Gene Cloning,Expression And Purification Of Recombinant 4-valent Group A Streptococcal Vaccine And Preliminary Researches On Immunogenity

M protein is the most important protein antigen in the cell wall of Group A Streptococcus. More than 150 serotypes have been identified according to difference of antigenicity of M proteins. Antibodies against M protein are protective, which are evoked within several weeks or months after infection of GAS and can persist for as long as 1-2 years usually, even 10-30 years. Type-specific M protein antibodies can prevent body form being infected again by the same serotype GAS. However, it is not protective against the other serotypes. At the same time, M protein shares epitopes with a number of host proteins including, cardiac and skeletal myosin, tropomyosin, laminin and keratin. As a result, some serotypes GAS infections may lead to some autoimmune diseases such as rheumatic fever, rheumatic heart disease and acute post-streptococal glomerulonephritis. So extracted native M proteins and compete M protein molecule can not be used as protective antigens of GAS vaccine.Recently, some researches indicated that M protein exists as an dimer consisting of the same two polypeptide chains complexed into an alpha-helical coiled-coil configuration that anchored in the cell membrance. Each polypeptide may consist of up to three repeat regions A-C from N-terminus to C-terminus. A-repeat region is hypervariable, type-specific and protective epitopes are located in it. B-repeat region is semi-variable, tissue cross-reactive epitopes are located in B-repeat region and the regions which are joined to B region in the A and C region. C-repeat region is highly conserved among GAS strains. There are some protective and not cross-reactive epitopes in A and C region. Consequently, it is available to design a type-specific GAS vaccine on the basic of A region of different serotype GAS M proteins. The vaccine can not only avoid tissue cross-reactive, but also induce type-specific protective antibodies.Part 1 The type-specific amino acid sequences of M protein of GAS M1, M3, M6, M18 were obtained from the CDC emm typing center website. The fragments without homologies with human tissue proteins were selected from every type-specific sequence thorough the documents and Bioinformatics methods. A recombinant protein was designed by linking the selected fragments and a common conservative sequences J14 which was not crossreactive with human tissue proteins in tandem in the order of M1-3-6-18-J14. Homologies with human tissue proteins were analyzed by BlastP. hydrophility, second structure,3D structure and B-cell epitopes were predicted by online software. The result indicated that the designed recombinant protein was not significant homology with human tissue proteins and had similar second structure and 3D structure with the naive M protein and was hydrophilic. B cell epitopes in every fragment containing different serotype sequences had not been changed. A group of Oligonucleotides were designed by softwere DNAWORK 2.0 and DNA sequence of the designed recombinant protein was synthesized and amplified thorough Overlap PCR. The amplified DNA sequence was cloned into PUC18 cloning vector to construct recombinant cloning vector PUC18-strep4 and sequenced. The result indicated that the synthesized DNA sequence was identical with the designed sequence. It laid a foundation of prokaryotic expression of recombinant protein.Part2 To construct an expressing vector which expresses the recombinant protein, Useing the constructed cloning vector PUC18-Strep4 as the template, gene of the recombinant protein were amplified by PCR. The purified target DNA fragment was digested with BamHI/HindⅢand cloned into the expressing vector PQE30. The recombinant expressing vector was transformed to E.coil M15, the positive clone were selected and identified by digestion of BamHI/HindⅢand sequenced. Result indicated that the prokaryotic expressing vector PQE30-strep4 was constructed successfully. E.coil M15/PQE30-Strep4 was induced by IPTG. The expressed products were analyzed by SDS-PAGE. The result showed that the recombinant protein Strep4 had a relative molecular weight of about 24KD and was expressed in solubility. It is consistent to the predicted result. The recombinant protein were purified by Ni2+-NTA chromatography and reached a purity of more than 90%. 9.46mg purified recombinant protein was obtained every 1 liter LB.Part3. To preliminary researches on immunogenity of recombinant protein, three rabbits were immunized subcutaneously with 500μg of recombinant protein emulsified in complete Freund's adjuvant in equal volume, and the same dose of recombinant protein emulsified in incomplete Freund's adjuvant were repeated at 4 and 8 weeks. Serum was obtained prior to the first injection and at 2-weeks interval thereafter. The rabbits were killed at 12 weeks. Enzyme-linked immunosorbent assays (ELISA) were performed to detect the change of titer of antibodies against recombinant protein in immunized sera. Type-specific antibodies against M protein in immunized sera were detected by indirect immunofluorescence assays (IFA). Bactericidal assays were performed to determine whether immunized sera were bactericidal. The result showed that the high-titer antibodies against recombinant protein were evoked in three immunized sera. There were type-specific bactericidal antibodies against GAS M1, M3, M6 in three immunized sera. However, it was uncertain that Type-specific bactericidal antibodies against GAS M18 were evoked because there were type-specific bactericidal antibodies against GAS M18 in three preimmune sera.Indirect immunofluorescence assays between mice sera immunized with recombinant protein and GAS M18 indicated that type-specific antibodies against GAS M18 could be evoked by immunization of recombinant protein. However, bactericidal assays of immune mice sera against GAS M18 could not be sure that type-specific bactericidal antibodies against GAS M18 were evoked by immunization of recombinant protein. 120 BALB/c mice (male, 6-week old) were randomly divided into two groups, 60 mice in every group. Mice in one group were injected subcutaneously with 50μg /200μl of recombinant protein emulsified in complete Freund's adjuvant in equal volume as the immunized group, the same dose were were repeated at 14 and 28 days. Mice in the other group were injected subcutaneously with 200ul PBS emulsified in complete Freund's adjuvant in equal volume at 0, 14, 28 days, as the control group. At 14 days after final immunization, mice in two groups were randomly divided into five groups respectively, 10 mice in every group. Immunized mice and control mice were challenged intraperitoneally with GAS M1, M3, M6, M18, M5. Mice survival rates were observed to the tenth day. All of the survival rates in the immunized group challenged with GAS M1, M3, M6, M18, M5 were higher than that of the control group. However, it was not significantly different between the immunized group and the control group (P>0.05) except challenged by GAS M1 (P<0.05). The results suggested that it is not remarkable that mice immunized by recombinant protein were protected from GAS challenge.Immune mice sera were tested for the presence of tissue-cross-reactive antibodies by immunohistochemical method using sections of human cardiac muscle tissue. No specific stain was found in human cardiac muscle tissue reacted with immune sera or normal sera. It showed that there were not common antigens between recombinant protein and human cardiac muscle.