CN101036791A

CN101036791A - Mutans streptococci gene vaccines pcDNA3-pac and the preparing method

Info

Publication number: CN101036791A
Application number: CNA2006100205064A
Authority: CN
Inventors: 刘天佳; 杨锦波; 刘建国; 郭丽宏; 杨德琴; 曹福娴; 贾文祥; 李继遥; 周学东; 谭红
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2006-03-17
Filing date: 2006-03-17
Publication date: 2007-09-19

Abstract

The invention provides a gene vaccine of Streptococcus mutans glucosyltransferase and preparation method thereof. The invention also provides a medicine combination, comprising the said vaccine and pharmaceutically acceptable carrier and excipient. The efficacy experiment indicates that the said vaccine can continually induce the immune response against Streptococcus mutans surface protein at a high level, and has remarkable curative effect of the diseases associated with Streptococcus mutans such as caries. The said vaccine may not integrate with genome of host cell after detecting, therefor it is safe and in conformity to clinical requirement, without adverse effects such as losing weight, having good application prospect.

Description

Mutans streptococci gene vaccines pcDNA 3-pac and preparation method thereof

Technical field

The present invention relates to the genetic engineering field, relate to a kind of mutans streptococci gene vaccines pcDNA 3-pac and preparation method thereof particularly.

Background technology

Dental caries (Caries) is the main oral disease of harm humans health, is classified as one of disease of three big serious harm human healths by WHO.The trend that rising is arranged in China's dental caries incidence rate at present, child and old people's sickness rate even up to more than 80%, the effective measures of therefore finding out the control dental caries are current urgent tasks.

Because dental caries is a kind of bacterial disease, (Streptococcus mutans S.mutans) is the main cariogenic bacteria of dental caries to Streptococcus mutans, and it is in the adhesion of facing and to gather be the prerequisite that dental caries takes place.Glucosyltransferase (glucosyltransferase, GTFs) be one of the main adhesion virulence factor of mutans streptococcus, mediation Streptococcus mutans cell sticks the sucrose dependency of facing, in the developing of dental caries, play pivotal role (ChiaJS, Lin RH, Chen JY et al.Inhibition of glucosyltransferase activities of Streptococcus mutans by amonoclonal antibody to a subsequence peptide.Infect Immun, 1993; 61 (11): 4689-95).The GTFs of mutans streptococcus has three kinds of forms, and promptly GtfB, GtfC and GtfD are wherein particularly important in the sucrose dependency of mutans streptococcus adheres to the GtfB of synthetic water insoluble glucan.The GtfB molecular weight is 165.8KDa, its full length gene is 4428bp, the protein of synthetic about 1400 amino acid residues of coding, the specific location, functional areas and distribution (the Eto A of epitope are arranged in the protein molecular, Saido TC, Fukushima K et al.Inhibitory effect of aself-derived peptide on glucosyltransferase of Streptococcus mutans.Possible novel anticariesmeasures.J biol chem, 1999; 274 (22): 15797-802).

Immune protection is one of effective means of control dental caries.Carry out immunity with regard to attempting with the attenuation whole-bacterial-vaccine of main cariogenic bacteria mutans streptococcus in the seventies in 20th century abroad, found that the full bacterium polyvalent vaccine of Streptococcus mutans can reduce the dental caries incidence rate, but but some antigenic component induce antibody of Streptococcus mutans and human heart tissue generation cross reaction.In recent years, successively adopt means such as subunit vaccine, polypeptide vaccine to carry out immune anticaries, but still had shortcomings such as vaccine production difficulty, cost height, immunogenicity are weak, immune efficacy is short.

At present, be badly in need of developing the novel Streptococcus mutans vaccine that can overcome existing vaccine shortcoming.

Summary of the invention:

First purpose of the present invention provides the vaccine of a kind of treatment and prevention Streptococcus mutans relevant disease.This vaccine contain shown in the sequence SEQ ID No.3 Streptococcus mutans glucosyltransferase B encoding gene.

Further, above-mentioned Streptococcus mutans glucosyltransferase B encoding gene fragment is loaded on the expression vector.

Preferably, described expression vector is a carrier for expression of eukaryon.

Further preferred, described carrier for expression of eukaryon is a plasmid.

Wherein, above-mentioned Streptococcus mutans relevant disease is a dental caries.

Second purpose of the present invention is to provide a kind of method for preparing above-mentioned vaccine.This method may further comprise the steps:

Specificity downstream primer downstream primer 5 '-ACT ACT CGA GTT AGA ACC ATT GAC CCT GAG CATTGC-3 ' shown in specificity forward primer 5 '-ATA TGG TAC CATGAC CGA AGC GAC ATC TAA GCA AGA-3 ' shown in a, the composition sequence SEQ ID No.1 and the sequence SEQ ID No.2, with this to the primer Streptococcus mutans glucosyltransferase B encoding gene shown in the preparation sequence SEQ ID No.3 that from the mutans streptococcus genomic DNA, increases;

B, gained Streptococcus mutans glucosyltransferase B encoding gene is loaded on the expression vector.

The 3rd purpose of the present invention is to provide a kind of pharmaceutical composition.This pharmaceutical composition: contain above-mentioned vaccine and pharmaceutically acceptable carrier and excipient.

Further, aforementioned pharmaceutical compositions also contains immunological adjuvant.

Preferably, above-mentioned immunological adjuvant is aluminium hydroxide, liposome or choleratoxin B subunit.

Wherein, the dosage form of aforementioned pharmaceutical compositions is oral formulations, buccal lozenge or injection.

The carrier that is used to prepare gene vaccine of the present invention is known in the art, as: recombinant phage, plasmid, adenovirus vector and other expression vector, the especially carrier for expression of eukaryon that after processing, can load the Streptococcus mutans protein coding gene.Simultaneously, need to prove: above production and the concrete technical method of operating recombinant vector disclosed by the invention and pharmaceutical composition are well known by persons skilled in the art, and can finish according to the technology of having described.

Beneficial effect of the present invention is: gene vaccine of the present invention has that preparation method is easy, with low cost, antigen intracellular expression and pathogen natural infection similar process, can effective body fluid of successive induction and advantages such as cellullar immunologic response, safety height, can remedy defectives such as existing medicine costliness, body internal stability difference, can increase administration time the interval, significantly reduce dosage, alleviate user financial burden and improve quality of life of patient.The effect experiment has proved that also gene vaccine of the present invention has tangible curative effect to Streptococcus mutans relevant diseases such as dental caries, and can not be integrated into the genome of host cell after testing, can not cause the body weight reduction to wait other harmful effects to the experimenter yet, has good safety, meet the medication requirement, have good market prospect.

Description of drawings

1% sepharose electrophoresis figure of the gtfB genetic fragment that Fig. 1 goes out for pcr amplification, wherein M is dna molecular amount Marker λ-EcoT14I, 1 and 2 all is the gtfB gene (4.7Kb) of amplification gained.

Fig. 2 is the structure flow chart of recombiant plasmid pcDNA3-gtfB.

Fig. 3 is the 1% agarose gel electrophoresis figure that recombiant plasmid pcDNA3-gtfB enzyme action is identified, M is dna molecular amount Marker λ-EcoT14I, and 1 and 2 is the KpnI enzyme action, and 3 and 4 is the XhoI enzyme action, and 5,6 and 7 is the KpnI+XhoI double digestion.

The invention will be further described below in conjunction with the description of accompanying drawing by the specific embodiment, but this is not a limitation of the present invention, those skilled in the art are according to basic thought of the present invention, can make various modification or improvement, only otherwise break away from basic thought of the present invention, all within the scope of the present invention.

The specific embodiment:

Embodiment one Streptococcus mutans glucosyltransferase B encoding gene gtfB obtains

The used specific PCR primer of synthetic amplification Streptococcus mutans glucosyltransferase B encoding gene gtfB:

Forward primer (SEQ ID No.1) 5 '-ATA TGG TAC CAT GAC CGA AGC GAC ATCTAA GCAA GA-3 ';

Downstream primer (SEQ ID No.2) 5 '-ACT ACT CGA GTT AGA ACC ATT GAC CCTGAG CAT TGC-3 '.

5 ' end at the upstream and downstream primer adds KpnI and XhoI restriction enzyme digestion sites respectively.5 ' end at restriction enzyme site respectively adds 4 protectiveness bases.Forward primer adds atg start codon ATG in the upstream of encoding gene, and downstream primer adds termination codon TTA in the downstream of encoding gene.

With Streptococcus mutans GS-5 complete genome DNA is template, adopt the Pyrobest DNA synzyme (TakaRa of high reliability, Dalian), primer with above design, with Gene Amp PCR System 9600 type PCR instrument (Perking Elmer, the U.S.) increase, obtain gtfB genetic fragment (904-4578bp).Amplification condition: adopt two step method PCR, 94 ℃ of degeneration 30Sec, 68 ℃ of 8min that anneal and extend, 30 circulations are stored the PCR products for 4 ℃.Adopting in a small amount, glue reclaims test kit (Watson, Shanghai) recovery and purification gtfB genes of interest.

The genes of interest gtfB that amplification is obtained detects with 1% sepharose electrophoresis, testing result is single band, the specific amplification height, molecular weight is and the 4.7Kb (see figure 1), sequence and expectation identical sequence and expectation identical (nucleotide sequence of Streptococcus mutans protein gene is seen SEQ ID No.3, and its amino acid sequence coded is seen SEQ ID No.4).

The structure of embodiment two reorganization plasmid pcDNA3-gtfB

1, the structure of recombiant plasmid pcDNA3-gtfB (make up flow process and see Fig. 2):

Carrier for expression of eukaryon pcDNA3 and genes of interest gtfB are respectively in the general buffer of KpnI and XhoI enzyme, 37 ℃ of double digestion 2hr, the double digestion product reclaims test kit purification double digestion product with a small amount of glue behind 1% low melting-point agarose gel (TakaRa, Dalian) electrophoresis.With dna ligation kit (TakaRa, Dalian), under the effect of T4 dna ligase, 16 ℃ connect 3hr with genes of interest gtfB and carrier for expression of eukaryon pcDNA3.Connect product transformed competence colibacillus cell colibacillus JM109 immediately,, obtain transformant with amicillin resistance LB plate screening positive clone strain.

2, the enzyme action of recombiant plasmid is identified and order-checking:

It is the single fragment of 9.0kb through KpnI, XhonI single endonuclease digestion that the gained recombiant plasmid is carried out enzyme action, is the gtfB target gene fragment of 3.6kb and the unloaded plasmid fragment of pcDNA3 of 5.4kb through KpnI, XhonI double digestion.The result shows that recombiant plasmid pcDNA3-gtfB successfully constructs (see figure 3).

Show insertion gene length 36756p among the recombiant plasmid pcDNA3-gtfB through the dna sequencing result, identical with Shiroza etc. 1986 with the gtfB gene order of the Streptococcus mutans GS-5 of double deoxidating chain termination measuring, and that inserts is in the right direction, do not change the reading frame of gtfB gene yet, guaranteed the correctness of encoding amino acid sequence.

The preparation of embodiment three mutans streptococci gene vaccines pcDNA 3-pacs of the present invention

1, the transformant with embodiment two gained is inoculated in the LB fluid medium, puts in the air shaking table case, and 37 ℃ of joltings are cultivated (250rpm) and spent the night, and extract test kit with plasmid and extract recombiant plasmid.

2,, and be prepared as gene vaccine with conventional method purification recombiant plasmid pcDNA3-gtfB.

The preparation of embodiment four gene vaccine injections of the present invention

This injection can prepare by the following method: get water for injection under the aseptic condition, add raw material recombiant plasmid pcDNA3-gtfB again, mix homogeneously under aseptic condition, be diluted to 0.1mg/ml, and under aseptic condition mix homogeneously, regulate pH value to 7.0 back aseptic filtration with buffer and be distributed into injection, cryopreservation, promptly.

One expression of mutans streptococci gene vaccines pcDNA 3-pac of the present invention in eukaryotic cell of test example

With liposome-mediated gene vaccine transfection COS-7 cell of the present invention, carry out every detection after cultivation a period of time, transcribe and translate activity to have behind definite gene vaccine transfecting eukaryotic cells of the present invention.

Test item and result:

1, detects the transcription product that inserts gene gtfB with the RT-PCR method

In the COS-7 of gene vaccine stable transfection of the present invention cell, extract total RNA, after the RT-PCR amplification, carry out electrophoresis detection, the visible molecular weight band consistent on gel with gtfB, showing has the gene transcription of insertion product.

2, detect exogenous gene gtfB at intracellular expression product with LSAB immunohistochemical staining

Experimental result shows, the endochylema of the COS-7 cell of gene vaccine stable transfection of the present invention is brown, do not see obvious brown colouration in the endochylema of the COS-7 cell of pcDNA3 empty carrier stable transfection, show the expression that Streptococcus mutans glucosyltransferase B is arranged in the born of the same parents of COS-7 cell of gene vaccine stable transfection of the present invention.

3, detect the expression product of exogenous gene on cell membrane with flow cytometer, have Streptococcus mutans glucosyltransferase B to exist on the cell membrane of the COS-7 cell of gene vaccine stable transfection of the present invention as a result.

4, detect the expression product of exogenous gene in the supernatant of extracellular with the Western immunoblotting, the result shows in the extracellular supernatant of COS-7 cell of gene vaccine stable transfection of the present invention has Streptococcus mutans glucosyltransferase B to express.

Have behind the mutans streptococci gene vaccines pcDNA 3-pac transfecting eukaryotic cells of the present invention and transcribe and translate activity, expressed protein has distribution in born of the same parents, on the after birth and outside the born of the same parents, and molecular weight is identical with expectation, and can with the corresponding antibody specific bond.

The zoopery of test example two gene vaccine injection immune effects of the present invention

Experiment grouping and immunity: 24 of the of the right age healthy rats that body weight is suitable are divided into pcDNA3-gtfB injection group, the full bacterium group of Streptococcus mutans JBP deactivation, pcDNA3 empty carrier group, PBS group at random, every group of rat goes respectively injects in bilateral submaxillary gland week, every each 100ul (1ug/ul), immunity is 3 times altogether.

Subsequently, inoculate Streptococcus mutans respectively, lure dental caries to experiment to finish (totally 90 days) with causing dental caries food to each group.

Anticaries action detects index: serum IgG and saliva S-IgA; Saliva Streptococcus mutans counting; The bacterial plaque of tooth surface index; Dental caries score: score according to the Keyes standards of grading.

Experimental result: the experimental result of above-mentioned effect experiment shows, mutans streptococci gene vaccines pcDNA 3-pac of the present invention has all produced significant anti-caries effect through all injecting immune rats of submaxillary gland gland, and effect is suitable substantially with existing sterilization whole-bacterial-vaccine.

The safety experiment of test example three gene vaccine injections of the present invention

1, gene vaccine injection of the present invention is to the influence of the body weight of being tried rat

Test example two finished by examination after, measure the body weight of respectively organizing rat.

The body weight of the full bacterium group of deactivation rat is obviously than other groups light (P＜0.01), and mutans streptococci gene vaccines pcDNA 3-pac group of the present invention is compared no significant difference with pcDNA3 empty carrier group with the PBS group, show the basic avirulence of gene vaccine of the present invention, safety obviously is better than existing sterilization whole-bacterial-vaccine.

2, whether gene vaccine of the present invention is integrated into the confirmatory experiment of host cell gene group

By the PCR experiment mutans streptococci gene vaccines pcDNA 3-pac of the present invention being integrated into the genomic probability of being tried rat detects.The PCR experimental result shows, in 10000 histiocytes nuclear, just can detect under the detection level that a pcDNA3-Pac copies and repeatedly test, do not find that all gene vaccine of the present invention is integrated into the host cell gene group in each group of immunity, promptly its integrate probability and be lower than ten thousand/.Show that mutans streptococci gene vaccines pcDNA 3-pac of the present invention has good biological safety.

By above example as seen, easy, with low cost, good immune effect, safe in utilization that mutans streptococci gene vaccines pcDNA 3-pac of the present invention prepares has good market prospect.

The above more excellent specific embodiment is that the present invention is further illustrated, but be not limitation of the scope of the invention, those skilled in the art are according to basic thought of the present invention, can make various modification or improvement, only otherwise break away from basic thought of the present invention, all within the scope that spirit of the present invention and claims of being enclosed define.

Mutans streptococci gene vaccines pcDNA 3-pac and preparation method thereof .ST25

SEQUENCE?LISTING

＜110〉Sichuan University

＜120〉mutans streptococci gene vaccines pcDNA 3-pac and preparation method thereof

<130>A060038

<160>4

<170>PatentIn?version?3.2

<210>1

<211>36

<212>DNA

<213>Artificial

<220>

＜223〉the PCR primer 1

<400>1

atatggtacc?atgaccgaag?cgacatctaa?gcaaga 36

<210>2

<211>36

<212>DNA

<213>Artificial

<220>

＜223〉PCR primer 2

<400>2

actactcgag?ttagaaccat?tgaccctgag?cattgc 36

<210>3

<211>4428

<212>DNA

<213>Streptococcus?mutans

<400>3

atggacaaga?aagtgcgtta?taaactgcgc?aaagttaaaa?aaagatgggt?gacagtatct 60

gttgcatctg?ctgtgatgac?tttaactaca?ctttcgggtg?gcttggttaa?agcagattct 120

aatgaatcga?aatcccaaat?ttctaatgat?tctaatacca?gtgttgttac?tgctaatgaa 180

gaatctaatg?taataaccga?agcgacatct?aagcaagaag?ctgctagtag?tcaaactaat 240

catacagtaa?cgacaagcag?tagctctact?tcggtagtta?atcccaaaga?ggttgtaagt 300

aatccttata?ctgttgggga?aacagcttct?aatggtgaaa?agcttcaaaa?tcaaacaact 360

acagttgaca?aaacttctga?agctgctgct?aataatatta?gtaaacaaac?aaccgaagct 420

gatacagatg?ttattgatga?tagcaatgca?gccaatctac?aaatattgga?aaaacttccc 480

aatgtaaaag?aaattgatgg?taagtattat?tattatgaca?ataacggcaa?agttcgtact 540

aattttacat?taattgctga?tggcaaaatt?ttacattttg?atgaaactgg?cgcttatact 600

gatacatcaa?ttgacactgt?aaataaagat?atcgtcacaa?caagaagtaa?tctatacaaa 660

aaatataatc?aagtttatga?tcgctctgca?cagagctttg?agcatgttga?tcattatttg 720

acagctgaga?gttggtatcg?tcctaagtac?atcttgaagg?atggcaaaac?atggacacag 780

tcaacagaaa?aagatttccg?tcccttattg?atgacatggt?ggcctgacca?agaaacgcag 840

cgtcaatatg?ttaactacat?gaatgcacag?cttggcatta?acaagactta?tgatgataca 900

agtaatcaat?tgcaattaaa?tattgcagct?gcaactattc?aagcaaaaat?tgaggccaaa 960

attacaactt?taaagaatac?tgattggctg?cgtcagacta?tttccgcatt?tgttaagaca 1020

cagtcagctt?ggaacagtga?cagcgaaaaa?ccgtttgatg?atcatttaca?aaatggagca 1080

gtgctttacg?ataatgaagg?aaaattaacg?ccttatgcta?attccaacta?ccgtatctta 1140

aatcgcaccc?cgaccaatca?aaccggaaag?aaagatccaa?ggtatacagc?tgataacact 1200

atcggcggtt?atgaattcct?tttggccaac?gatgtggata?attctaatcc?tgtcgtgcag 1260

gccgaacaat?tgaactggct?acattttctc?atgaactttg?gtaacattta?tgccaatgat 1320

ccggatgcta?actttgattc?cattcgtgtt?gatgcggtag?ataatgtgga?tgctgacttg 1380

ctccaaattg?ctggggatta?cctcaaagct?gctaagggga?tccataaaaa?tgataaggct 1440

gctaatgatc?atttgtctat?tttagaggca?tggagtgaca?acgacactcc?ttaccttcat 1500

gatgatggcg?acaatatgat?taatatggac?aataagctgc?gtttgtctct?attattttca 1560

ttagctaaac?ccttaaatca?acgttcaggc?atgaatcctc?tgatcactaa?cagtttggtg 1620

aatcgtactg?atgataatgc?tgaaactgcc?gcagtccctt?cttattcctt?catccgtgcc 1680

catgacagtg?aagtgcagga?tttgattgct?gatatcatca?aggcagaaat?caatcctaat 1740

gttgtcgggt?attcattcac?tatggaggaa?atcaagaagg?ctttcgagat?ttacaacaaa 1800

gacttattag?ctacagagaa?gaaatacaca?cactataata?cggcactttc?ttatgccctg 1860

cttttaacca?acaaatccag?tgtgccgcgt?gtctattatg?gggatatgtt?tacagatgac 1920

gggcaataca?tggctcataa?gacgatcaat?tacgaagcca?tcgaaaccct?gcttaaagct 1980

cgtattaagt?atgtttcagg?cggtcaagcc?atgcgcaatc?aacaggttgg?caattctgaa 2040

atcattacgt?ctgtccgcta?tggtaaaggt?gctttgaaag?caacggatac?aggggaccgc 2100

accacacgga?cttcaggagt?ggccgtgatt?gaaggcaata?acccttcttt?acgtttgaag 2160

gcttctgatc?gcgtggttgt?caatatggga?gcagcccata?agaaccaagc?ttaccgacct 2220

ttactcttga?ccacagataa?cggtatcaag?gcttatcatt?ccgatcaaga?agcggctggt 2280

ttggtgcgct?acaccaatga?cagaggggaa?ttgatcttca?cagcggctga?tattaaaggc 2340

tatgccaacc?ctcaagtttc?tggctattta?ggtgtctggg?ttccagtagg?cgctgcgctg 2400

atcaagatgt?tcgcgttgcg?gctagcacgg?ccccatcaac?agatggcaag?tgtgcatcaa 2460

aatgcggccc?ttgattcacg?cgtcatgttt?gaaggtttct?ctaatttcca?agctttcgcc 2520

actaaaaaag?aggaatatac?caatgttgtg?attgctaaga?atgtggataa?gtttgcggaa 2580

tggggggtca?cagactttga?aatggcaccg?cagtatgtgt?cttcaacgga?tggttctttc 2640

ttggattctg?tgatccaaaa?cggctatgct?tttacggacc?gttatgattt?gggaatttcc 2700

aaacctaata?aatacgggac?agccgatgat?ttggtgaaag?ccatcaaagc?gttacacagc 2760

aagggcatta?aggtaatggc?tgactgggtg?cctgatcaaa?tgtatgcttt?ccctgaaaaa 2820

gaagtggtaa?ctgcaacccg?tgttgataag?tatgggactc?ctgttgcagg?aagtcagatc 2880

aaaaacaccc?tttatgtagt?tgatggtaag?agttctggta?aagatcaaca?agccaagtat 2940

gggggagctt?tcttagagga?gctgcaagcg?aagtatccgg?agctttttgc?gagaaaacaa 3000

atttccacag?gggttccgat?ggatccttct?gttaagatta?agcaatggtc?tgccaagtac 3060

tttaatggga?caaatatttt?agggcgcgga?gcaggctatg?tcttaaaaga?tcaggcaact 3120

aatacttact?ttaatatttc?agateataaa?gaaataaact?tccttcctaa?aacattgtta 3180

aaccaagata?gtcaagttgg?tttctcttat?gacggtaaag?gttatgttta?ttattcaacg 3240

agtggttacc?aagccaaaaa?tactttcatc?agcgaaggtg?ataaatggta?ttattttgat 3300

aataacggtt?atatggtcac?tggtgctcaa?tcaattaacg?gtgttaatta?ttatttctta 3360

tcaaatggcc?tacagctcag?agatgctatt?cttaagaatg?aagatggaac?ttacgcttat 3420

tatggaaatg?acggtcgccg?ttatgaaaat?ggttattatc?aattcatgag?tggtgtatgg 3480

cgtcacttca?ataatggtga?aatgagtgtt?ggattaactg?taattgatgg?tcaggttcaa 3540

tactttgatg?aaatgggcta?tcaagccaaa?ggaaaatttg?taacaactgc?cgatggtaaa 3600

ataagatatt?ttgataagca?atctgggaac?atgtaccgta?atcgttttat?tgaaaacgaa 3660

gaaggtaaat?ggctgtatct?cggtgaagat?ggtgcagcag?tgacaggatc?tcaaaccatt 3720

aacggtcaac?acctgtactt?tagagcaaac?ggtgttcagg?tcaagggtga?atttgtcact 3780

gaccaccacg?gccgtatcag?ctattacgac?ggcaattcag?gggatcaaat?ccgcaaccgc 3840

tttgtccgca?atgctcaggg?tcaatggttc?tactttgata?acaatggcta?tgccgtaacc 3900

ggtgccagaa?ccattaacgg?tcaactccta?tactttagag?caaacggtgt?tcaggtcaag 3960

ggtgaatttg?tcactgaccg?ctacggccgt?atcagctatt?acgacggcaa?ttcaggggat 4020

caaatccgca?accgctttgt?ccgcaatgct?cagggtcaat?ggttctactt?tgataacaat 4080

ggctatgccg?taaccggtgc?cagaaccatt?aacggtcaac?acctatactt?tagagcaaac 4140

ggtgttcagg?tcaagggtga?atttgtcact?gaccgccacg?gccgtatcag?ctattacgac 4200

ggcaattcag?gggatcaaat?ccgcaaccgc?tttgtccgca?atgctcaggg?tcaatggttc 4260

tactttgata?acaatggcta?tgccgtaacc?ggtgccagaa?ccattaacgg?tcaacaccta 4320

tactttagag?caaacggtgt?tcaggtcaag?ggtgaatttg?tcactgaccg?ctacggccgt 4380

atcagttatt?acgatgctaa?ctctggagaa?cgagttcgga?ttaactaa 4428

<210>4

<211>1475

<212>PRT

<213>Streptococcus?mutans

<400>4

Met?Asp?Lys?Lys?Val?Arg?Tyr?Lys?Leu?Arg?Lys?Val?Lys?Lys?Arg?Trp

1 5 10 15

Val?Thr?Val?Ser?Val?Ala?Ser?Ala?Val?Met?Thr?Leu?Thr?Thr?Leu?Ser

20 25 30

Gly?Gly?Leu?Val?Lys?Ala?Asp?Ser?Asn?Glu?Ser?Lys?Ser?Gln?Ile?Ser

35 40 45

Asn?Asp?Ser?Asn?Thr?Ser?Val?Val?Thr?Ala?Asn?Glu?Glu?Ser?Asn?Val

50 55 60

Ile?Thr?Glu?Ala?Thr?Ser?Lys?Gln?Glu?Ala?Ala?Ser?Ser?Gln?Thr?Asn

65 70 75 80

His?Thr?Val?Thr?Thr?Ser?Ser?Ser?Ser?Thr?Ser?Val?Val?Asn?Pro?Lys

85 90 95

Glu?Val?Val?Ser?Asn?Pro?Tyr?Thr?Val?Gly?Glu?Thr?Ala?Ser?Asn?Gly

100 105 110

Glu?Lys?Leu?Gln?Asn?Gln?Thr?Thr?Thr?Val?Asp?Lys?Thr?Ser?Glu?Ala

115 120 125

Ala?Ala?Asn?Asn?Ile?Ser?Lys?Gln?Thr?Thr?Glu?Ala?Asp?Thr?Asp?Val

130 135 140

Ile?Asp?Asp?Ser?Asn?Ala?Ala?Asn?Leu?Gln?Ile?Leu?Glu?Lys?Leu?Pro

145 150 155 160

Asn?Val?Lys?Glu?Ile?Asp?Gly?Lys?Tyr?Tyr?Tyr?Tyr?Asp?Asn?Asn?Gly

165 170 175

Lys?Val?Arg?Thr?Asn?Phe?Thr?Leu?Ile?Ala?Asp?Gly?Lys?Ile?Leu?His

180 185 190

Phe?Asp?Glu?Thr?Gly?Ala?Tyr?Thr?Asp?Thr?Ser?Ile?Asp?Thr?Val?Asn

195 200 205

Lys?Asp?Ile?Val?Thr?Thr?Arg?Ser?Asn?Leu?Tyr?Lys?Lys?Tyr?Asn?Gln

210 215 220

Val?Tyr?Asp?Arg?Ser?Ala?Gln?Ser?Phe?Glu?His?Val?Asp?His?Tyr?Leu

225 230 235 240

Thr?Ala?Glu?Ser?Trp?Tyr?Arg?Pro?Lys?Tyr?Ile?Leu?Lys?Asp?Gly?Lys

245 250 255

Thr?Trp?Thr?Gln?Ser?Thr?Glu?Lys?Asp?Phe?Arg?Pro?Leu?Leu?Met?Thr

260 265 270

Trp?Trp?Pro?Asp?Gln?Glu?Thr?Gln?Arg?Gln?Tyr?Val?Asn?Tyr?Met?Asn

275 280 285

Ala?Gln?Leu?Gly?Ile?Asn?Lys?Thr?Tyr?Asp?Asp?Thr?Ser?Asn?Gln?Leu

290 295 300

Gln?Leu?Asn?Ile?Ala?Ala?Ala?Thr?Ile?Gln?Ala?Lys?Ile?Glu?Ala?Lys

305 310 315 320

Ile?Thr?Thr?Leu?Lys?Asn?Thr?Asp?Trp?Leu?Arg?Gln?Thr?Ile?Ser?Ala

325 330 335

Phe?Val?Lys?Thr?Gln?Ser?Ala?Trp?Asn?Ser?Asp?Ser?Glu?Lys?Pro?Phe

340 345 350

Asp?Asp?His?Leu?Gln?Asn?Gly?Ala?Val?Leu?Tyr?Asp?Asn?Glu?Gly?Lys

355 360 365

Leu?Thr?Pro?Tyr?Ala?Asn?Ser?Asn?Tyr?Arg?Ile?Leu?Asn?Arg?Thr?Pro

370 375 380

Thr?Asn?Gln?Thr?Gly?Lys?Lys?Asp?Pro?Arg?Tyr?Thr?Ala?Asp?Asn?Thr

385 390 395 400

Ile?Gly?Gly?Tyr?Glu?Phe?Leu?Leu?Ala?Asn?Asp?Val?Asp?Asn?Ser?Asn

405 410 415

Pro?Val?Val?Gln?Ala?Glu?Gln?Leu?Asn?Trp?Leu?His?Phe?Leu?Met?Asn

420 425 430

Phe?Gly?Asn?Ile?Tyr?Ala?Asn?Asp?Pro?Asp?Ala?Asn?Phe?Asp?Ser?Ile

435 440 445

Arg?Val?Asp?Ala?Val?Asp?Asn?Val?Asp?Ala?Asp?Leu?Leu?Gln?Ile?Ala

450 455 460

Gly?Asp?Tyr?Leu?Lys?Ala?Ala?Lys?Gly?Ile?His?Lys?Asn?Asp?Lys?Ala

465 470 475 480

Ala?Asn?Asp?His?Leu?Ser?Ile?Leu?Glu?Ala?Trp?Ser?Asp?Asn?Asp?Thr

485 490 495

Pro?Tyr?Leu?His?Asp?Asp?Gly?Asp?Asn?Met?Ile?Asn?Met?Asp?Asn?Lys

500 505 510

Leu?Arg?Leu?Ser?Leu?Leu?Phe?Ser?Leu?Ala?Lys?Pro?Leu?Asn?Gln?Arg

515 520 525

Ser?Gly?Met?Asn?Pro?Leu?Ile?Thr?Asn?Ser?Leu?Val?Asn?Arg?Thr?Asp

530 535 540

Asp?Asn?Ala?Glu?Thr?Ala?Ala?Val?Pro?Ser?Tyr?Ser?Phe?Ile?Arg?Ala

545 550 555 560

His?Asp?Ser?Glu?Val?Gln?Asp?Leu?Ile?Ala?Asp?Ile?Ile?Lys?Ala?Glu

565 570 575

Ile?Asn?Pro?Asn?Val?Val?Gly?Tyr?Ser?Phe?Thr?Met?Glu?Glu?Ile?Lys

580 585 590

Lys?Ala?Phe?Glu?Ile?Tyr?Asn?Lys?Asp?Leu?Leu?Ala?Thr?Glu?Lys?Lys

595 600 605

Tyr?Thr?His?Tyr?Asn?Thr?Ala?Leu?Ser?Tyr?Ala?Leu?Leu?Leu?Thr?Asn

610 615 620

Lys?Ser?Ser?Val?Pro?Arg?Val?Tyr?Tyr?Gly?Asp?Met?Phe?Thr?Asp?Asp

625 630 635 640

Gly?Gln?Tyr?Met?Ala?His?Lys?Thr?Ile?Asn?Tyr?Glu?Ala?Ile?Glu?Thr

645 650 655

Leu?Leu?Lys?Ala?Arg?Ile?Lys?Tyr?Val?Ser?Gly?Gly?Gln?Ala?Met?Arg

660 665 670

Asn?Gln?Gln?Val?Gly?Asn?Ser?Glu?Ile?Ile?Thr?Ser?Val?Arg?Tyr?Gly

675 680 685

Lys?Gly?Ala?Leu?Lys?Ala?Thr?Asp?Thr?Gly?Asp?Arg?Thr?Thr?Arg?Thr

690 695 700

Ser?Gly?Val?Ala?Val?Ile?Glu?Gly?Asn?Asn?Pro?Ser?Leu?Arg?Leu?Lys

705 710 715 720

Ala?Ser?Asp?Arg?Val?Val?Val?Asn?Met?Gly?Ala?Ala?His?Lys?Asn?Gln

725 730 735

Ala?Tyr?Arg?Pro?Leu?Leu?Leu?Thr?Thr?Asp?Asn?Gly?Ile?Lys?Ala?Tyr

740 745 750

His?Ser?Asp?Gln?Glu?Ala?Ala?Gly?Leu?Val?Arg?Tyr?Thr?Asn?Asp?Arg

755 760 765

Gly?Glu?Leu?Ile?Phe?Thr?Ala?Ala?Asp?Ile?Lys?Gly?Tyr?Ala?Asn?Pro

770 775 780

Gln?Val?Ser?Gly?Tyr?Leu?Gly?Val?Trp?Val?Pro?Val?Gly?Ala?Ala?Leu

785 790 795 800

Ile?Lys?Met?Phe?Ala?Leu?Arg?Leu?Ala?Arg?Pro?His?Gln?Gln?Met?Ala

805 810 815

Ser?Val?His?Gln?Asn?Ala?Ala?Leu?Asp?Ser?Arg?Val?Met?Phe?Glu?Gly

820 825 830

Phe?Ser?Asn?Phe?Gln?Ala?Phe?Ala?Thr?Lys?Lys?Glu?Glu?Tyr?Thr?Asn

835 840 845

Val?Val?Ile?Ala?Lys?Asn?Val?Asp?Lys?Phe?Ala?Glu?Trp?Gly?Val?Thr

850 855 860

Asp?Phe?Glu?Met?Ala?Pro?Gln?Tyr?Val?Ser?Ser?Thr?Asp?Gly?Ser?Phe

865 870 875 880

Leu?Asp?Ser?Val?Ile?Gln?Asn?Gly?Tyr?Ala?Phe?Thr?Asp?Arg?Tyr?Asp

885 890 895

Leu?Gly?Ile?Ser?Lys?Pro?Asn?Lys?Tyr?Gly?Thr?Ala?Asp?Asp?Leu?Val

900 905 910

Lys?Ala?Ile?Lys?Ala?Leu?His?Ser?Lys?Gly?Ile?Lys?Val?Met?Ala?Asp

915 920 925

Trp?Val?Pro?Asp?Gln?Met?Tyr?Ala?Phe?Pro?Glu?Lys?Glu?Val?Val?Thr

930 935 940

Ala?Thr?Arg?Val?Asp?Lys?Tyr?Gly?Thr?Pro?Val?Ala?Gly?Ser?Gln?Ile

945 950 955 960

Lys?Asn?Thr?Leu?Tyr?Val?Val?Asp?Gly?Lys?Ser?Ser?Gly?Lys?Asp?Gln

965 970 975

Gln?Ala?Lys?Tyr?Gly?Gly?Ala?Phe?Leu?Glu?Glu?Leu?Gln?Ala?Lys?Tyr

980 985 990

Pro?Glu?Leu?Phe?Ala?Arg?Lys?Gln?Ile?Ser?Thr?Gly?Val?Pro?Met?Asp

995 1000 1005

Pro?Ser?Val?Lys?Ile?Lys?Gln?Trp?Ser?Ala?Lys?Tyr?Phe?Asn?Gly

1010 1015 1020

Thr?Asn?Ile?Leu?Gly?Arg?Gly?Ala?Gly?Tyr?Val?Leu?Lys?Asp?Gln

1025 1030 1035

Ala?Thr?Asn?Thr?Tyr?Phe?Asn?Ile?Ser?Asp?Asn?Lys?Glu?Ile?Asn

1040 1045 1050

Phe?Leu?Pro?Lys?Thr?Leu?Leu?Asn?Gln?Asp?Ser?Gln?Val?Gly?Phe

1055 1060 1065

Ser?Tyr?Asp?Gly?Lys?Gly?Tyr?Val?Tyr?Tyr?Ser?Thr?Ser?Gly?Tyr

1070 1075 1080

Gln?Ala?Lys?Asn?Thr?Phe?Ile?Ser?Glu?Gly?Asp?Lys?Trp?Tyr?Tyr

1085 1090 1095

Phe?Asp?Asn?Asn?Gly?Tyr?Met?Val?Thr?Gly?Ala?Gln?Ser?Ile?Asn

1100 1105 1110

Gly?Val?Asn?Tyr?Tyr?Phe?Leu?Ser?Asn?Gly?Leu?Gln?Leu?Arg?Asp

1115 1120 1125

Ala?Ile?Leu?Lys?Asn?Glu?Asp?Gly?Thr?Tyr?Ala?Tyr?Tyr?Gly?Asn

1130 1135 1140

Asp?Gly?Arg?Arg?Tyr?Glu?Asn?Gly?Tyr?Tyr?Gln?Phe?Met?Ser?Gly

1145 1150 1155

Val?Trp?Arg?His?Phe?Asn?Asn?Gly?Glu?Met?Ser?Val?Gly?Leu?Thr

1160 1165 1170

Val?Ile?Asp?Gly?Gln?Val?Gln?Tyr?Phe?Asp?Glu?Met?Gly?Tyr?Gln

1175 1180 1185

Ala?Lys?Gly?Lys?Phe?Val?Thr?Thr?Ala?Asp?Gly?Lys?Ile?Arg?Tyr

1190 1195 1200

Phe?Asp?Lys?Gln?Ser?Gly?Asn?Met?Tyr?Arg?Asn?Arg?Phe?Ile?Glu

1205 1210 1215

Asn?Glu?Glu?Gly?Lys?Trp?Leu?Tyr?Leu?Gly?Glu?Asp?Gly?Ala?Ala

1220 1225 1230

Val?Thr?Gly?Ser?Gln?Thr?Ile?Asn?Gly?Gln?His?Leu?Tyr?Phe?Arg

1235 1240 1245

Ala?Asn?Gly?Val?Gln?Val?Lys?Gly?Glu?Phe?Val?Thr?Asp?His?His

1250 1255 1260

Gly?Arg?Ile?Ser?Tyr?Tyr?Asp?Gly?Asn?Ser?Gly?Asp?Gln?Ile?Arg

1265 1270 1275

Asn?Arg?Phe?Val?Arg?Asn?Ala?Gln?Gly?Gln?Trp?Phe?Tyr?Phe?Asp

1280 1285 1290

Asn?Asn?Gly?Tyr?Ala?Val?Thr?Gly?Ala?Arg?Thr?Ile?Asn?Gly?Gln

1295 1300 1305

Leu?Leu?Tyr?Phe?Arg?Ala?Asn?Gly?Val?Gln?Val?Lys?Gly?Glu?Phe

1310 1315 1320

Val?Thr?Asp?Arg?Tyr?Gly?Arg?Ile?Ser?Tyr?Tyr?Asp?Gly?Asn?Ser

1325 1330 1335

Gly?Asp?Gln?Ile?Arg?Asn?Arg?Phe?Val?Arg?Asn?Ala?Gln?Gly?Gln

1340 1345 1350

Trp?Phe?Tyr?Phe?Asp?Asn?Asn?Gly?Tyr?Ala?Val?Thr?Gly?Ala?Arg

1355 1360 1365

Thr?Ile?Asn?Gly?Gln?His?Leu?Tyr?Phe?Arg?Ala?Asn?Gly?Val?Gln

1370 1375 1380

Val?Lys?Gly?Glu?Phe?Val?Thr?Asp?Arg?His?Gly?Arg?Ile?Ser?Tyr

1385 1390 1395

Tyr?Asp?Gly?Asn?Ser?Gly?Asp?Gln?Ile?Arg?Asn?Arg?Phe?Val?Arg

1400 1405 1410

Asn?Ala?Gln?Gly?Gln?Trp?Phe?Tyr?Phe?Asp?Asn?Asn?Gly?Tyr?Ala

1415 1420 1425

Val?Thr?Gly?Ala?Arg?Thr?Ile?Asn?Gly?Gln?His?Leu?Tyr?Phe?Arg

1430 1435 1440

Ala?Asn?Gly?Val?Gln?Val?Lys?Gly?Glu?Phe?Val?Thr?Asp?Arg?Tyr

1445 1450 1455

Gly?Arg?Ile?Ser?Tyr?Tyr?Asp?Ala?Asn?Ser?Gly?Glu?Arg?Val?Arg

1460 1465 1470

Ile?Asn

1475

Claims

1, the vaccine of a kind of treatment and prevention Streptococcus mutans relevant disease is characterized in that: contain shown in the sequence SEQ IDNo.3 Streptococcus mutans glucosyltransferase B encoding gene.

2, vaccine according to claim 1 is characterized in that: described Streptococcus mutans glucosyltransferase B code cDNA is loaded on the expression vector.

3, vaccine according to claim 2 is characterized in that: described expression vector is a carrier for expression of eukaryon.

4, vaccine according to claim 3 is characterized in that: described carrier for expression of eukaryon is a plasmid.

5, according to the described vaccine of claim 1～4, it is characterized in that: described Streptococcus mutans relevant disease is a dental caries.

6, a kind of method for preparing the described vaccine of claim 2 is characterized in that may further comprise the steps:

Specificity forward primer shown in a, the design composition sequence SEQ ID No.1 and the specificity downstream primer shown in the sequence SEQ ID No.2, with this primer increase from the mutans streptococcus genomic DNA prepares Streptococcus mutans glucosyltransferase B encoding gene;

B, gained Streptococcus mutans glucosyltransferase B encoding gene is reprinted on expression vector.

7, a kind of pharmaceutical composition is characterized in that: contain each described vaccine of claim 1～5 and pharmaceutically acceptable carrier and excipient.

8, pharmaceutical composition according to claim 7 is characterized in that also containing immunological adjuvant.

9, pharmaceutical composition according to claim 8 is characterized in that described immunological adjuvant is aluminium hydroxide, liposome or choleratoxin B subunit.

10, according to each described pharmaceutical composition of claim 7～9, the dosage form that it is characterized in that this pharmaceutical composition is oral formulations, buccal lozenge or injection.