CN102772792A - Vaccine for preventing decayed tooth and preparation method thereof - Google Patents

Abstract

The invention discloses a vaccine for preventing decayed tooth and a preparation method thereof. Active ingredients of the vaccine for preventing a decayed tooth are proteins shown as 1) or 2) or 3): wherein 1) a protein is composed of amino acid sequences shown as a sequence 2 in a sequence table1; 2) a protein is derived by 1), relative to the prevention of the decayed tooth and formed by the fact that one or more amino acids is/are substituted and/or lost and/or added on amino acid sequences of the sequence 2 in the sequence table; and 3) a protein is composed of amino acid sequences shown as 9th position to 419th position of the sequence 2 in the sequence table. The vaccine has the advantages that the vaccine has a solubility expression and is easy to purify, the purity is high, the preparation method is simple and convenient and the like, and besides, the vaccine has remarkable economic benefits and can be used as a candidate vaccine for preventing the decayed tooth caused by streptococcal infections.

Description

Vaccine of a kind of caries prevention and preparation method thereof

Technical field

The present invention relates to the medical biotechnology field, particularly vaccine of a kind of caries prevention and preparation method thereof.

Background technology

Dental caries is one of the most general human disease, and sickness rate is high, and WHO is listed as the mankind's three big keypoint control diseases (Petersen PE.Community Dent Oral Epidemiol.2003,31Suppl 1:3-23.) with itself and cancer and cardiovascular disease.In " the global oral health report " of the up-to-date issue of World Health Organization (WHO); (Daniel J S.2010 to estimate have 5,000,000,000 people to suffer from dental caries by the whole world 6,300,000,000 populations; 9 (1): 1-3), 60%～90% school age population and most adults were suffered from dental caries in developed country, and adult's sickness rate is 50% in China; The child is then up to 70%～90%, and it becomes the global problem that threatens human oral health already.

Dental caries is to be the destructive disease of dental hard tissue's chronic progressive external under the multifactor combined effect of leading with the oral microorganism.Antibacterial only just causes the dental caries effect after forming dental plaque, and carbohydrate sucrose particularly in the food is the main cause that causes dental caries.Especially adhesive food is attached on the tooth, and the oral bacterial fermentation produces acid, thereby makes the enamel top layer inorganic matter dissolving of tooth, enamel and the continuous mineralising of dentin inorganic matter, and the soft thereupon forfeiture of hard tooth tissue forms cavity then.Streptococcus mutans (S.Mutans) is the human main dental caries microorganism that causes, the generation of its field planting and dental caries in the oral cavity closely related (Zhang Zhi is willing to. " Oral Science ", People's Health Publisher, the 7th edition,, 48 in 2008; Yue Songling. " modern cariology ", scientific and technical literature publishing house, 2009,53-54.).

Have now and mainly take fluoride, antibiotic and limiting sugar etc. for the prevention of dental caries.The fluoride preventing decayed tooth is through suppressing demineralization, promote remineralization to disturb the cariogenic bacteria metabolism, suppresses the multiple metabolic enzyme of cariogenic bacteria and reaches anti-caries effect.But the use of fluoride has potential toxicity, and unsuitable preschooler uses.Antibiotic can reach the effect of control bacterial plaque, but life-time service exists drug resistance and toxic and side effects, and oral microorganism is not had the inhibition of selection, also suppresses probiotics simultaneously the kill harmful bacterium, generally not as first-selected preventing decayed tooth method.The absorption of control sugar and the use of sugar substitute also can be played preventive effect, though reduce the dental caries incidence rate to a certain extent, can not fundamentally block dental caries and take place.Therefore develop a kind of safe and effective dental caries vaccine and seemed particularly necessary.

Since the eighties in 20th century, people just begin to prepare the subunit anti-caries vaccine with effective antigenic component of Streptococcus mutans.Though traditional deactivation killed vaccine and attenuated live vaccine can suppress the adhesion of Streptococcus mutans to facing; But antibody and the heart nephridial tissue of full cell induction have cross reaction, and attenuated live vaccine has the insufficient or virulence answer of attenuation then can be caused and all be difficult to be applied to clinical to the injury of body.Existing multiple anti-caries vaccine (like Streptococcus mutans protein vaccine, polypeptide vaccine and nucleic acid vaccine) is conducting a research, but up to now, does not still have the dental caries vaccine listing that prevention S.Mutans infects field planting both at home and abroad.

The multiple anti-caries vaccine that is the basis with S.Mutans antigen can significantly reduce animal dental caries incidence rate; Antigen mainly contains: surface protein antigen I/II (Ag I/II), glucosyltransferase (GTF) and glucan-binding protein antigen molecule (Hajishengallis G.Infect Immun such as (GBP); 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 DJ.Infect Immun, 2005,73 (5): 2797-2804.).SMU 862 is a kind of albumen that the S.Mutans genome sequencing is found, Unknown Function, and theoretical prediction is a memebrane protein, is assumed to hyaluronidase.This protein sequence high conservative, in S.Mutans UA159 that accomplishes genome sequencing and S.Mutans NN2025 bacterial strain, similarity reaches 99%, and in S.Mutans LJ23 bacterial strain, similarity reaches 100% (NCBI Blast); Simultaneously specificity is high, all is lower than 55% with the similaritys of other antibacterials.It is not appeared in the newspapers as antigenic research.

Anti-caries vaccine needs to induce body in saliva, to continue to produce specificity IgA antibody, influences the stick field planting of S.Mutans at facing, thus the generation of prevention dental caries.Single soluble protein antigen or polypeptide antigen are difficult to reach this purpose, must be aided with the adjuvant that can effectively bring out mucosa-immune and come enhancing body to be directed against antigenic immunne response ability, could produce a large amount of secretory IgAs, obtain effective immune protective effect.Cholera toxin (CT) and E.coli LT (LT) have been proved has very strong mucosa-immune adjuvant effect, but is restricted using because of its toxicity.

In prokaryotic expression system, the application of glutathione s-transferase (GST) expression and purification system is very general in recent years, and its source is the GST albumen of the 25kDa size of Schistosoma japonicum.The GST tag system has high, the expression product convenient purification of protein expression productive rate, and is beneficial to characteristics and advantage such as GST Antibody Preparation.Gst fusion protein is solvable in aqueous solution, can from bacterial lysate, extract, and under the condition of invariance, obtains through affinity chromatograph.Gst fusion protein can be by the locus specificity protease cracking, thereby removes GST albumen.Just because of above advantage, commercial gst fusion protein expression system and GST tag antibody system still are widely used so far.

Summary of the invention

An object of the present invention is to provide a kind of vaccine of caries prevention.

The vaccine of caries prevention provided by the present invention, its active component are following 1) or 2) or 3) shown in protein:

1) protein that the aminoacid sequence shown in the sequence 2 is formed in the sequence table;

2) in sequence table the aminoacid sequence of sequence 2 through replacement and/or disappearance and/or add one or several aminoacid and relevant with caries prevention by 1) deutero-protein;

3) protein that the aminoacid sequence shown in the 9th to the 419th is formed in the sequence 2 in the sequence table.

Said vaccine also comprises adjuvant; Said adjuvant is a mucosal adjuvants.

Said mucosal adjuvants is LT _S63K

Said vaccine is by albumen and the LT shown in the sequence in the sequence table 2 _S63KAdjuvant is formed, said albumen and said LT _S63KThe mass ratio of adjuvant is 1:1 ~ 25:1, preferred 5:1.

Said dental caries is by the dental caries due to the S.Mutans infection.

Another object of the present invention provides the method for preparing of the vaccine of caries prevention.

The method for preparing of the vaccine of caries prevention provided by the present invention is characterized in that: with following 1) or 2) or 3) shown in protein as active component, and the acceptable adjuvant of medicine combination:

1) protein that the aminoacid sequence shown in the sequence 2 is formed in the sequence table;

2) in sequence table the aminoacid sequence of sequence 2 through replacement and/or disappearance and/or add one or several aminoacid and relevant with caries prevention by 1) deutero-protein;

3) protein that the aminoacid sequence shown in the 9th to the 419th is formed in the sequence 2 in the sequence table.

The method for preparing of the vaccine of said caries prevention comprises the steps:

(1) through PCR method, from S.mutans UA159 genome amplification smu_862 genetic fragment;

(2) said smu_862 genetic fragment is building up on the expression vector that contains gst gene, forms fusion gene with GST, order-checking is identified, is obtained recombiant plasmid;

(3) said recombinant plasmid transformed is expressed bacterium, the abduction delivering recombination fusion protein is also identified;

(4) the said recombination fusion protein of purification is through obtaining vaccine antigen protein behind the enzyme action removal GST purification tag;

(5) with said vaccine antigen protein and mucosal adjuvants LT _S63KMix, promptly obtain the vaccine of said caries prevention.

The expression vector that contains gst gene in the said step (2) is the pGEX-6P-2 plasmid;

Expression bacterium in the said step (3) is Ecoli BL21 (DE3).

Another purpose of the present invention provides a kind of albumen and in the vaccine of preparation caries prevention, uses.

A kind of albumen provided by the present invention is used in the vaccine of preparation caries prevention, and said albumen is following 1) or 2) or 3) shown in:

1) protein that the aminoacid sequence shown in the sequence 2 is formed in the sequence table;

2) in the sequence table aminoacid sequence of sequence 2 through replacement and/or disappearance and/or add one or several aminoacid and relevant with caries prevention by 1) deutero-protein;

3) protein that the aminoacid sequence shown in the 9th to the 419th is formed in the sequence 2 in the sequence table.

Said dental caries is by the dental caries due to the S.Mutans infection.

The recombinant protein vaccine of dental caries due to prevention S.Mutans provided by the present invention infects; With a kind of S.Mutans memebrane protein SMU 862 as antigen; Said recombiant protein derives from Streptococcus mutans memebrane protein SMU_862; Adopt gene engineering method to remove and stride the film district, keep the film outskirt, have immunity protection function.Said SMU_862 recombiant protein aminoacid sequence is shown in sequence in the sequence table 2 shown in the 9th to the 419th.

The present invention clones recombination through molecular biology method from S.Mutans UA159, make up recombinant expression plasmid, expresses and obtains recombination fusion protein, removes purification tag through the enzyme action mode behind the fusion rotein purification.Can find out that from the result of embodiment 2 this recombination fusion protein has good immunogenicity, can the stimulation test Mus produce the specific immune response of anti-SMU_862, and Al (OH) ₃Injection groups and PBS group do not produce the antibody of anti-SMU_862.

Those skilled in the art can construct recombiant plasmid, the engineering bacteria of express recombinant protein vaccine according to gene order provided by the invention, are used for the recombinant protein vaccine of production requirement protection of the present invention.

Because the recombinant protein vaccine of dental caries was mainly used in prevention S.Mutans and infects due to prevention provided by the invention S.Mutans infected, and considered that the mucosa-immune adjuvant is used for the toxicity problem of human body, so the present invention preferably adopts to be proved to be and is LT nontoxic and that tool is higher _S63KAs adjuvant.With recombiant protein SMU_862 antigen and LT _S63KThe vaccine immunity rat of adjuvant preparation; Rat can generate specific antibody (Fig. 9 of anti-S.Mutans; 10) and antigen-specific antibodies secretory cell (Figure 11); And can reduce rat dental caries incidence rate and dental caries and decrease degree (table 1, table 2), so the present invention can be used as prevention S.Mutans infect due to the candidate vaccine of dental caries.

In sum, the present invention utilizes S.Mutans memebrane protein SMU_862 as antigen, through GST expression and purification systems produce vaccine antigen, with LT non-toxic mutant LT _S63KAs adjuvant, obtain preventing S.Mutans infect due to the vaccine of dental caries.This vaccine can effectively be induced the body mucosal immune response through the mucosal route immunity, produces specificity IgA antibody.This vaccine has solubility expression, is prone to advantages such as purification, purity is high, method for preparing is easy, has remarkable economic efficiency.Can be used as the prevention Streptococcus mutans infect due to the candidate vaccine of dental caries.

Description of drawings

Fig. 1 is pcr amplification SMU_862 agarose gel electrophoresis figure;

Wherein swimming lane l is that (TIANGEN, MD115), swimming lane 2 is a SMU_862 PCR product (1263bp) to nucleic acid (DNA) molecular weight standard.

Fig. 2 is the enzyme action qualification result figure of Sma I and Not I double digestion SMU_862 DNA and pGEX-6P-2 plasmid;

Wherein swimming lane l be nucleic acid (DNA) molecular weight standard (Thermo, #SM0311/2/3*), swimming lane 2 is a pGEX-6P-2 plasmid double digestion product (4971bp), swimming lane 3 is a smu_862 double digestion product (1241bp).

Fig. 3 is the enzyme action qualification result that Sma I and Not I double digestion are identified recombiant plasmid;

Wherein swimming lane l is that (Thermo, #SM0311/2/3*), swimming lane 2 is a recombiant plasmid pGEX-6P-2/smu_862 double digestion product (4971bp, 1241bp) to nucleic acid (DNA) molecular weight standard.

Fig. 4 identifies the figure as a result that recombination engineering is expressed for the SDS-PAGE electrophoresis;

Wherein swimming lane l be empty carrier contrast bacterium E coli BL21 (DE3) (pGEX-6P-2) induce before; Swimming lane 2 is induced 12h for empty carrier contrast bacterium, and swimming lane 3 induces 12h to break the bacterium supernatant for empty carrier contrast bacterium, and swimming lane 4 is induced the broken bacterium deposition of 12h for empty carrier contrast bacterium; Swimming lane 5 is protein molecular weight standard (Fermentas; #SM0671), swimming lane 6 for recombination engineering EcoliBL21 (DE3) (pGEX-6P-2/smu_862) induce before, swimming lane 7 is induced 12h for recombination engineering; Swimming lane 8 is induced 12h for recombination engineering and is broken the bacterium supernatant, and swimming lane 9 is induced the broken bacterium deposition of 12h for recombination engineering.Swimming lane 7,8 arrow indications are recombination fusion protein.

Fig. 5 is the proteic figure as a result of SDS-PAGE electrophoresis purification Identification;

Wherein swimming lane l ~ 3 are recombiant protein SMU_862 behind the PreScission Protease enzyme action, and swimming lane 4 is that (Fermentas, #SM0671), swimming lane 5 is fusion rotein GST-SMU_862 before the enzyme action to protein molecular weight standard.

Fig. 6 detects the immunoreactive figure as a result of recombiant protein for Western;

Swimming lane l ~ 3 are recombiant protein SMU_862.

Fig. 7 is that Nde I and BamH I double digestion are identified recombiant plasmid pET11c/LT _S63KThe enzyme action qualification result;

Wherein swimming lane l is that (Thermo, #SM0311/2/3*), swimming lane 2 is recombiant plasmid pET11c/LT to nucleic acid (DNA) molecular weight standard _S63KDouble digestion product (5.6kb, 1.2kb).

Fig. 8 identifies LT for the SDS-PAGE electrophoresis _S63KThe figure as a result of purifying protein;

Wherein swimming lane l is that recombination engineering induces 4h to break the bacterium supernatant, and swimming lane 2,4 is purification LT _S63KThe albumen sample-loading buffer does not contain DTT, and swimming lane 3 is purification LT _S63KThe albumen sample-loading buffer contains DTT, swimming lane 5 be protein molecular weight standard (Fermentas, #SM0671).

Fig. 9 is the result that saliva antigenic specificity sIgA detects;

Through statistical analysis (t check), vaccine immunity group saliva antigenic specificity sIgA level compares LT _S63KAdjuvant group and PBS group be rising (*: with LT significantly _S63KThe adjuvant group is compared, p<0.01; #: compare p with the PBS group<0.01).

The result that Figure 10 detects for the serum antigen specific IgG;

Through statistical analysis (t check), vaccine immunity group serum antigen specific IgG level compares LT _S63KAdjuvant matched group and the PBS matched group (*: that significantly raises with LT _S63KThe adjuvant group is compared, p<0.01; #: compare p with the PBS group<0.01).

The result that Figure 11 detects for the antigen-specific antibodies secretory cell

Through statistical analysis (t check), vaccine immunity group antigen-specific antibodies secretory cell (ASC) number compares LT _S63KAdjuvant group and PBS group be rising (*: with LT significantly _S63KThe adjuvant group is compared, p<0.01; #: compare p with the PBS group<0.01).

The specific embodiment

The antigen protein of the vaccine of dental caries due to embodiment 1, preparation prevention S.Mutans infect

One, pcr amplification smu_862 DNA

1. design of primers reaches synthetic

With " smu_862 " nucleotide sequence (812323..813030) design primer in the S.mutans UA159 genome (GenBank:AE014133.1) of GenBank announcement; Forward primer 5 ' end is introduced Sma I restriction enzyme site, and downstream primer 5 ' end is introduced the NotI restriction enzyme site.

Design of primers is following:

smu_862-F?5’-TCC

GCTATTTTGATAGTATTAGGCC-3’

Sma I restriction enzyme site

smu_862-R?5’-TTTTCCTTTT TCACCTCTTTTCATTAGCCTTA-3’

Not I restriction enzyme site

Primer is synthetic by Sangon Biotech (Shanghai) Co., Ltd..

2.PCR amplification target DNA

1) preparation of template

(TIANGEN, DP302) extracting S.mutans UA159 is (available from the American Type Culture Collection of Unite States Standard type culture collection institute, Streptococcus mutans with " bacterial genomes DNA extraction test kit " 700610 ^TM) genome, the operation by specification carries out.

2) pcr amplification

With S.mutans UA159 genomic DNA is template, uses smu_862-F and smu_862-R primer amplification smu_862 respectively.Adopt precious biological engineering (Dalian) company limited (RaKaRa) high-fidelity DNA polymerase

HSDNA Polymerase; Code:DR010A) carry out pcr amplification, reaction system is following:

The PCR reaction condition is: 94 ℃ of preparatory degeneration 5min; 94 ℃ of degeneration 30s, 60 ℃ of annealing 30s, 72 ℃ are extended 1min 30s, 30 circulations; 72 ℃ are extended 5min fully.Get 3 μ l PCR product after reaction finishes, 2.0% agarose gel electrophoresis detects (Fig. 1).Behind the agarose gel electrophoresis, reclaim the purpose fragment, obtain smu_862 DNA, its nucleotide sequence as in the sequence table 3 the 709th to shown in 1941.

Two, construction recombination plasmid pGEX-6P-2/smu_862

1. enzyme action

Respectively with the smu_862 DNA that obtains and pGEX-6P-2 plasmid (GE, Code:28-9546-50), with restriction endonuclease sma I (TaKaRa, Code:D1085A) and Not I (TaKaRa Code:D1166A) does double digestion, operates to specifications.The endonuclease reaction system is prepared as follows:

Mixing, 37 ℃ of enzyme action 1h.The enzyme action qualification result is as shown in Figure 2.Agarose gel electrophoresis reclaims 4.9kb carrier segments and 1.3kb SMU_862 genetic fragment respectively.

2. connect

Through UV spectrophotometer measuring smu_862 genetic fragment and pGEX-6P-2 carrier segments concentration; Be generally the principle of l:2～10 according to exogenous segment and carrier mole ratio; Carry out coupled reaction, the coupled reaction system (TaKaRa, Code:D6022A) as follows:

Smu_862 genetic fragment enzyme action reclaims product 4 μ l; The pGEX-6P-2 enzyme action reclaims product l μ l; Connect solution (Solution I) 5 μ l.Cumulative volume 10 μ l, 16 ℃ of coupled reactions 2 hours.

3. transform

To connect product, and Transformed E coli DH5a (available from TIANGEN, Code:CB101), LB plate screening positive recombinant.

4. evaluation recombiant plasmid

Single bacterium colony on the picking ammonia benzyl resistance screening flat board is cultivated, and the extracting plasmid is done double digestion with Sma I and Not I and identified, identifies that correctly genetic fragment (Fig. 3) is reclaimed in the back.Plasmid send Beijing Liuhe Huada Genomics Technology Co., Ltd's order-checking; The gene order of fusion gene (gst-smu_862) is shown in sequence in the sequence table 3; Be the gst gene sequence wherein from 5 ' the 1st to 660 of ends; The 661st to 684 is PreScission Protease restriction enzyme site; The 685th to 702 is the calling sequence for ease of plasmid construction and expression, and the 709th to 1941 is the smu_862 DNA sequence, its encoded protein sequence and expectation (seeing sequence 1 in the sequence table) in full accord.

Three, recombiant plasmid pGEX-6P-2/smu_862 Transformed E coli BL21 (DE3) and expression are identified

1. transform

Get recombiant plasmid pGEX-6P-2/smu_862, Transformed E coli BL21 (DE3) competent cell (, Code:CB105), carries out ammonia benzyl resistance LB plate screening positive recombinant available from TIANGEN by operating instruction.

2. the recombined engineering dientification of bacteria

Single bacterium colony on the picking ammonia benzyl resistance screening flat board is cultivated, and the extracting plasmid is done double digestion with Sma I and Not I and identified.After identifying correctly, express evaluation.

3. express and identify

1) recombination engineering induces

Get and identify that correct recombination engineering is inoculated in 10ml and contains in the LB culture fluid of 0.1mg/ml ampicillin, 37 ℃ of shaking tables are cultivated (200r/min) and are spent the night.Plant the recombination engineering of incubated overnight in 100ml LB culture fluid in 1% ratio commentaries on classics next day, and 37 ℃ of shaking tables are cultivated (200r/min) and treated that OD600 reaches at 0.6 ~ 0.8 o'clock, and the adjustment cultivation temperature is 16 ℃.Adding IPTG is that 0.5mmol/L induces to final concentration, induces 12 hours results, and SDS-PAGE detects Recombinant Protein Expression.Induce with the E coli BL21 (DE3) that only contains the pGEX-6P-2 plasmid simultaneously and do the empty carrier contrast.

2) SDS-PAGE identifies Recombinant Protein Expression

Get the bacterium liquid that 1m1 induced 12 hours, the centrifugal 2min of 12000g, the resuspended deposition of 100 μ l distilled waters, add 25 μ l5 * SDS-PAGE sample-loading buffer (Beyotime, Code:P0015), mixing, boiling water bath 5 minutes.SDS-PAGE electrophoresis (4% concentrates glue 10% separation gel); Coomassie brilliant blue staining; With albumen Marker (Fermentas; Code:#SM0671) the check analysis electrophoresis result is visible, and recombination engineering is induced can obtain the protein expression that conforms to the recombination fusion protein GST-SMU_862 theoretical molecular (71kd) that designs behind the 12h, and empty carrier contrast bacterium does not obtain this recombination fusion protein (Fig. 4) after inducing.

Four, recombinant protein purification and evaluation

1. recombinant protein purification

Get the centrifugal 5min of 1000ml bacterium liquid 5000g of abduction delivering, and deposition adding 100ml lysis buffer (90ml 10mM PBS (pH7.2-7.4, China fir Golden Bridge in Beijing, ZLI-9062); 10ml 10% Triton (Amresco, CAS 9002-93-1,0694-100ML); 1ml 100mM PMSF (Beyotime ST506-2), 20 μ l 100mM Hemisulfate Leupeptine (Applichem, Lot 8B011312); 100 μ l 75IU/mlAprotinin (Applichem, Code A2132,0100); 16 μ l10mM Pepstetin A (Applichem, Lot 0Q000719)), ice-water bath carrying out ultrasonic bacteria breaking; The centrifugal 15min of 12000g gets supernatant and 5ml Glutathione Sepharose 4B (GE, Code 10049253) and mixes, and 25 ℃ of vibrations combine 1 ~ 2h; With the mixed liquor gradation with the small-sized chromatographic column void column of Column (Sangon Biotech (Shanghai); Code SD6608) filters 100mlPBST (100ml 10mM PBS, 250 μ l Tween, 20 (Amresco; Lot 2042B201)) after the flushing; Reuse 50ml 10mMPBS flushing is put and is done filtrating, closes outlet; Add 10ml enzyme action liquid (10ml 10mM PBS, 500 μ l 500U Prescission ^TMProtease (GE Healthcare, Lot 4662320)), 4 ℃ of vibration enzyme action 12h; Open outlet, collect filtrating; Close outlet, add 10ml PBS, 25 ℃ of vibration 15min open outlet, collect filtrating.The filtrating that merges twice collection; The SDS-PAGE electrophoresis is identified; The result is as shown in Figure 5, and visible from figure, the recombiant protein behind the purification conforms to the recombiant protein theoretical molecular (44.6kd) of design; With the reorganization of the recombiant protein called after behind this purification SMU 862 albumen, its aminoacid sequence is shown in sequence in the sequence table 2.

2. antigenicity detects

1) Western detects the immunoreactivity of recombiant protein

Identify the proteic immunoreactivity of reorganization SMU_862 with the full bacterium antiserum of the anti-S.mutans of rabbit: SDS-PAGE electrophoresis (12% separation gel, 5% concentrates glue) SMU_862 protein sample, electricity be transferred to pvdf membrane (Roche company, Code:03010040001); 37 ℃ of sealings of 5% (W/V) defatted milk powder/TBST Buffer (20mmol/L Tris-HCl, 150mmol/L NaCl, 0.05% (V/V) Tween20) 2h; TBST Buffer rinsing 2min adds the anti-S.mutans serum of rabbit (acquisition of the full bacterium injecting immune of S.mutans deactivation rabbit) with 5% (W/V) defatted milk powder/TBST Buffer 1:500 dilution, 4 ℃ of slow shaken over night; TBSTBuffer rinsing 4 times, each 10min, add the HRP labelling that dilutes with 5% (W/V) defatted milk powder/TBST Buffer 1:5000 goat anti-rabbit igg (China fir Golden Bridge in Beijing, ZDR-5306), 37 ℃ are slowly shaken, hatch 1h; TBST Buffer rinsing 4 times; Each 10min; Drip

West Dura Extended Duration Substrate (Thermo Scientifc; Code#34075), film exposure sheet 15 seconds dries behind the developing fixing.The result shows that reorganization SMU_862 albumen and the full bacterium antiserum of the anti-S.mutans of rabbit have good immunoreactivity (Fig. 6).

2) zoopery detects the immunogenicity of recombiant protein

Get reorganization SMU_862 albumen 50 μ g+Al (OH) ₃100 μ g mixings, intramuscular injection immunity SD rat (available from big level ground hospital of Third Military Medical University aseptic sursery institute medical experiment animal center, SPF level, credit number: SCXK (Chongqing) 2007-0005), 1 time weekly, totally 3 times.21d after the last immunity, the socket of the eye vein is got blood, separation of serum.Encapsulate (100ng/ hole) ELISA Plate with reorganization SMU_862 albumen, ELISA detects the anti-SMU_862 protein I of SD rat blood serum gG antibody.Reorganization SMU_862 albumen+Al (OH) ₃Injection groups specific serum IgG tires and reaches 1:640, and 000, and Al (OH) ₃(phosphate buffer, pH7.0) group SMU_862 specific serum IgG level does not have significant change for injection groups and PBS.The result shows that reorganization SMU_862 albumen has good immunogenicity.

The vaccine of dental caries due to embodiment 2, preparation prevention S.mutans infect

One, preparation LT _S63K

(1) the synthetic LT of full gene _S63KGene

Synthetic by Sangon Biotech (Shanghai) Co., Ltd..LT _S63KGene order is a Nde I restriction enzyme site sequence from 5 ' the 1st to 6 of ends wherein shown in sequence in the sequence table 4, and the 7th to 60 is the LTA signal peptide sequence, and the 61st to 783 is LT _S63KThe A gene order, the 780th to 842 is the LTB signal peptide sequence, and the 843rd to 842 is the LTB gene order, and the 1155th to 1160 are BamH I restriction enzyme site sequence (seeing sequence 4 in the sequence table).

(2) recombiant plasmid pET11c/LT _S63KMake up

1. enzyme action

Respectively with the synthetic LT of full gene _S63K(NOVAGEN, Code:69438-3), (TaKaRa, Code:D1161A) (TaKaRa Code:D1010A) does double digestion, to specifications operation with BamH I with restricted enzyme Nde I for gene and pET11c plasmid.The endonuclease reaction system is prepared as follows:

Mixing, 37 ℃ of enzyme action 1h.Agarose gel electrophoresis reclaims 5.6kb pET11c carrier segments and 1.2kb LT respectively _S63KGenetic fragment.

2. connect

Through UV spectrophotometer measuring LT _S63KGenetic fragment and pET11c fragment concentrations are generally the principle of l:2～10 according to exogenous segment and carrier mole ratio, carry out coupled reaction, the coupled reaction system (TaKaRa, Code:D6022A) as follows:

LT _S63KProduct 4 μ l are received in the gene enzyme switchback; The pET11c enzyme action reclaims product l μ l; Connect solution (Solution I) 5 μ l.Cumulative volume 10 μ l, 16 ℃ of coupled reactions 2 hours.

3. transform

To connect product, Transformed E coli BL21 (DE3) competent cell (, Code:CB105), carries out ammonia benzyl resistance LB plate screening positive recombinant available from TIANGEN by operating instruction.

(3) recombination engineering E coli BL21 (DE3) (pET11c/LT _S63K) identify

Single bacterium colony on the picking ammonia benzyl resistance screening flat board is cultivated, and the extracting plasmid is identified (Fig. 7) with Nde I and BamH I double digestion.After identifying correctly, express evaluation.

(4) expression of recombinant proteins, purification and evaluation

1. recombination engineering is expressed

Get and identify that correct recombination engineering is inoculated in 10ml and contains in the LB culture fluid of 0.1mg/ml ampicillin, 37 ℃ of shaking tables are cultivated (200r/min) and are spent the night.Plant the recombination engineering of incubated overnight in 100ml LB culture fluid in 1% ratio commentaries on classics next day, and 37 ℃ of shaking tables are cultivated (200r/min) and treated that OD600 reaches at 0.6 ~ 0.8 o'clock, and adding IPTG is that 0.5mmol/L induces to final concentration, induces 4 hours results.

2. recombinant protein purification

Get the centrifugal 15min of bacterium liquid 5000g of abduction delivering, collect wet bacterium and weigh, the pH7.3TE buffer (50mmol/L Tris, 1mmol/L EDTA, 200mmol/L NaCl) that adds 10 times of bodies is resuspended.The ice-water bath carrying out ultrasonic bacteria breaking, the centrifugal 15min of 12000g gets supernatant.Last Immobilized D (+)-galactose affinity column (PIERCE, 20372) purification is fully after the flushing, with elution buffer (300mmol/L galactose (BIO BASIC INC, GB0215)+pH7.3TE) LT of elution of bound on post _S63K, collect eluting peak.The SDS-PAGE electrophoresis identifies that the result is as shown in Figure 8, and the result is as shown in Figure 8, confirms the LT of purification through the SDS-PAGE electrophoresis _S63KBe LTA monomer and part six aggressiveness and LTB oligomer (shown in the hollow arrow).Sample-loading buffer with containing DTT becomes LTA and LTB monomer after boiling processing.

Two, the vaccine production and the effect of dental caries due to prevention S.mutans infects

1. vaccine production

Reorganization SMU_862 albumen and LT _S63K(phosphate buffer, pH7.0) dilution is desired concn all to use PBS during preparation.Get reorganization SMU_862 albumen and LT respectively _S63KMixing is subsequent use, and its final concentration is respectively:

SMU_862（mg/ml）	LT S63K（mg/ml）	Mass ratio
			6.25	0.25	25:1
1.25	0.25	5:1
			0.25	0.25	1:1

2. vaccine immunity

Get 3 age in week 120 of female SPF SD rats, be divided into 5 groups at random, 30/group.Specifically be divided into experiment I group: SMU_862250 μ g+LT _S63K10 μ g; Experiment II group: SMU_86250 μ g+LT _S63K10 μ g; Experiment III group: SMU_86210 μ g+LT _S63K10 μ g; Adjuvant group: LT _S63K10 μ g; PBS group: PBS.The immunity of employing collunarium, 40 μ l/ Mus.Immunity is 1 time weekly, totally 3 times.

3.S.mutans counteracting toxic substances

Get S.mutans UA159 (available from ATCC, 700610) inoculation BHI culture medium (available from BD company, REF:237500), 37 ℃ of little aerobic cultivations 24h, adjusting bacterial concentration is 2.0 * 10 ⁹CFU dips in aseptic cotton carrier and gets S.mutans bacterium liquid, smears rat oral cavity and facing, inoculates every day 2 times, and each 30min at interval inoculates back 1 hour and prohibits drink fasting, vaccinization 3 days.

Begin to feed from counteracting toxic substances and cause dental caries feedstuff (whole wheat flour 6%, sucrose 56%, refine milk powder 28%, alfalfa leaf meal 3%, the full hepar siccatum 1% that dewaters, yeast 4%, salt 1%).

4. immunoprotection detects

(1) saliva antigenic specificity sIgA detects

The antibacterial counteracting toxic substances is after 8 weeks, and lumbar injection 50 μ l (5 μ g) pilocarpine nitrate is collected excretory saliva after 3 ~ 5 minutes.Get the about 0.3ml of rat saliva, the centrifugal 10min of 14000g gets supernatant.With antibody diluent 1:5 dilution saliva sample, add with reorganization SMU_862 albumen by 100 μ l/ holes and to encapsulate (100ng/ hole) ELISA Plate, carry out the ELISA detection (the goat-anti rat IgA of HRP labelling, BETHYL, Code:A110-102P).

Through statistical analysis (t check), vaccine immunity group and LT _S63KAdjuvant group and PBS group relatively, vaccine immunity group saliva antigenic specificity sIgA level significantly raise (Fig. 9).

(2) the serum antigen specific IgG detects

The antibacterial counteracting toxic substances is after 8 weeks, etherization, and the socket of the eye vein is got blood, separation of serum.Doubly dilute serum to be checked with antibody diluent 1:200, carry out ELISA detect (the goat-anti rat IgG of HRP labelling, ZSGB-BIO, Code:ZB-2307).

Through statistical analysis (t check), vaccine immunity group and adjuvant group and PBS group relatively, vaccine immunity group serum antigen specific IgG level significantly raise (Figure 10).

(3) the antigen-specific antibodies secretory cell detects

After 8 weeks of microbionation, etherization, rat is put to death in the cervical vertebra dislocation.Get Rat Parotid and submandibular gland, put 200 order steel meshes and grind.The physiological saline solution flushing, ficoll liquid isolated lymphocytes, the centrifugal 5min of 250g draws buffy coat, and normal saline 10ml/ is all over washing 3 times.Counting, with RPMI1640 culture fluid re-suspended cell, the adjustment cell concentration is 107/ml.Add the 96 porocyte culture plates that encapsulate with SMU_862 (400ng/ hole), 5%CO by every hole 0.1ml ₂Behind 37 ℃ of cultivations of incubator 4h, PBS washes 4 times.Every hole adds goat-anti rat IgA (1:500) antibody of 100 μ l HRP labellings, hatches 1h for 37 ℃, and PBST washes 4 times.Every hole adds 25 ℃ of lucifuge colour developings of 100 μ LAEC colour developing liquid 20min.The ELISPOT plate is dried naturally, place Biosys Bioreader to read automatically in the plate appearance, the speckle counting.

Through statistical analysis (t check), vaccine immunity group and adjuvant group and PBS group compare, and vaccine immunity group antigen-specific antibodies secretory cell quantity significantly increases (Figure 11).

(4) dental caries scoring

After 8 weeks of microbionation, etherization, rat is put to death in the cervical vertebra dislocation.Get head and put HCS pot processing 5 minutes, remove soft tissue, mandibular bone in the separation cleans the back drying at room temperature.Place the dyeing of 40g/L murexide solution to spend the night dried jawbone, thoroughly clean back room temperature lucifuge drying.Cut along occlusal surface middle-distant direction sheet, carry out the dental caries scoring under the stereomicroscope, each sample repeats 3 times, is accomplished by same experimenter.Press Keyes dental caries standards of grading, lower molar on the SD rat is divided into some facing units.Decrease degree according to dental caries and can be divided into level Four: (enamel E) refers to the bad enamel that only involves of dental caries to enamel caries; Slight carious dentin (DS) refers to that dental caries badly involve in enamel and the dentin outer 1/4; Moderate carious dentin (DM) refers to that dental caries badly involve between the outer 1/4-3/4 of enamel and dentin; Severe carious dentin (DX) refers to bad enamel and the dentin thickness 3/4 of involving of dental caries.For ease of statistics, lead each face of grinding one's teeth in sleep to be divided into some facing units up and down rat, decrease the damage envelope statistics according to the every facing dental caries of every experimental group and be different unitss, be the dental caries score value, with the dental caries score value addition of every identical facing of animal total dental caries branch of facing for this reason.

Through statistical analysis (t check), vaccine immunity group and adjuvant group and PBS group relatively, dental caries scoring there were significant differences (*: with LT _S63KThe adjuvant group is compared, p<0.01; #: compare p with the PBS group<0.01), (table 1).The result shows: vaccine immunity group rat dental caries incidence rate and the dental caries degree of decreasing significantly is lower than adjuvant group and PBS group, the effect that has caries prevention to take place.

Table 1 rat immunity and through the metainfective dental caries score value of S.mutans (total dental caries divide mean+SD)

*: with LT _S63KThe adjuvant group is compared, p<0.01; #: compare p with the PBS group<0.01

Claims (10)

1. the vaccine of a caries prevention, its active component is following 1) or 2) or 3) shown in protein:

1) protein that the aminoacid sequence shown in the sequence 2 is formed in the sequence table;

2) in sequence table the aminoacid sequence of sequence 2 through replacement and/or disappearance and/or add one or several aminoacid and relevant with caries prevention by 1) deutero-protein;

3) protein that the aminoacid sequence shown in the 9th to the 419th is formed in the sequence 2 in the sequence table.

2. vaccine according to claim 1 is characterized in that:

Said vaccine also comprises adjuvant; Said adjuvant is a mucosal adjuvants.

3. vaccine according to claim 2 is characterized in that:

Said mucosal adjuvants is LT _S63K

4. vaccine according to claim 3 is characterized in that:

Said vaccine is by albumen and the LT shown in the sequence in the sequence table 2 _S63KAdjuvant is formed, said albumen and said LT _S63KThe mass ratio of adjuvant is 1:1 ~ 25:1.

5. vaccine according to claim 4 is characterized in that: said dental caries is by the dental caries due to the S.Mutans infection.

6. the method for preparing of the said vaccine of claim 1 is characterized in that: with following 1) or 2) or 3) shown in protein as active component, and the acceptable adjuvant of medicine combination:

1) protein that the aminoacid sequence shown in the sequence 2 is formed in the sequence table;

2) in sequence table the aminoacid sequence of sequence 2 through replacement and/or disappearance and/or add one or several aminoacid and relevant with caries prevention by 1) deutero-protein;

3) protein that the aminoacid sequence shown in the 9th to the 419th is formed in the sequence 2 in the sequence table.

7. the method for preparing of the said vaccine of claim 5 comprises the steps:

(1) through PCR method, from S.mutans UA159 genome amplification smu_862 genetic fragment;

(2) said smu_862 genetic fragment is building up on the expression vector that contains gst gene, forms fusion gene with GST, order-checking is identified, is obtained recombiant plasmid;

(3) said recombinant plasmid transformed is expressed bacterium, the abduction delivering recombination fusion protein is also identified;

(4) the said recombination fusion protein of purification is through obtaining vaccine antigen protein behind the enzyme action removal GST purification tag;

(5) with said vaccine antigen protein and mucosal adjuvants LT _S63KMix, promptly obtain the vaccine of said caries prevention.

8. method according to claim 7 is characterized in that:

The expression vector that contains gst gene in the said step (2) is the pGEX-6P-2 plasmid;

Expression bacterium in the said step (3) is E coli BL21 (DE3).

9. an albumen is used in the vaccine of preparation caries prevention, and said albumen is following 1) or 2) or 3) shown in:

1) protein that the aminoacid sequence shown in the sequence 2 is formed in the sequence table;

2) in the sequence table aminoacid sequence of sequence 2 through replacement and/or disappearance and/or add one or several aminoacid and relevant with caries prevention by 1) deutero-protein;

3) protein that the aminoacid sequence shown in the 9th to the 419th is formed in the sequence 2 in the sequence table.

10. application according to claim 9 is characterized in that:

Said dental caries is by the dental caries due to the S.Mutans infection.

Images