CN101474400A - Human streptococcus mutans genetic engineering vaccine for decayed tooth and preparation method thereof - Google Patents
Human streptococcus mutans genetic engineering vaccine for decayed tooth and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the biopharmaceutical field and relates to a method for constructing and preparing human streptococcus mutans gene engineering vaccine for tooth decay. In the method, the genes at a glucan binding domain at the end of glucosyltransferase C and at an N end immunodominance domain of glucan-binding protein B which are major protective antigens of streptococcus mutans of human primary cariogenic bacteria and the mucous membrane immunologic adjuvant heat-labile toxin B subunit are used for constructing fusion engineering bacteria by a gene recombination method, and fusion protein molecular vaccine with high purity is obtained by high density fermentation and a series of purifying procedures. The technology for preparing the vaccine is simple and easy for amplifying and has good repeatability; the obtained protein has high purity; primary animal experiments prove that the fusion protein can stimulate body to produce efficient immunity response; and the fusion protein is approved as good candidate antigens of gene engineering vaccine for tooth decay and has good application prospect.
Description
Technical field
The present invention relates to the medical biotechnology field, relate in particular to a kind of human streptococcus mutans genetic engineering vaccine for decayed tooth and preparation method thereof.
Background technology
Streptococcus mutans (S.Mutans) is the human main dental caries microorganism that causes, and the generation of its field planting in the oral cavity and dental caries is closely related.And dental caries is the most a kind of disease of human sickness rate at present, and WHO in 2003 just classify it as the disease of the third need keypoint control after cardiovascular disease and cancer.The child in the whole world 60% to 90% and adult once were subjected to the puzzlement (The World Oral Health Report2003) of dental caries.National for the third time oral health epidemiological investigation in 2007 shows: China's dental caries sickness rate is up to 70%-80%.Because dental caries is made slow progress, and does not jeopardize patient's life usually, therefore do not come into one's own in developing country." toothache is not a disease, and pain is got up and very will be killed! " in fact dental caries is very big to the harm that the mankind cause; the deciduous teeth dental caries has a strong impact on the growth and the normal masticatory function of teenager teeth, and the Streptococcus mutans of infection may cause rheumatoid arthritis, nephritis, rheumatic heart disease etc. with human body generation cross-immune reaction.Existing preventive measure such as fluoride, Mechanical Method, antibacterials and limit sugared diet preventing decayed tooth etc. all can only reduce the dental caries occurred level to a certain extent may not blocked the popular of dental caries.And develop safely and effectively anti-caries vaccine is the simplest, economic, the means efficiently of caries prevention.
S.Mutans is as the main cariogenic bacteria of the mankind, and it causes dental caries mechanism and is that it can stick and is colonizated in facing that fermentation sucrose produces acid, and possesses the survival and reproduction ability under sour environment.And it facing stick field planting and bacterial plaque form in most important two protein ingredients: glucosyltransferase (glucosyltransferase, GTF) and glucan-binding protein (glucan-binding proteins Gbps) is the key factor that dental caries takes place.GTF has glucosyl transferase activity and glucosan simultaneously in conjunction with activity, and it causes the Streptococcus mutans of coding GTF gene delection dental caries power and obviously reduce (Yamashita, Y and Bowen, W.H., Burne, R.A.Infect.Immun.1993 61:3811 3817.).Childers etc. are that the anti-caries vaccine of main immunogens development has carried out a large amount of clinical experimental studies with GTF, finding that this vaccine can be induced experimenter IgA to produce by the immunity of intranasal chamber and be excited keeps body to the antigenic secondary immune response of S.Mutans, reduce the incidence rate of dental caries, also confirm simultaneously the safety (1.Childers that the isolating natural GTF of S.Mutans uses at human body as immunogen, N.K. and Tong, G, Michalek, S.M.Oral Microbiol.Immunol.1997.12:329 335; 2.Childers NK and Li F, Kirk K.J Dent Res2003; 82 (special issue); 3.Dasanayake AP and Li Y, Kirk K.Oral MicrobiolImmunol 2003; 18:271 277; 4.Childers NK and Li F, Dasanayake AP.OralMicrobiol Immunol.2006 21 (5): 309-13).And (glucan-binding region GLU) can combine with glucosan, and mediation S.Mutans plays a significant role in the bacterial plaque maturation process the gathering of facing, and is the principal element that dental caries takes place in the glucosan land of GTF c-terminus.GLU has good immunogenicity and immune protective, and it can efficiently induce anti-GTF antibody, thereby suppresses the ability of the synthetic glucosan of GTF, reduces the formation of bacterial plaque.(1.Q.A.Xu and F.Yu, M.W.Fan.Vaccine 2007 25:11911195; 2.Jespersgaard C and Hajishengallis G, Greenway TE.Infect Immun1999 67 (2): 810-81; 3.Martin A.Taubman and Cynthia J, Holmberg.Infect.Immun 2001:69,4,210 4216)
Gbps initially sticks and gathers in conjunction with the dependent S.Mutans of glucosan mediation sucrose, plays a significant role in the bacterial plaque maturation.S.Mutans produces 4 class Gbps promptly at least: GbpA, GbpB, GbpC and GbpD.But have only GbpB can bring out strong protective immunity at dental caries; subcutaneous injection or intranasal immunization route by the saliva district all can be induced high-caliber IgA level, suppress (Smith, DJ and the Taubman of sticking of S.Mutans; MA, Infect-Immun.1996 64 (8): 3069-73.).Epidemiological study simultaneously is presented at great majority and does not infect the saliva IgA that has anti-GbpB in the S.Mutans children's salivary, the saliva IgA level that anti-GbpB is described plays key effect (Nogueira in sticking at the initial stage of suppressing S.Mutans, R.D and A.C.Alves, M.H.Napimoga.Infect.Immun.73:5675 5684.).Smiths etc. are by finding that to the analysis of gene sequences of GbpB there are three sections immunodominant region polypeptide epitopes that closely link to each other at GbpB albumen n end 1/3rd places, wherein two sections polypeptide (SYI and QGQ) have been synthesized, and in zoopery, detect its immunizing potency, all in the rat body, induce the serum IgG and the saliva IgA antibody of high concentration, and this antibody can both with GbpB albumino reaction (Daniel J.Smith and William F.King, Leigh A.Barnes.Infect Immun 2,003 71 (3): 11791184).Nogueira etc. analyze IgA antibody response in the children's salivary, confirm that GbpB protein N terminal 52～132 amino acids sequences have good immunoreactivity, closely related with anti-GbpB antibody horizontal in the saliva, play key effect (Ruchele D.Nogueira and Alessandra C.Alves in may sticking at the initial stage of suppressing S.Mutans, William F.King.Clin Vaccine Immunol, June 2007,14 (6) p.804807).We think that this section polypeptide is the main immunodominant region of GbpB (GbpB Immunodominant Regions, GIR), epitope is concentrated, even can cause the antibody response stronger than GbpB whole protein, a large amount of irrelevant polypeptide have been removed simultaneously, reduce the danger of cross reaction, can be used as an ideal anti-caries vaccine candidate antigens.
The ideal goal of anti-caries vaccine is can induce to produce IgA antibody response special, effective, that continue in saliva, disturbs 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, could produce a large amount of secretory IgAs, obtain effective immune protective effect.Mucosa-immune adjuvant commonly used at present mainly contains colitoxin B subunit (LTB) and choleratoxin B subunit (CTB), and Comparatively speaking, LTB toxicity is lower, is more conducive to the clinical development of vaccine.In the S.Mutans course of infection; owing to there is complicated mechanism of action between host and the pathogenic bacterium; the vaccine that single antigen component makes up is difficult to produce effective protective effect; with the intramolecularly Adjuvanted vaccines of fusion gene mode with multi-resistance ultimate constituent and mucosa-immune adjuvant structure; can the stronger immunne response of excitating organism; simultaneously both reduced the purification cost, can guarantee again that the fusion rotein quality was stable, differences between batches are little.
Summary of the invention
The present invention is direct at cause the protectiveness subunit antigen molecule that plays a significant role in the dental caries pathogenesis at S.Mutans in the selection of vaccine antigen; the combination of emphasizing the critical function district of a plurality of virulence factor protective antigens and immundominance section is to induce more fully immunological effect; and in conjunction with the work of mucosa-immune adjuvant in order to excite stronger persistent protective effect, a kind of human streptococcus mutans genetic engineering vaccine for decayed tooth and preparation method thereof is provided.
Human streptococcus mutans genetic engineering vaccine for decayed tooth provided by the invention comprises N end two kinds of immune protective antigens of immunodominant region GIR and the mucosa-immune adjuvant E.coli LT B subunit LTB of glucosan land GLU, the glucan-binding protein B of Streptococcus mutans glucosyltransferase C-terminal at least.
The present invention connects N end immunodominant region GIR, the E.coli LT B subunit LTB of glucosan land GLU, the glucan-binding protein B of Streptococcus mutans glucosyltransferase C-terminal construction of fusion protein at gene level, and then obtains the vaccine antigen of different protective antigen subunit antigen amalgamation modes.Preferably, its Build Order is GLU-GIR-LTB.
Preferential following connected mode and the connexon of adopting of the present invention:
1. the N of glucan-binding protein B end immunodominant region GTR with E.coli LT B subunit LTB connected mode is: the encoding gene of GIR is connected with the LTB gene by one section nucleotide sequence of coding connexon, thereby obtains fusion rotein GIR-LTB.The gene order of encoding said fusion protein can be nucleotide sequence shown in Figure 1 or the nucleotide sequence that has the genetic code degeneracy with it.Described encoding gene with GIR with the mode that the LTB gene is connected is: GIR is positioned at 5 ' end, (glycine-serine-glycine-glycine-serine-glycine, one section sequence GSGGSG) are connected in 3 ' end of GIR gene to the encoding gene of LTB by the coding connexon.
2. the preferred connected mode of the N of glucosan land GLU, the glucan-binding protein B of glucosyltransferase C-terminal end immunodominant region GIR, E.coli LT B subunit LTB is: above-mentioned fusion rotein GIR-LTB is connected with the GLU gene by one section nucleotide sequence of coding connexon, thereby obtains fusion rotein GLU-GIR-LTB.The gene order of encoding said fusion protein is the coding nucleotide sequence shown in Figure 3 or the nucleotide sequence that has the genetic code degeneracy with it of coding same protein.The structure of described fusion rotein is the aminoacid sequence shown in the SEQ ID NO:4.Describedly with the GIR-LTB fusion gene with the optimal way that the GLU gene is connected be: the GIR-LTB fusion gene is positioned at 3 ' end, (tyrosine-alanine-proline-glutamine-aspartic acid-proline, one section nucleotide sequence YAPQDP) are connected in 5 ' end of GIR-LTB fusion gene to the GLU encoding gene by the coding connexon.Connect 6 polyhistidyl coded sequences at GLU-GIR-LTB E.coli LT B subunit LTB gene 3 ' end.The fusion rotein that obtains has higher expression, has set up the purifying process of fusion rotein GLU-GIR-LTB, and preliminary animal experiment proves that this fusion rotein can stimulate body to produce immunne response efficiently.
The aminoacid sequence of the fusion rotein of preferred human streptococcus mutans genetic engineering vaccine for decayed tooth provided by the present invention is the aminoacid sequence shown in the SEQ ID NO:4.
The present invention also provides a kind of method for preparing human streptococcus mutans genetic engineering vaccine for decayed tooth, and it may further comprise the steps:
1) cultivates Streptococcus mutans, obtain its genomic DNA;
2) encoding gene of clone GLU of Streptococcus mutans and GIR and be template clone LTB encoding gene with the pET-11c-LTB plasmid that this chamber makes up and preserves;
3) GIR and the LTB encoding gene to obtain to obtain, gene fusion construct GIR-LTB expression plasmid;
4) GLU and the GIR-LTB to obtain, gene fusion construct GLU-GIR-LTB expression plasmid;
5) transform the host bacterium with fusion gene GLU-GIR-LTB expression plasmid, obtain destination protein; And
6) separate also purification destination protein, make genetic engineering vaccine for decayed tooth
Wherein, gene fusion construct GIR-LTB expression plasmid may further comprise the steps:
1) obtains GIR and LTB encoding gene with the PCR method amplification;
2) obtain the encoding gene of fusion gene GIR-LTB with the method for overlap extension PCR;
3) cloned plasmids of usefulness restriction enzyme digestion GIR-LTB;
4) the enzyme action product separates through agarose gel electrophoresis;
5) the target DNA electrophoresis band on the cutting-out agarose gel;
6) the recovery product cloning of GIR-LTB genes of interest is gone into procaryotic cell expression carrier pET28a (+), obtain recombiant plasmid pET28a (+)-GIR-LTB, and
7) will contain pET28a (+)-GIR-LTB transformed into escherichia coli;
Described gene fusion construct GLU-GIR-LTB expression plasmid can may further comprise the steps:
1) obtains the GLU encoding gene with the PCR method amplification;
2) with restriction enzyme digestion GLU and recombiant plasmid pET28a (+)-GIR-LTB;
3) the enzyme action product separates through agarose gel electrophoresis;
4) the target DNA electrophoresis band on the cutting-out agarose gel;
5) the recovery product enzyme with GLU and recombiant plasmid pET28a (+)-GIR-LTB connects, and obtains to contain the recombiant plasmid of GLU-GIR-LTB gene, and
6) will contain the recombinant plasmid transformed escherichia coli of GLU-GIR-LTB gene.
The gene order of fusion gene GIR-LTB is shown in SEQ ID NO:1, and the aminoacid sequence of fusion rotein GIR-LTB is shown in SEQ ID NO:2; The gene order of fusion gene GLU-GIR-LTB is shown in SEQ IDNO:3, and fusion rotein GLU-GIR-LTB aminoacid sequence is shown in SEQ ID NO:4.
Gene engineering recombinant bacterium of the present invention and recombination fusion protein have following fundamental characteristics: 1. the host bacterium is BL21, and expression vector is pET-28a; 2. recombiant protein is with the inclusion body formal representation, and molecular weight is 55kDa; 3. the Recombinant Protein Expression amount is more than 35%; The recombiant protein of purification can produce higher titer antibody by induced animal, and good immunoprotective effect is arranged.
The present invention adopts the genetic engineering means construction expression to comprise adjuvant amalgamation protein vaccine in two kinds of protective antigen sub-unit molecules, and income earner's streptococcus mutans genetic engineering vaccine for decayed tooth is convenient to separation and purification, stimulates body to produce immunne response efficiently.
For above and other objects of the present invention, feature and advantage can be become apparent, preferred embodiment cited below particularly, and conjunction with figs. are described in detail below:
Description of drawings
Fig. 1 recombiant plasmid pET28a (+)-GIR-LTB construction strategy sketch map;
Fig. 2 recombiant plasmid pET28a (+)-GLU-GIR-LTB construction strategy sketch map;
Fig. 3 is the PCR clonal expansion of the object of the invention gene GIR and LTB
The result shows that the PCR clonal expansion of genes of interest GIR and LTB is respond well;
Fig. 4 is the overlap extension PCR clonal expansion of fusion gene GIR-LTB
The arrow indication is the pcr amplification product (588bp) of fusion gene GIR-LTB in the swimming lane 1;
Although the result shows at the 300bp place assorted taking out of now arranged, still obtained fusion gene GIR-GIR overlap extension PCR clone product;
Fig. 5 is that the enzyme action of recombiant plasmid pET28a (+)-GIR-LTB is identified
The endonuclease bamhi size is consistent with design, tentatively proves the construction of recombinant plasmid success, and genes of interest connects correct;
Fig. 6 is the PCR clonal expansion of genes of interest GLU
The result shows that the PCR clonal expansion of genes of interest GIR and LTB is respond well;
Fig. 7 is that the enzyme action of recombiant plasmid pET28a (+)-GLU-GIR-LTB is identified
The endonuclease bamhi size is consistent with design, tentatively proves the construction of recombinant plasmid success, and genes of interest connects correct;
Fig. 8 is a recombination engineering GIR-GIR-LTB/BL21 abduction delivering PAGE electrophoretogram
(arrow is depicted as recombination fusion protein GLU-GIR-LTB)
Gene recombination bacterium is through the protein expression band of increase is arranged at molecular weight 55KDa place after inducing, and is consistent with the destination protein molecular weight.Analyze through UVP image scanning, induce 4 hours destination protein expressions about 35%;
Fig. 9 is a destination protein GLU-GIR-LTB purification effect PAGE electrophoretogram
The result shows through purification step, the purity of destination protein be improved significantly, the destination protein peak of results reaches 92.5% through UVP scanning analysis purity;
Figure 10 and Figure 11 are respectively fusion gene GIR-LTB and GLU-GIR-LTB sequencing result figure.
The specific embodiment
Below in conjunction with drawings and Examples the present invention is further described; these examples are used for the preparation method of a kind of vaccine of the present invention is described; all the other compound modes similarly; be understood that; these examples are to be used for the present invention; rather than limitation of the present invention, under design prerequisite of the present invention,, all belong to the scope of protection of present invention to the simple modifications of preparation method of the present invention
The present invention adopts the technology path and the key step of technique scheme:
1.GIR-LTB the structure of fusion gene
1) the GIR encoding gene of clone Streptococcus mutans UA159 (Medical University Of Anhui's stomatological hospital) and LTB encoding gene (the pET-11c-LTB plasmid that this chamber makes up and preserves).
1. cultivate Streptococcus mutans UA159
2. with the GIR encoding gene of PCR method amplification Streptococcus mutans UA159 and the encoding gene of LTB.
3. the clone of PCR product.
2) fusion gene of structure GIR and LTB.
1. be template with GIR and LTB, with overlap extension pcr amplification fusion gene GIR-LTB.
2. the PCR product separates through agarose gel electrophoresis.
3. downcut the target DNA electrophoresis band on the agarose gel.
4. the recovery product cloning of GIR-LTB genes of interest is gone into procaryotic cell expression carrier pET28a (+), obtain recombiant plasmid pET28a (+)-GIR-LTB, and
The recombinant plasmid transformed escherichia coli that 5. will contain the GIR-LTB gene;
3) sequence of mensuration GIR-LTB fusion gene.
2. the structure of prokaryotic expression plasmid pET28a (+)-GLU-GIR-LTB.
1) fusion gene of structure GLU-GIR-LTB.
1. obtain the GLU encoding gene of Streptococcus mutans with the PCR method amplification;
2. with restriction enzyme digestion GLU and the recombiant plasmid that contains the GIR-LTB gene;
2. the enzyme action product separates through agarose gel electrophoresis;
3. downcut the target DNA electrophoresis band on the agarose gel;
4. the enzyme action recovery product with GLU and recombiant plasmid pET28a (+)-GIR-LTB is connected, and obtains to contain the recombiant plasmid of GLU-GIR-LTB gene, and
The recombinant plasmid transformed escherichia coli that 5. will contain the GLU-GIR-LTB gene;
2) sequence of mensuration GLU-GIR-LTB fusion gene.
3) abduction delivering GLU-GIR-LTB fusion gene obtains destination protein GLU-GIR-LTB.
4) purifies and separates destination protein GLU-GIR-LTB.
5) purity of evaluation destination protein GLU-GIR-LTB.
This destination protein GLU-GIR-LTB is the recombinate glucosan land (glucan-binding region of Streptococcus mutans glucosyltransferase C-terminal of pattern of fusion people, GLU) and the N-terminal immunodominant region of glucan-binding protein B (GbpB Immunodominant Regions, GIR) and the genetic engineering vaccine for decayed tooth of mucosa-immune adjuvant LTB.
Embodiment 1The structure of the expression vector of fusion gene GIR-LTB
1. design of primers and pcr amplification
1) design of primers is as follows: gene and heat-labile toxin B subunit LTB gene according to the S.Mutans that finds from GenBank (UA159 type strain) coding GIR, utilize software Primer Premier5.0 to carry out primer design and analysis.Linker (frame district part) sequence is designed respectively in 5 of upstream gene (GIR) ' end and introducing BamHI site, 3 ' end is introduced linker (frame district part) sequence, introduce linker sequence (frame district part) at 5 of downstream gene LTB ' end simultaneously, 3 ' end is introduced the XhoI site, utilizes the overlap extension PCR method that GIR gene and LTB gene are coupled together.Primer is synthetic by the handsome company in Shanghai.
GIR: forward primer P1:SEQ ID NO:5
5’-
CAAGCACAAGTTAAT-3’(BamHI)
Downstream primer P2:SEQ ID NO:6
5’-
ATCAGAAACTGATTT-3’
LTB: forward primer P3:SEQ ID NO:7
5’-
GCTCCCCAGTCTATT-3’
Downstream primer P4:SEQ ID NO:8
5’-CTCGAGGTTTTCCATGCTGATTGC-3’(XhoI)
2) pcr amplification of genes of interest
The recombiant plasmid pET-11c-LTB that makes up and preserve with human streptococcus mutans UA159 genomic DNA (boiling brokenly the bacterium supernatant) and this chamber is a template respectively.94 ℃ of pre-degeneration 5 minutes were carried out 30 circulations in 1 minute according to 94 ℃ 30 seconds → 55 ℃ 30 seconds → 72 ℃, and last 72 ℃ were extended amplification GIR and LTB genetic fragment 10 minutes.(amplification as shown in Figure 3)
3) overlap extension pcr amplification GIR-LTB fusion gene
With 2) in amplification PCR to reclaim product be template, with P1, P4 is a primer, 94 ℃ of pre-degeneration 5min carry out 35 circulations according to 94 ℃ of 30s → 57 ℃ 30s → 72 ℃ of 1min, last 72 ℃ are extended 10min, amplify the GIR-LTB fusion gene.(amplification as shown in Figure 4)
2.GIR-LTB the structure of integrative gene expression vector (the plasmid construction strategy as shown in Figure 1)
1) clone of GIR-LTB fusion gene
With 3) in the GIR-LTB fusion gene fragment cloning that obtains of amplification go into pMD18-T, Transformed E .coliDH5 α, ammonia benzyl resistance screening positive recombinant, the extracting plasmid is done double digestion with NcoI and XhoI and is identified, identifies correct back recovery genetic fragment.
2) structure of GIR-LTB integrative gene expression vector
With 1) in the GIR-LTB fusion gene fragment that reclaims be implemented in expression vector pET-28a (+), do the double digestion evaluation with BamHI and XhoI.(the enzyme action result as shown in Figure 5)
3) enzyme action is identified errorless gene recombination plasmid is converted into host bacterium E.coli BL21 (DE3), obtained recombination engineering
4) recombination engineering GIR-LTB/BL21 serves the order-checking of extra large handsome company, and sequencing result as shown in figure 10.
Embodiment 2The construction and expression of GLU-GIR-LTB integrative gene expression vector and recombinant expressed type engineering bacteria
1. design of primers and pcr amplification
1) design of primers is as follows: the gene according to the S.Mutans that finds from GenBank (UA159 type strain) coding GLU, utilize software Primer Premier5.0 to carry out primer design and analysis.Linker (frame district part) sequential design at 3 of GLU encoding gene ' end, and is introduced NcoI and BamHI site at the upstream and downstream primer respectively,, can correctly be connected with fusion gene (GIR-LTB) with the sticky end of BamHI enzyme action.Primer is synthetic by the handsome company in Shanghai.
GLU: forward primer P1:SEQ ID NO:9
5’CCATGGATGAAATGGGCTATCAAGC-3’
Downstream primer P2:SEQ ID NO:10
5’-
CCGAACTCGTTCTCCAG-3’
2) pcr amplification of genes of interest
With Streptococcus mutans UA159 genomic DNA (boiling brokenly the bacterium supernatant) is template.94 ℃ of pre-degeneration 5 minutes were carried out 30 circulations in 1 minute according to 94 ℃ 30 seconds → 58 ℃ 30 seconds → 72 ℃, and last 72 ℃ were extended 10 minutes, and expanded the GLU target gene fragment.(amplification procedure makes up the result as shown in Figure 6 as shown in Figure 2)
2.GLU-GIR-LTB the structure of integrative gene expression vector and recombinant expressed type engineering bacteria
1) clone of PCR product
The GLU gene fragment clone that obtains is gone into pMD18-T, Transformed E .coli DH5 α, ammonia benzyl resistance screening positive recombinant, the extracting plasmid is done double digestion with NcoI and BamHI and is identified, identifies that correctly genetic fragment is reclaimed in the back.
2) structure of GLU-GIR-LTB integrative gene expression vector
With 1) in the GLU fusion gene fragment that reclaims be implemented in integrative gene expression vector pET-28a (+)-GIR-LTB, carry out 2 double digestions respectively and identify: NcoI and BamHI; NcoI and XhoI.(the enzyme action result as shown in Figure 7)
3) enzyme action is identified errorless gene recombination plasmid is converted into host bacterium E.coli BL21 (DE3), obtained recombination engineering.
4) recombination engineering GLU-GIR-LTB/BL21 serves the order-checking of extra large handsome company, and sequencing result as shown in figure 11.
3. the abduction delivering of recombination engineering and form are identified
Recombination engineering is cultivated in the LB fluid medium that contains kanamycin (50 μ g/ml), to bacterium liquid OD
600Add during ≈ 0.6 IPTG to final concentration be 1.0mmol/L, induce different time to collect bacterium liquid, ultrasound wave break bacterium, differential centrifugation is left and taken cleer and peaceful precipitation respectively, carries out gel electrophoresis evaluation expression-form and expression after the processing.(abduction delivering and form are identified as shown in Figure 8)
4. the purification of fusion rotein GLU-GIR-LTB
German B.Braun 10L fermentation tank is adopted in the fermentation of reorganization bacterium, carries out according to this laboratory engineering bacterium fermentation common process.Centrifugal collection antibacterial after the fermentation ends, the back of weighing is frozen standby.The resuspended antibacterial of TE buffer with pH value 7.8, break bacterium through the high-pressure homogenization instrument, differential centrifugation is collected inclusion body, use the TE buffer that contains 1%Triton X100 respectively and contain 1M, 2M carbamide TE buffer washing inclusion body, with 8M carbamide dissolving inclusion body, utilize after the dialysis albumen with 6 His labels, adopt the method for affinity chromatograph to carry out protein purification.
5. purified target protein is carried out SDS-PAGE, examines and determine its purity, and destination protein purity reaches (purification result as shown in Figure 9) more than 90%
Embodiment 3The immunogenicity pre-test of fusion rotein
1. immune mouse
Destination protein GLU-GIR-LTB is through the Balb/c mice in 4～5 ages in week of immunity behind the purification, and 100ug//time, 100 μ L antigens mix with equivalent Fu Shi Freund's complete adjuvant, injection mouse web portion and the subcutaneous immunity of groin.Immune programme for children is: 0,1,2 weeks, totally 3 times, 1st, add the Fu Shi Freund's complete adjuvant 2 times, do not add adjuvant the 3rd time, be inoculated in mouse web portion and groin is subcutaneous, the amount of injections of antigens and adjuvant is about 0.2ml, and is weekly, the 3rd immunity docking blood sampling in back 6 days, ELISA detects the change of serological specificity antibody titer.
2. the detection of specific immune response.
(1) preparation of ELISA antigen coated microplate:
GLU antigen is diluted to 5 μ g/ml with coating buffer, and wrap by elisa plate in 100 μ l/ holes, and 4 ℃ are spent the night.Cleaning mixture is washed 4 times.Every hole adds 300 μ l 1%BSA, 37 ℃ of sealing 2h, and after cleaning mixture was washed 4 times, 4 ℃ of preservations were standby.
(2) collection of mice serum specimen:
Serum is gathered: respectively at before the immunity and after the immunity 2,3 times 7 days, will every about 100 μ l of mice docking blood sampling, and the aseptic EP pipe of 1.5ml is collected, and room temperature is placed 1h, 5000g * 10min, collection serum, packing ,-70 ℃ of preservations
(3) detection of serological specificity IgG antibody:
The detection of serum antigen specific IgG: from 1:5000, with antibody diluent doubling dilution serum to be checked, 1:100 dilutes negative serum, 60min is hatched for 37 ℃ in 100 μ l/ holes, and cleaning mixture is washed 4 times, add the sheep anti-mouse igg (1:20000) of HRP labelling, 30min is hatched for 37 ℃ in 100 μ l/ holes, cleaning mixture is washed 4 times, add OPD substrate colour developing liquid, room temperature lucifuge reaction 30min, 50 μ l stop buffer cessation reactions, make blank with the PBS hole, 492nm measures each hole OD value.
3. result
Antibody positive rate after the GLU-GIR-LTB immunity 3 times is 92%, and the antibody positive rate after the immunity 4 times reaches 95%.Prompting fusion rotein GLU-GIR-LTB can effectively stimulate body to produce stronger immunne response, is a kind of good dental caries vaccine candidate antigens.
Though the present invention discloses as above with preferred embodiment; right its is not in order to limit the present invention; any person of ordinary skill in the field; without departing from the spirit and scope of the present invention; when can doing a little change and improvement, so protection scope of the present invention is as the criterion when looking the claim person of defining.
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＜110〉Military Medical Univ No.3, P.L.A
＜120〉a kind of human streptococcus mutans genetic engineering vaccine for decayed tooth and preparation method thereof
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<400>9
<210>10
<211>34
<212>DNA
＜213〉GLU downstream primer sequence
<400>10
Claims (7)
1. human streptococcus mutans genetic engineering vaccine for decayed tooth; it is characterized in that, comprise N end immunodominant region fragment GIR and three kinds of immune protective antigens of E.coli LT B subunit LTB of glucosan land GLU, the glucan-binding protein B of Streptococcus mutans glucosyltransferase C-terminal at least.
2. recombinant vaccine according to claim 1, it is characterized in that its N end immunodominant region fragment GIR and E.coli LT B subunit LTB for glucosan land GLU, the glucan-binding protein B of described Streptococcus mutans glucosyltransferase C-terminal connects the fusion rotein that structure forms at gene level.
3. recombinant vaccine according to claim 2, the Build Order that it is characterized in that described fusion rotein is GLU-GIR-LTB.
4. recombinant vaccine according to claim 3, the aminoacid sequence that it is characterized in that described fusion rotein are the aminoacid sequence shown in the SEQ ID NO:4.
5. method for preparing human streptococcus mutans genetic engineering vaccine for decayed tooth is characterized in that comprising step:
1) cultivates Streptococcus mutans, obtain its genomic DNA;
2) encoding gene of clone GLU of Streptococcus mutans and GIR and be template clone LTB encoding gene with the pET-11c-LTB plasmid;
3) based on the encoding gene of the GIR that obtains, gene fusion construct GIR-LTB expression plasmid;
4) GLU and the GIR-LTB to obtain, gene fusion construct GLU-GIR-LTB expression plasmid;
5) fusion gene GLU-GIR-LTB expression plasmid transforms the host bacterium, obtains destination protein;
6) separate also purification destination protein GLU-GIR-LTB, make genetic engineering vaccine for decayed tooth.
6. genetic engineering vaccine for decayed tooth preparation method according to claim 5 is characterized in that, described gene fusion construct GIR-LTB expression plasmid may further comprise the steps:
1) obtains GIR and LTB encoding gene with the PCR method amplification;
2) obtain the encoding gene of fusion gene GIR-LTB with the method for overlap extension PCR;
3) cloned plasmids of usefulness restriction enzyme digestion GIR-LTB;
4) the enzyme action product separates through agarose gel electrophoresis;
5) the target DNA electrophoresis band on the cutting-out agarose gel;
6) the recovery product cloning of GIR-LTB genes of interest is gone into procaryotic cell expression carrier, obtain to contain the recombiant plasmid of GIR-LTB gene, and
7) will contain the recombinant plasmid transformed escherichia coli of GIR-LTB gene;
7. genetic engineering vaccine for decayed tooth preparation method according to claim 5 is characterized in that, described gene fusion construct GLU-GIR-LTB expression plasmid may further comprise the steps:
1) obtains the GLU encoding gene with the PCR method amplification;
2) with restriction enzyme digestion GLU and the recombiant plasmid that contains the GIR-LTB gene;
3) the enzyme action product separates through agarose gel electrophoresis;
4) the target DNA electrophoresis band on the cutting-out agarose gel;
5) the recovery product cloning of GLU is gone into procaryotic cell expression carrier, obtain to contain the recombiant plasmid of GLU-GIR-LTB gene, and
6) will contain the recombinant plasmid transformed escherichia coli of GLU-GIR-LTB gene.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2009101030840A CN101474400A (en) | 2009-01-19 | 2009-01-19 | Human streptococcus mutans genetic engineering vaccine for decayed tooth and preparation method thereof |
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|---|---|---|---|
| CNA2009101030840A CN101474400A (en) | 2009-01-19 | 2009-01-19 | Human streptococcus mutans genetic engineering vaccine for decayed tooth and preparation method thereof |
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| CNA2009101030840A Pending CN101474400A (en) | 2009-01-19 | 2009-01-19 | Human streptococcus mutans genetic engineering vaccine for decayed tooth and preparation method thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102772792A (en) * | 2012-06-19 | 2012-11-14 | 重庆原伦生物科技有限公司 | Vaccine for preventing decayed tooth and preparation method thereof |
| CN104940920A (en) * | 2015-06-12 | 2015-09-30 | 武汉大学 | Dental caries protein vaccine and preparation method thereof |
-
2009
- 2009-01-19 CN CNA2009101030840A patent/CN101474400A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102772792A (en) * | 2012-06-19 | 2012-11-14 | 重庆原伦生物科技有限公司 | Vaccine for preventing decayed tooth and preparation method thereof |
| CN102772792B (en) * | 2012-06-19 | 2014-02-26 | 重庆原伦生物科技有限公司 | Vaccine for preventing decayed tooth and preparation method thereof |
| CN104940920A (en) * | 2015-06-12 | 2015-09-30 | 武汉大学 | Dental caries protein vaccine and preparation method thereof |
| CN104940920B (en) * | 2015-06-12 | 2018-02-09 | 武汉迈伦口腔科技有限责任公司 | A kind of anti-caries protein vaccine and preparation method thereof |
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