilution refolding, final concentration of protein after dilution controls at 0.05-0.1mg/ml, 13000g is centrifuged 30 minutes, abandons precipitation, column purification on supernatant；

5) affinitive layer purification: select protein purification system, under urea-containing PB buffer conditions, destination protein is purified, after destination protein is combined with filler, carry out enzyme action eluting with the PB buffer of containing sodium fluoride, collect eluent and be the Glu albumen of chromatography purification；Described filler is Profinity eXact fusion tag system；

6) dialysis concentrates: be placed in bag filter by the Glu albumen of purification, sealing, is suspended in equipped with in urea-containing PB buffer, and stirring is dialysed, and takes out bag filter high molecular polymer and is sprinkling upon outside bag filter, after volume concentration, takes out；

Method the most according to claim 1, it is characterised in that step 3) is particularly as follows: every gram of inclusion body 19ml PBS containing 8M carbamide is resuspended, and 4 DEG C of gentle agitation 12h, 13000G is centrifuged 30 minutes, abandons precipitation, supernatant 4 DEG C preservation.

Method the most according to claim 1, it is characterized in that, step 5) is particularly as follows: select Profinity eXactTM protein purification system to be purified, use and under the pH 7.0 100mmol/L PB buffer conditions containing 2mol/L carbamide, target protein is purified, after destination protein is combined with filler, carry out enzyme action eluting with the PB buffer containing 100mmol/L sodium fluoride, collect eluent and be the Glu albumen of chromatography purification.

Method the most according to claim 1, it is characterised in that step 6), particularly as follows: be placed in the bag filter of MW10000 by the Glu albumen of purification, sealed, is suspended in equipped with the 100mmol/L containing 1mol carbamide pH 7.0 In PB buffer, 4 DEG C of stirrings are dialysed 10 hours；Changing buffer is the most urea-containing 100mmol/L PB buffer, and 4 DEG C of stirrings are dialysed 10 hours；Taking out bag filter high molecular polymer or sucrose is sprinkling upon outside bag filter, after 20 times of volumes of volume concentration, take out, described high molecular polymer is Polyethylene Glycol or polyvinylpyrrolidone.