| A self-designed and synthesized mRNA transcribable and untranslatable functional protein molecule Ag85A DNA of full-length fragment encoded genes was cloned into eukaryotic vector pIRES2-EGFP used for follow up preparation of Ag85A-M vaccine, and for studying double strand Ag85 DNA vaccine whether it could present antigen information, and induce inherent and adaptable immune responses. Ag85A-M gene was designed and replaced 114th and 230th base C mutated into G in encoded Ag85A gene (891 bp), to make it contain double translation terminator TAG and TGA. The Ag85A(M) full-length gene fragment was demerged common PCR into 332 bp (A), 332 bp (B), and 264 bp (C) three small fragments respectively, and 488 bp (D), 423 bp (E) two small fragments and carried out proliferation synthesis. And constructed pIRES2-EGFP-Ag85A-M vector through EcoR I/BamH I double restriction site to insert pIRES2-EGFP vector, and confirmed the gene expression in DC2.4 by double enzyme cutting and identifiedwith gene sequencing. The results showed that the synthesized Ag85-M complete sequence (891 bp) mutation sites and bases adopted by common PCR method amplified, synthesized and obtained fragment by fragment accorded with the length of A, B, C, D, and E small fragments were corrected. And the average positive clone ratio of three demerged fragments (A, B, and C small fragments) was 87.67%, the gene fidelity was 90.33%. And the average positive clone ratio of two demerged fragments (D and E small fragments) was 81.5%, the gene fidelity was 23%. The results suggested that this study had obtained that could be used in the follow up study on full length Ag85A-M DNA fragment containing 114 and 230 mutation sites, the fidelity of 332 bp obtained by three-fragment demerging method and below gene obtained by PCR synthesis was significantly higher than those 423 bp and above fragments obtained by two-fragment demerging method. |
