Prokaryotic Expression、 Purification, And Immunogenicity Analysis Of The Specific Protein Of Mycobacterium Tuber-culosis

Mycobacterium tuberculosis(Mycobacterium tuberculosis, MTB), commonly known as Mycobacterium tuberculosis is the cause of tuberculosis(TB) pathogenic bacteria.At present,it still has a high mortality rate of chronic infectious diseases. In 1998, whole genome of Mycobacterium tuberculosis sequencing, which found two core protein family PE and PPE, about 10% of the entire genome of open reading frame encoding the two families was finished.Because of its N-terminal Pro- Glu, with conservative PE and Pro- Pro- Glu, PPE motif, we called PE/PPE protein family.PE family have 99 members, each member has a conserved sequence composed of 110 amino acid residues at the N-terminal. Through bioinformatics analysis of the characteristics of the PE protein family, we found that the main protein family are rich in GGXGG motif, so we speculated that GGXGG as the main antigen epitope.PPE family have 69 members, each member has a conserved sequence composed of 180 amino acid residues at the N-terminal. At present,the structure and function of the two families are unclear, but speculation is associated with bacterial virulence, and plays an important role in the process of antigenic variation and immune escape. At present, the identification of all kinds of MTB specific antigenlaid a good foundation on the detection of tuberculosis antibody。 In this study, a variety of MTB specific antigen were prokaryotic expressed to analyze and compare the specificity and sensitivity, and then compare the different tuberculosis specific antigen and serum antibody reaction model. This research is divided into the following two parts:Part I: PE and PPE family related genes of Mycobacterium tuberculosis in vitro synthesis, prokaryotic expression and immunogenicity analysis.Mycobacterium tuberculosis in PE and the PPE protein family chose a highlyconserved sequence was obtained from Gen Bank, translated into the e. coli codon optimization sequence of three PE-PGRS33(110 aa), PPE38(180 aa), PE-PGRS52(637-731aa),,build and prokaryotic express the three sections of the sequence. Specific methods are as follows: respectively- PGRS33 PE(110aa), PPE38(180aa), PE- PGRS52(637-731-aa) gene, using the OE- PCR principle design specific primers for in vitro gene synthesis of PE- PGRS33(110aa), PPE38(180 aa), PE- PGRS52(637-731aa).Three pieces of synthetic after using the method of T/A clone cloning sequencing to p MD18-T carrier identification. Prokaryotic expression vector to sequencing the right sequence of PET-32-a connection, construct its prokaryotic expression plasmid: PET32a/PE-PGRS33(110aa), PET32a/PPE38(180aa), PET32a/PEPGRS52(637-637aa), the above three kinds of prokaryotic expression plasmid respectively into e. coli BL21(DE3), expressed by IPTG induction, among them, the PET32a/PE-PGRS33(110aa), PET32a/PPE38(180aa) is not successful, PET32a/PE-PGRS52(637-731aa), small amount can be obviously express a stripe, induced the relative molecular weight of about 28 kd, respectively with Ni- NTA fusion protein affinity chromatography purification purposes. By 12% sds-page electrophoresis analysis, respectively is the molecular weight of about 28 kd about specific bands, size is consistent with the theoretical value. Indicates that this research successfully build and expresses the PET32a/PE-PGRS52(637-731-aa) fusion protein. The purified protein immune BALB/c mice, the purpose of the preparation of antiserum, ELISA tests showed PET32a/PE-PGRS52(637-731-aa) and PEPTIDE/PE- PGRS52(637-731-aa) and immune rabbit serum show specific reaction, the Ig G antibody degrees respectively 000, 000 and 1:32 1:16 PET32 a protein only weak binding reaction. That we design this section for GGNGG antigen epitope sequences in vitro synthetic gene expression and purification of proteins has good immunogenicity, and also reflects the GGNGG is antigen decided to center. For future TB early diagnostic kit and vaccine research to provide the reference.Part II: Specific proteins of Mycobacterium tuberculosis gene cloning, prokaryotic expression and immunogenicity analysisThe 6 kinds of mycobacterium tuberculosis nucleotide sequence was obtained from Gen Bank, according to the query sequence of nucleotide amplification primer design, in the upstream and downstream primers, respectively, add the appropriate enzyme site and enzyme site protection bases, H37 Rv genome as template above 6 gene amplification, respectively. Would expand product recovery and purification, and then connected to the p MD- 18 t carrier, PCR identification and sequencing, the sequence right of prokaryotic expression plasmid vector PET32 a connection, construct prokaryotic expression plasmid, turn the plasmids to e. coli BL21(DE3), expressed by IPTG induction, in the right way to purify the 7 kinds of proteins. ELISA test results showed that in 80 TB patients and 96 normal adults serum as sample, by indirect ELISA method to detect the protein fragment antigenicity, according to the results of PET32a/PE-PGRS52(637-731aa) specificity of 97.9%, the sensitivity is 37.5%;And RV0934,RV0831 C joint detection, sensitivity increased significantly; This suggests that the PET32a/PE- PGRS52(637-731aa), as a single antigen for serological detection significance is not big, but can be diagnosed as tuberculosis joint alternative antigen. Summary:1. This research successfully build three kinds of mycobacterium tuberculosis specificity prokaryotic expression plasmid and induced by E.coli,obtained 1 kind of mycobacterium tuberculosis PE family a truncated protein.2. The PE-PGRS52(637-731aa) protein better retains the immunogenicity.3. This study successfully built and expressed the 6 kinds of mycobacterium tuberculosis specific protein, the restructuring of 6 kinds of proteins by indirect ELISA method analyzed the immunogenicity, preliminary research results for further analysis and selection of clinical used to detect tuberculosis(TB) provides the experimental basis.