CN1419938A - Fusion anticaries DNA vaccine and prepring method thereof - Google Patents

Abstract

The invention discloses a fusion anti-caries DNA vaccine and a preparation method thereof, Escherichia coli (Esherichiacoli) JM109/pGLUA-P CCTCC NO: M201042. Using the PCR method, the sequence GLU encoding the glucan binding region was amplified from the plasmid pYNB13 carrying the glucosyltransferase GTF-I gene gtfB of Streptococcus mutans GS-5 strain, and cloned into the eukaryotic vector pCI , construct the eukaryotic expression plasmid pGLU. The correctness of the inserted sequence was confirmed by DNA sequence determination. Then the A-P fragment sequences of the A region and P region of the mutans surface protein PAc in the pCIA-P plasmid were connected with the pGLU plasmid to construct the GTF-PAc fusion anti-caries DNA vaccine pGLUA-P. The method of the invention is simple, high in safety, low in production cost, durable in immune response and effective in caries prevention.

Description

A fusion anti-caries DNA vaccine and its preparation method

Technical field

The invention belongs to the technical field of oral preventive medicine, and in particular relates to a preparation method of PAc and GTFs fusion anti-caries DNA vaccine.

Background technique

Caries is a kind of bacterial infectious disease. Many scholars have proved that the genus Mutans Streptococci is highly related to caries, among which Streptococcus mutans and Streptococcus sobribus are the most important pathogenic bacteria that cause human caries. Glucosyltransferases (glucosyltransferaesGTFs) and surface protein (surface protein antigen PAc) (also known as antigen B, I/II, IF, P1) are important cariogenic virulence factors of Streptococcus mutans. GTF _s synthesized glucan and mediated the dextran-dependent adhesion of Streptococcus mutans on the tooth surface, and PAc participated in mediating the non-glucan-dependent adhesion of bacteria to the acquired membrane on the tooth surface. The alanine-rich A region and the proline-rich P region in the PAc protein are the main adhesion functional regions and immunologically active regions; _GTFs contain two immunologically active regions and functional regions: catalytic region (catalytic region) CAT) and glucan binding region (glucan binding region GLU). Streptococcus mutans mainly synthesizes three _GTFs : GTF-I, GTF-SI, and GTF-S, among which GTF-I mainly synthesizes water-insoluble glucan mainly composed of α-1,3 glycosidic bonds, and its coding gene is gtfB .

Taubman et al. mixed the short peptide CAT synthesized according to the 448-457 amino acid sequence of the GTF-I catalytic functional region of Streptococcus downei and the short peptide GLU synthesized from the 1303-1324 amino acid sequence of the dextran binding functional region with complete Freund's adjuvant SD rats after immunization can elicit significant serum and saliva anti-Streptococcus sobrinus glucosyltransferase antibodies (Taubman, M.A., D.J.Smith, C.J.Holmberg, and J.W.Eastcott.2000. Coimmunization with complementary glucosyltransferase peptides results in enhanced immunogenicity stdental carinies protection . Infect Immun 68:2698-703.). Jespersgaard et al. immunized rabbits with CAT and GLU polypeptides to obtain specific antibodies that could significantly inhibit GTF synthesis of glucan by Streptococcus mutans. Intranasal instillation of CAT and GLU polypeptides into mice induced a significant mucosal IgA immune response (Jespersgaard, C., G.Hajishengallis, T.E.Greenway, D.J.Smith, M.W.Russell, and S.M.Michalek.1999.Functional and Immunogenic characterization of two cloned regions of Streptococcus mutans glucosyltransferase I. Infect Immun 67:810-6.). Saito et al. used the mutans streptococcus surface protein antigen (PAc) and the mucosal adjuvant cholera toxin A subunit mutant (mCT E112K) to immunize mice through the nasal mucosa. As a result, obvious specificity was detected in saliva and nasal secretions. Secreted anti-PAc antibodies, and a large number of cells secreting specific secreted anti-PAc antibodies were detected in the submandibular gland and nasal cavity. After re-stimulation in vitro, CD4(+) cells in cervical lymph nodes were found to proliferate significantly. Moreover, after immunizing mice, the accumulation of oral Streptococcus mutans can be significantly reduced. Explain that PAc and mCT E112K complex are expected to become effective mucosal anti-caries vaccine (Saito, M., S.Otake, et al. (2001). "Protective Immunity to Streptococcus mutans Induced by Nasal Vaccination with Surface Protein Antigen and Mutant Cholera Toxin Adjuvant . "J Infect Dis 183(5): 823-826.). Oishi et al. believe that immunizing animals with a 13 peptide derived from the A region of the PAc protein alone can hardly produce antibodies, but adding amino acid residues, especially lysine fragments, between the dimer peptides can significantly enhance the 13 peptide in the A region of the PAc protein. Antigenicity of peptides (Oishi, Y., A. Onozuka, et al. (2001). "The effect of amino acid spaces on the antigenicity of dimeric peptide-inducing cross-reacting antibodies to a cell surface protein antigen of Streptococcus mutans."Oral Microbiol Immunol 16(1):40-44.). Oho et al constructed a fusion protein vaccine comprising the GLU region of Streptococcus mutans surface protein antigen (PAc) and glucosyltransferase (GTF), and immunized cows to obtain specific antibodies containing anti-PAc and GTF, which can inhibit Adhesion of Streptococcus mutans to saliva-coated hydroxyapatite beads. (Oho, T., Y. Shimazaki, M. Mitoma, M. Yoshimura, Y. Yamashita, K. Okano, Y. Nakano, H. Kawagoe, M. Fukuyama, N. Fujihara, and T. Koga. 1999. Bovine milkantibodies against cell surface protein antigen PAc-glucosyltransferase fusion protein suppress cell adhesion and alter glucan synthesis of Streptococcus mutans. J Nutr129: 1836-41.)

However, there are many deficiencies in the above various anti-caries peptide vaccines. Firstly, the preparation and purification of these peptides is difficult, generally takes several weeks and is expensive, and special conditions are required for storage; secondly, peptide anti-caries vaccines stimulate the body to produce effective immunity. The ability to respond is poor, and it is often necessary to add various immune adjuvants that are harmful to the human body; peptide anti-caries vaccines, like other peptide vaccines, generally have a short duration of induction of specific immune responses, and repeated vaccinations are required to achieve satisfactory results In addition, peptide vaccines generally cannot induce immune responses in infants. (Kowalczyk DW, Ertl HCJ. 1999. Immune responses to DNA vaccines. CMLS, Cell. Mol. Life Sci., 55:751-770).

Contents of Invention

The purpose of the present invention is to provide a fusion anti-caries DNA vaccine, the sequence GLU of the glucan-binding region of the gene gtfB of the amplified coding Streptococcus mutans GS-5 strain glucosyltransferase GTF-I is cloned into a true The pGLUA-P fusion anti-caries DNA vaccine was obtained from the nuclear expression plasmid pCIA-P.

Another object of the present invention is to provide a preparation method for the fusion anti-caries DNA vaccine, which has a simple production method, low cost, simple purification and high product purity.

Escherichia coli (Escherichia coli) JM109/pGLUA-P provided by the present invention, CCTCC NO: M201042. It is characterized in that it uses the method of polymerase chain reaction (PCR), adopts positive strand primer: 5'TCACTCGAGGTACCGGAGAAATGGGCTATCAA3'; The sequence GLU encoding the glucan-binding region was amplified from the plasmid pYNB13 of the gene gtfB of -I, and cloned into the eukaryotic vector pCI to construct the eukaryotic expression plasmid pGLU. The correctness of the inserted sequence was confirmed by DNA sequence determination. Then, the A-P fragment sequences of the A region and the P region of the mutans surface protein PAc in the pCIA-P plasmid were connected with the pGLU plasmid to construct the GTF-PAc fusion anti-caries DNA vaccine pGLUA-P. Compared with other DNA vaccines, pGLUA-P has the early promoter/enhancer (CMV IE Pro/En) of cytomegalovirus CMV, and the ability of this promoter to enhance expression is significantly higher than other such as Rous sarcoma virus promoter LTR, Promoters such as Simian Virus 40 (SV40). In addition, the auxiliary enhanced expression component of pGLUA-P also contains the intron sequence of human β-globin gene. Cell transfection and expression experiments show that the intron sequence can significantly increase gene expression, compared with only CMV promoter. Another excellent eukaryotic expression plasmid pcDNA3 (Invitrogen Company) without this intron, the expression ability of pCI is 10 to 40 times stronger. The ampicillin resistance gene (AmpR) in the pGLUA-P plasmid contains two 5'-AACGTT-3' copies, which belong to the immunostimulatory DNA sequence (ImmunostimulatoryDNA sequence, ISS), which can induce the body to produce cytokines such as IFN, promote The body's immune response to an antigen.

Compared with peptide anti-caries vaccines, pGLUA-P DNA anti-caries vaccines have the following advantages: ① pGLUA-P plasmids have stable physical and chemical properties, can be stored at room temperature, and are convenient for transportation and use, while peptide vaccines generally require special conditions such as low-temperature refrigeration; ②pGLUA-P simplifies the production steps of the vaccine, only needs to complete the upstream part of the genetic engineering, saves the expression and purification process of the downstream recombinant polypeptide, and can obtain a large number of plasmids in a short time, and the purification method of pGLUA-P plasmid is relatively simple and The purity of the product is very high, but the preparation and purification of peptide vaccines is relatively difficult and complicated, and generally takes several weeks; ③The ability of peptide anti-caries vaccines to stimulate the body to produce an immune response is poor, and various immune adjuvants need to be added, but these adjuvants often cause Severe inflammatory reactions greatly limit the application of polypeptide vaccines, and the pGLUA-P plasmid DNA vaccine itself contains CpG immunostimulatory sequences, which can be used as immune adjuvants to significantly improve the body's immune response to pGLUA-P, avoiding the use of various harmful substances Adjuvant; ④ Polypeptide vaccines generally cannot induce immune responses in infants and young children, but pGLUA-P vaccines can induce specific anti-PAc antibodies in suckling mice, which is very beneficial to protect children who are susceptible to dental caries; ⑤ pGLUA-P is as Like other DNA vaccines, after being infused into the body by any means, the GLUA-P fusion protein may be expressed in a small number of transfected cells for a long time, continuously stimulating the body's immune system and inducing a long-term immune response. On the contrary, peptide vaccines are very unstable in the body and are easily degraded by proteases. Therefore, a large amount of vaccines must be inoculated to cause an obvious immune response, and multiple inoculations are required to maintain the effect. In addition, pGLUA-P can express two important virulence factors of Streptococcus mutans. Cell and animal experiments have proved that pGLUA-P can induce the body to produce specific antibodies and inhibit the occurrence and development of dental caries. The DNA vaccine of the A-P fragment is preferred.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic diagram of the structure of PAc and GTF _s fusion anti-caries DNA vaccine CMV immediate-early enhancer/promoter Chimeric intron human β globin gene intron sequence T ₇ promoter T ₇ bacteriophage Promoter GLUA-P fragment GLUA-P recombinant DNA fragment SV40 late polyA signal SV40 late polyA signal Phage f1 region Filamentous phage f1 replicon AmpR Ampicillin resistance gene SalI (1957) Restriction endonuclease SalI digestion Site XhoI(1964) Restriction endonuclease XhoI cut site KpnI(1075) Restriction endonuclease KpnI cut site HindIII(748) Restriction endonuclease HindIII cut site

PAc and GTF _s fusion anti-caries DNA vaccine pGLUA-P carries the gene sequence of two important virulence factors of Streptococcus mutans, namely, the glucan-binding domain (GLU ) sequence and surface protein PAc encode A region and P region (AP) sequence, and have a variety of control elements that can initiate and enhance gene expression in eukaryotic cells, including: cytomegalovirus early promoter/enhancer, human beta globule Intron sequence of protein gene, late tailing signal of SV40 virus. The ability of the early promoter/enhancer of cytomegalovirus CMV (CMV immediate-early ehancer/promoter) to enhance expression is significantly higher than that of other promoters such as Rous sarcoma virus promoter LTR and monkey virus 40 (SV40); The intron sequence of the protein gene can help enhance expression. The ampicillin resistance gene (AmpR) contains two copies of 5'-AACGTT-3', which belong to the immunostimulatory DNA sequence (Immunostimulatory DNAsequence, ISS), which has the ability to induce the body Produce cytokines such as IFN to promote the body's immune response to antigens. The late polyA signal of SV40 can terminate transcription under the action of RNA polymerase II and add about 200 to 250 adenine residues to the 3' end of the RNA transcript. This polyA structure enhances RNA stability and subsequent translation. The f1 replicon is the replication source of the filamentous phage f1, which facilitates the replication of the plasmid in E. coli. T ₇ phage promoter is an excellent prokaryotic promoter, which can regulate the expression of AP fragments. Figure 2 Schematic diagram of enzyme digestion and identification of pGLUA-P1 GeneRuler 1Kb DNA Ladder2 pGLUA-P digested with MluI3 pCI digested with SalI4 pGLUA-P digested with SalI and XhoI5 pGLUA-P digested with SalI6 Lambda DNA /HindIII Marker Figure 3 Schematic diagram of the expression of GLUA-P fragments in Escherichia coli BL-21 (DE3) 1. Protein molecular weight marker 2. BL-21 containing pGLUA-P (IPTG induction) 3. BL-21 containing pGLUA-P (IPTG induction) 4. BL-21 containing pGLUA-P (no induction) 5. BL-21 containing pCI (IPTG induction) 6. BL-21 (IPTG induction) Figure 4 Recombinant plasmid pGLUA-P transfection Schematic diagram of primary mouse myoblasts (SABC method, hematoxylin staining, ×100) Figure 5 Schematic diagram of the results of ELISA detection of anti-PAc specific antibody levels

seraIgG Serum anti-PAc IgG antibody

sera IgA Serum anti-PAc IgA antibody

saliva IgG saliva anti-PAc IgG antibody

saliva IgA saliva anti-PAc IgA antibody grouping description see attached table 1 Figure 6 Schematic diagram of dental caries score analysis results

E is enamel caries, D _s is mild dentine caries, and D _m is moderate dentine caries.

Detailed ways

Below in conjunction with accompanying drawing, the present invention is described in further detail:

According to Figure 1-6, it can be seen that professional molecular biology software is used: DNASIS comprehensive DNA sequence analysis software, Gene Construction kit 2.0 gene simulation cloning software, Omiga comprehensive molecular biology analysis software, BLAST homologous sequence analysis software, Oligo primer design and analysis software, etc. The software analyzes and designs GTF-PAc fusion anti-caries DNA vaccine and simulates the whole process of cloning. The primers for the GLU region were designed as follows: positive-strand primer: 5'TCACTCGAGGTACCGGAGAAATGGGCTATCAA3'; negative-strand primer: 5'CCCGGGTTAGTCGACAATCCGAACTCGTTC3', KpnI and SalI restriction sites were introduced at the 5' ends of the positive-strand and negative-strand primers, respectively. Using the polymerase chain reaction (PCR) method, the plasmid pYNB13 carrying the gene gtfB of the glucosyltransferase GTF-I of Streptococcus mutans GS-5 strain (gifted by Kuramitsu, State University of New York) was amplified to encode dextran The sequence GLU of the sugar-binding region was cloned into the eukaryotic vector pCI (Promega Company) through KpnI and SalI multiple cloning sites to construct the eukaryotic expression plasmid pGLU. The correctness of the inserted sequence was confirmed by DNA sequence determination. Then, the pCIA-P plasmid carrying the AP fragment sequence of the A region and the P region of the surface protein PAc of Streptococcus mutans was digested with XhoI, and the end was filled with a large fragment of DNA polymerase Klenow and _dNTPs , and digested with HindIII The linearized pCIA-P plasmid with part of the vector sequence cut off was obtained; pGLU was digested with HindIII and SmaI to obtain a blunt-ended fragment containing part of the vector sequence and the entire GLU sequence; the two fragments were digested with T ₄ DAN ligase The GTF-PAc fusion anti-caries DNA vaccine pGLUA-P was constructed by connection (see Figure 1). Enzyme digestion analysis of pGLUA-P: a single band with a molecular weight of 7.1kb after single digestion with Mlu I; After digestion, three bands appeared, with molecular weights of 4.0kb, 2.2kb and 0.9kb, respectively, which were consistent with the expected results, confirming that the recombinant plasmid pGLUA-P carried GLU and AP fragments (Figure 2). Due to the connection of the 5' blunt end of SmaI and the cleaved end of XhoI after filling in, it has a completely correct reading frame, which ensures that both the AP fragment and the GLU fragment can be correctly expressed, and a correct GLUA-P fusion protein is produced.

Inoculate BL-21(DE3) Escherichia coli containing pGLUA-P into LB medium containing Amp and culture overnight at 37°C. The next day, inoculate 50 μl of the saturated culture into 5 ml of the same medium, incubate at 37°C for 2 hours, add IPTG (final concentration: 1 mM) and place on a shaker at 37°C for 5 hours to induce, centrifuge at 12,000 rpm for 1 minute, and resuspend the pellet In 40 μl of 2×SDS gel loading buffer, heat at 100°C for 3 minutes, and take another BL-21(DE3) that contains only the blank vector pCI after induction, BL-21(DE3)/pCIA-P that has not been induced Culture, BL-21 (DE3) bacteria liquid served as control. Prepare a 15% separating gel and a 5% stacking gel, and add samples in sequence, add 15 μl to each of the four samples, and add 20 μl to the standard protein marker. After electrophoresis, the gel was stained with Coomassie brilliant blue staining solution, decolorized with methanol/acetic acid solution, and finally recorded by photography. The results showed that a protein band of about 115KDa appeared between Marker's 175KDa and 83KDa, which was consistent with the molecular weight of the protein product expected to be expressed by the cloned fragment (3129bp, encoding a protein containing 1043 amino acids), confirming the identity of the fusion protein GLUA-P expression (Figure 3).

Take an appropriate amount of purified recombinant plasmid pGLUA-P and dilute it in 200 μl serum-free DMEM medium (hereinafter referred to as DMEM-SF), and take another 12 μl of cationic liposome DOSPER and dilute it in 188 μl DMEM-SF, and mix the two gently. Place it at room temperature for 15 minutes, then add it dropwise to the cell culture plate, incubate in the cell culture incubator at 37°C, 5% CO ₂ for 5 hours, then add DMEM medium containing 10% calf serum, and incubate at 37°C for 24-48 Hours later, cells were subjected to immunohistochemical staining. The results of transfection and immunohistochemistry showed that about 10% of the cells appeared specific brown particles in the cytoplasm, which confirmed that the GLUA-P fusion protein could be correctly expressed in eukaryotic cells. (Figure 4).

Inoculate SD rats with subcutaneous injection of pGLUA-P around the quadriceps femoris and submandibular gland, and compare the immune effect of subcutaneous injection of pCIA-P around the submandibular gland. See Table 1 for the experimental groups.

Attached Table 1 Grouping and immunization routes of mice with certain bacteria

Grouping Vaccines Route of Inoculation Immunization Dose

A subcutaneous injection of pGLUA-P in the area around the submandibular gland 100μg/piece

B subcutaneous injection of pCIA-P in the area around the submandibular gland 100μg/piece

C pGLUA-P quadriceps muscle injection 100μg/body

D pCI Quadriceps muscle injection 100μg/body

E Normal saline Quadriceps muscle injection 100μl/body

The saliva, serum and feces samples were collected at the age of 70 days, and were stored at -20°C for testing after treatment. 10 μg/ml PAc standard freeze-dried product (gifted by Professor Takahiko Oho, School of Dentistry, Kyushu University, Japan) was coated on a 96-well ELISA plate, 100 μl per well, washed overnight at 4°C, and after the carrier was blocked, the serum, saliva and feces to be tested were respectively Dilute according to the concentration of 1:10, 1:5, 1:1, and add 100 μl of sample to each well. Add goat anti-rat IgA, IgG (US Sigma Company) at a concentration of 1:1000 and 5 μg/ml, incubate for 2 hours, add 1:5000 alkaline phosphatase-labeled rabbit anti-IgG (US Sigma Company), respectively, Color developed after 5 hours of incubation. The enzyme-linked detector records the sample OD value at a wavelength of 405nm. ELISA method detects antibody level result: the IgG antibody level of serum anti-PAc of A, B, C group is obviously higher than matched group (P＜0.05), and there is no significant difference among three groups (P＞0.05); Salivary anti-PAc IgA antibody levels were significantly higher than those in groups C, D, and E (P<0.05), indicating that pGLUA-P can stimulate the body to produce specific humoral and mucosal immune responses (Figure 5).

After the rats were sacrificed, their skulls were severed, and then their heads were placed in a high-pressure steam cooker, treated with 10 Ib/in ² for 5 minutes, the soft tissues were removed, the upper and lower jaws were separated, cleaned, and dried at room temperature. Soak the dried jawbone in 0.4% ammonium viurate staining solution for 12 hours, take it out and rinse it, and use a 0.1mm thick, 25mm diameter emery chip to sagittal hemisection along the occlusal surface of the upper and lower molars , after cleaning and drying, under a stereomicroscope, observe and evaluate the dental caries condition of the mouse molars according to Keyes' classic caries evaluation method. The results of Keyes dental caries score showed that the enamel caries and dentin superficial caries scores of group A were the lowest (P<0.05), indicating that the subcutaneous injection of pGLUA-P around the submandibular gland had the best caries prevention effect (Figure 6).

Sequence Listing

pGLUA-P融合防龋DNA疫苗的基因序列如下：TCAATATTGGCCATTAGCCATATTATTCATTGGTTATATAGCATAAATCAATATTGGCTATTGGCCATTGCATACGTTGTATCTATATCATAATATGTACATTTATATTGGCTCATGTCCAATATGACCGCCATGTTGGCATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCACTAGAAGCTTTATTGCGGTAGTTTATCACAGTTAAATTGCTAACGCAGTCAGTGCTTCTGACACAACAGTCTCGAACTTAAGCTGCAGAAGTTGGTCGTGAGGCACTGGGCAGGTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCTGATAGGCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAGGTGTCCACTCCCAGTTCAATTACAGCTCTTAAGGCTAGAGTACTTAATACGACTCACTATAGGCTAGCCTCGAGAATTCACGCGTGGTACCGGAGAAATGGGCTATCAAGCCAAAGGAAAATTTGTAACAACTGCCGATGGTAAAATAAGATATTTTGATAAGCAATCTGGGAACATGTACCGTAATCGTTTTATTGAAAACGAAGAAGGTAAATGGCTGTATCTCGGTGAAGATGGTGCAGCAGTGACAGGATCTCAAACCATTAACGGTCAACACCTGTACTTTAGAGCAAACGGTGTTCAGGTCAAGGGTGAATTTGTCACTGACCACCACGGCCGTATCAGCTATTACGACGGCAATTCAGGGGATCAAATCCGCAACCGCTTTGTCCGCAATGCTCAGGGTCAATGGTTCTACTTTGATAACAATGGCTATGCCGTAACCGGTGCCAGAACCATTAACGGTCAACTCCTATACTTTAGAGCAAACGGTGTTCAGGTCAAGGGTGAATTTGTCACTGACCGCTACGGCCGTATCAGCTATTACGACGGCAATTCAGGGGATCAAATCCGCAACCGCTTTGTCCGCAATGCTCAGGGTCAATGGTTCTACTTTGATAACAATGGCTATGCCGTAACCGGTGCCAGAACCATTAACGGTCAACACCTATACTTTAGAGCAAACGGTGTTCAGGTCAAGGGTGAATTTGTCACTGACCGCCACGGCCGTATCAGCTATTACGACGGCAATTCAGGGGATCAAATCCGCAACCGCTTTGTCCGCAATGCTCAGGGTCAATGGTTCTACTTTGATAACAATGGCTATGCCGTAACCGGTGCCAGAACCATTAACGGTCAACACCTATACTTTAGAGCAAACGGTGTTCAGGTCAAGGGTGAATTTGTCACTGACCGCTACGGCCGTATCAGTTATTACGATGCTAACTCTGGAGAACGAGTTCGGATTGTCGACCCTCGAGAAATGGCTGCCAATCAAGCAGCCTATCAAAAAGCCCTTGCTGCTTATCAGGCTGAACTGAAACGTGTTCAGGAAGCTAATGCAGCCGCCAAAGCCGCTTATGATACTGCTGTAGCAGCAAATAATGCCAAAAATACAGAAATTGCCGCTGCCAATGAAGAAATTAGAAAACGCAATGCAACGGCCAAAGCTGAATATGAGACTAAGTTAGCTCAATATCAAGCTGAACTAAAGCGTGTTCAGGAAGCTAATGCCGCAAACGAAGCAGACTATCAAGCTAAATTGACCGCCTATCAAACAGAGCTTGCTCGTGTTCAAAAAGCCAATGCGGATGCTAAAGCGACCTATGAAGCAGCTGTAGCAGCAAATAATGCCAAAAATGCGGCACTCACAGCTGAAAATACTGCAATTAAGCAACGCAATGAGAATGCTAAGGCGACTTATGAAGCTGCACTCAAGCAATATGAGGCCGATTTGGCAGCGGTGAAAAAAGCTAATGCCGCAAACGAAGCAGACTATCAAGCTAAATTGACCGCCTATCAAACAGAGCTCGCTCGCGTTCAAAAAGCCAATGCGGATGCTAAAGCGGCCTATGAAGCAGCTGTAGCAGCAAATAATGCCGCAAATGCAGCGCTCACAGCTGAAAATACTGCAATTAAGAAGCGCAATGCGGATGCTAAAGCTGATTACGAAGCAAAACTTGCTAAGTATCAAGCAGATCTTGCCAAATATCAAAAAGATTTAGCAGACTATCCAGTTAAGTTAAAGGCATACGAAGATGAACAAACTTCTATTAAAGCTGCACTGGCAGAACTTGAAAAACATAAAAATGAAGACGGAAACTTAACAGAACCATCTGCTCAAAATTTGGTCTATGATCTTGAGCCAAATGCGAACTTATCTTTGACAACAGATGGGAAGTTCCTTAAGGCTTCTGCTGTGGATGATGCTTTTAGCAAAAGCACTTCAAAAGCAAAATATGACCAAAAAATTCTTCAATTAGATGATCTAGATATCACTAACTTAGAACAATCTAATGATGTTGCTTCTTCTATGGAGCTTTATGGGAATTTTGGTGATAAAGCTGGCTGGTCAACGACAGTAAGCAATAACTCACAGGTTAAATGGGGATCGGTACTTTTAGAGCGCGGTCAAAGCGCAACAGCTACATACACTAACCTGCAGAATTCTTATTACAATGGTAAAAAGATTTCTAAAATTGTCTACAAGTATACAGTGGACCCTAAGTCCAAGTTTCAAGGTCAAAAGGTTTGGTTAGGTATTTTTACCGATCCAACTTTAGGTGTTTTTGCTTCTGCTTATACAGGTCAAGTTGAAAAAAACACTTCTATTTTTATTAAAAATGAATTCACTTTCTATCACGAAGATGAAAAACCAATTAATTTTGATAATGCCCTTCTCTCAGTGACTTCTCTTAACCGTGAACATAACTCTATTGAGATGGCTAAAGATTATAGTGGTAAATTTGTCAAAATCTCTGGTTCATCTATTGGTGAAAAGAATGGCATGATTTATGCTACAGATACTCTTAACTTTAAACAGGGTGAAGGTGGCTCTCGCTGGACTATGTATAAAAATAGTCAAGCTGGTTCAGGATGGGATAGTTCAGATGCGCCGAATTCTTGGTATGGAGCAGGGGCTATTAAAATGTCTGGTCCGAATAACCATGTTACTGTAGGAGCAACTTCTGCAACAAATGTAATGCCAGTTTCTGACATGCCTGTTGTTCCTGGTAAGGACAATACTGATGGCAAAAAACCAAATATTTGGTATTCTTTAAATGGTAAAATCCGTGCGGTTAATGTTCCTAAAGTTACTAAGGAAAAACCCACACCTCCGGTTAAACCAACAGCTCCAACTAAACCAACTTATGAAACAGAAAAGCCATTAAAACCGGCACCAGTAGCTCCAAATTATGAAAAGGAGCCAACACCGCCGACAAGGACACCGGATCAAGCAGAGCCAAACAAACCCACACCGCCGACCTATGAAACAGAAAAGCCGTTGGAGCCAGCACCTGTTGAGCCAAGCTATGAAGCAGAGCCAACACCGCCGACAAGGACACCGGATCAGGCAGAGCCAAATAAACCCACACCGCCGACCTATGAAACAGAAAAGCCGTTGGAGCCAGCACCTGTTGAGCCAAGCTATGAAGCAGAGCCAACGCCACCGACACCAACACCAGATCAACCAGAACCAAACAAACCTGTTGAGCCAACTTATGAGTAAGTCGACCCCGGGCGGCCGCTTCGAGCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTAAAATCGATAAGGATCCGGGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGAGCTTTACGGCACCTCGACCGCAAAAAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAATATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGGCTCGACAGATCT黑体部分为GLUA-P片段。

Claims (4)

1. A fusion anti-caries DNA vaccine, characterized in Escherichia coli Esherichia coliJM109/pGLUA-P, CCTCC NO: M201042. 2. A method for preparing a fusion anti-caries DNA vaccine according to claim 1, characterized in that positive-strand primers and negative-strand primers are used; -I gene gtfB plasmid pYNB13 amplifies the sequence GLU encoding the glucan binding region, and clones it into the eukaryotic vector pCI to construct the eukaryotic expression plasmid pGLU, and then the surface of Streptococcus mutans in the pCIA-P plasmid The A-P fragment sequences of the A region and the P region of the protein PAc are connected with the pGLU plasmid to construct the GTF-PAc fusion anti-caries DNA vaccine pGLUA-P. 3. A fusion anti-caries DNA vaccine according to claim 1, characterized in that the positive and negative strand primers of the fusion anti-caries DNA vaccine pGLUA-P: Positive strand primer: 5'TCACTCGAGGTACCGGAGAAATGGGCTATCAA3' Negative strand primer: 5'CCCGGGTTAGTCGACAATCCGAACTCGTTC3' 4、根据权利要求1所述的一种融合防龋DNA疫苗，其特征在于融合防龋DNA疫苗pGLUA-P的基因序列是：TCAATATTGGCCATTAGCCATATTATTCATTGGTTATATAGCATAAATCAATATTGGCTATTGGCCATTGCATACGTTGTATCTATATCATAATATGTACATTTATATTGGCTCATGTCCAATATGACCGCCATGTTGGCATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCACTAGAAGCTTTATTGCGGTAGTTTATCACAGTTAAATTGCTAACGCAGTCAGTGCTTCTGACACAACAGTCTCGAACTTAAGCTGCAGAAGTTGGTCGTGAGGCACTGGGCAGGTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCTGATAGGCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAGGTGTCCACTCCCAGTTCAATTACAGCTCTTAAGGCTAGAGTACTTAATACGACTCACTATAGGCTAGCCTCGAGAATTCACGCGTGGTACCGGAGAAATGGGCTATCAAGCCAAAGGAAAATTTGTAACAACTGCCGATGGTAAAATAAGATATTTTGATAAGCAATCTGGGAACATGTACCGTAATCGTTTTATTGAAAACGAAGAAGGTAAATGGCTGTATCTCGGTGAAGATGGTGCAGCAGTGACAGGATCTCAAACCATTAACGGTCAACACCTGTACTTTAGAGCAAACGGTGTTCAGGTCAAGGGTGAATTTGTCACTGACCACCACGGCCGTATCAGCTATTACGACGGCAATTCAGGGGATCAAATCCGCAACCGCTTTGTCCGCAATGCTCAGGGTCAATGGTTCTACTTTGATAACAATGGCTATGCCGTAACCGGTGCCAGAACCATTAACGGTCAACTCCTATACTTTAGAGCAAACGGTGTTCAGGTCAAGGGTGAATTTGTCACTGACCGCTACGGCCGTATCAGCTATTACGACGGCAATTCAGGGGATCAAATCCGCAACCGCTTTGTCCGCAATGCTCAGGGTCAATGGTTCTACTTTGATAACAATGGCTATGCCGTAACCGGTGCCAGAACCATTAACGGTCAACACCTATACTTTAGAGCAAACGGTGTTCAGGTCAAGGGTGAATTTGTCACTGACCGCCACGGCCGTATCAGCTATTACGACGGCAATTCAGGGGATCAAATCCGCAACCGCTTTGTCCGCAATGCTCAGGGTCAATGGTTCTACTTTGATAACAATGGCTATGCCGTAACCGGTGCCAGAACCATTAACGGTCAACACCTATACTTTAGAGCAAACGGTGTTCAGGTCAAGGGTGAATTTGTCACTGACCGCTACGGCCGTATCAGTTATTACGATGCTAACTCTGGAGAACGAGTTCGGATTGTCGACCCTCGAGAAATGGCTGCCAATCAAGCAGCCTATCAAAAAGCCCTTGCTGCTTATCAGGCTGAACTGAAACGTGTTCAGGAAGCTAATGCAGCCGCCAAAGCCGCTTATGATACTGCTGTAGCAGCAAATAATGCCAAAAATACAGAAATTGCCGCTGCCAATGAAGAAATTAGAAAACGCAATGCAACGGCCAAAGCTGAATATGAGACTAAGTTAGCTCAATATCAAGCTGAACTAAAGCGTGTTCAGGAAGCTAATGCCGCAAACGAAGCAGACTATCAAGCTAAATTGACCGCCTATCAAACAGAGCTTGCTCGTGTTCAAAAAGCCAATGCGGATGCTAAAGCGACCTATGAAGCAGCTGTAGCAGCAAATAATGCCAAAAATGCGGCACTCACAGCTGAAAATACTGCAATTAAGCAACGCAATGAGAATGCTAAGGCGACTTATGAAGCTGCACTCAAGCAATATGAGGCCGATTTGGCAGCGGTGAAAAAAGCTAATGCCGCAAACGAAGCAGACTATCAAGCTAAATTGACCGCCTATCAAACAGAGCTCGCTCGCGTTCAAAAAGCCAATGCGGATGCTAAAGCGGCCTATGAAGCAGCTGTAGCAGCAAATAATGCCGCAAATGCAGCGCTCACAGCTGAAAATACTGCAATTAAGAAGCGCAATGCGGATGCTAAAGCTGATTACGAAGCAAAACTTGCTAAGTATCAAGCAGATCTTGCCAAATATCAAAAAGATTTAGCAGACTATCCAGTTAAGTTAAAGGCATACGAAGATGAACAAACTTCTATTAAAGCTGCACTGGCAGAACTTGAAAAACATAAAAATGAAGACGGAAACTTAACAGAACCATCTGCTCAAAATTTGGTCTATGATCTTGAGCCAAATGCGAACTTATCTTTGACAACAGATGGGAAGTTCCTTAAGGCTTCTGCTGTGGATGATGCTTTTAGCAAAAGCACTTCAAAAGCAAAATATGACCAAAAAATTCTTCAATTAGATGATCTAGATATCACTAACTTAGAACAATCTAATGATGTTGCTTCTTCTATGGAGCTTTATGGGAATTTTGGTGATAAAGCTGGCTGGTCAACGACAGTAAGCAATAACTCACAGGTTAAATGGGGATCGGTACTTTTAGAGCGCGGTCAAAGCGCAACAGCTACATACACTAACCTGCAGAATTCTTATTACAATGGTAAAAAGATTTCTAAAATTGTCTACAAGTATACAGTGGACCCTAAGTCCAAGTTTCAAGGTCAAAAGGTTTGGTTAGGTATTTTTACCGATCCAACTTTAGGTGTTTTTGCTTCTGCTTATACAGGTCAAGTTGAAAAAAACACTTCTATTTTTATTAAAAATGAATTCACTTTCTATCACGAAGATGAAAAACCAATTAATTTTGATAATGCCCTTCTCTCAGTGACTTCTCTTAACCGTGAACATAACTCTATTGAGATGGCTAAAGATTATAGTGGTAAATTTGTCAAAATCTCTGGTTCATCTATTGGTGAAAAGAATGGCATGATTTATGCTACAGATACTCTTAACTTTAAACAGGGTGAAGGTGGCTCTCGCTGGACTATGTATAAAAATAGTCAAGCTGGTTCAGGATGGGATAGTTCAGATGCGCCGAATTCTTGGTATGGAGCAGGGGCTATTAAAATGTCTGGTCCGAATAACCATGTTACTGTAGGAGCAACTTCTGCAACAAATGTAATGCCAGTTTCTGACATGCCTGTTGTTCCTGGTAAGGACAATACTGATGGCAAAAAACCAAATATTTGGTATTCTTTAAATGGTAAAATCCGTGCGGTTAATGTTCCTAAAGTTACTAAGGAAAAACCCACACCTCCGGTTAAACCAACAGCTCCAACTAAACCAACTTATGAAACAGAAAAGCCATTAAAACCGGCACCAGTAGCTCCAAATTATGAAAAGGAGCCAACACCGCCGACAAGGACACCGGATCAAGCAGAGCCAAACAAACCCACACCGCCGACCTATGAAACAGAAAAGCCGTTGGAGCCAGCACCTGTTGAGCCAAGCTATGAAGCAGAGCCAACACCGCCGACAAGGACACCGGATCAGGCAGAGCCAAATAAACCCACACCGCCGACCTATGAAACAGAAAAGCCGTTGGAGCCAGCACCTGTTGAGCCAAGCTATGAAGCAGAGCCAACGCCACCGACACCAACACCAGATCAACCAGAACCAAACAAACCTGTTGAGCCAACTTATGAGTAAGTCGACCCCGGGCGGCCGCTTCGAGCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTAAAATCGATAAGGATCCGGGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGAGCTTTACGGCACCTCGACCGCAAAAAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAATATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGCCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGGCTCGACAGATCT

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