结核分支杆菌Mtb8.4抗原原核表达质粒的构建与鉴定

目的构建结核杆菌低分子质量抗原Mtb8.4基因的原核表达重组质粒,为其蛋白质的表达及免疫学研究打下基础.方法 PCR扩增目的基因,克隆到质粒pET-His T7启动子的下游,转化大肠杆菌TOp10F‘,进行重组子的筛选、鉴定.结果 Mtb8.4基因正向插入表达载体中,测序证实具有正确的读码框,基因序列与文献报道一致.结论重组原核表达质粒pET-His-Mtb8.4构建成功.

Construction of Prokaryotic recombinant plasmid encoding Mtb8.4 gene of M.tuberculosis and identification

[Objective] To construct prokaryotic expression recombinant plasmid encoding Mtb8.4 gene of M.tuberculosis. [Methods] The gene encoding protein Mtb8.4 was amplified by PCR technique. Clone the gene into the downstream of the T7 promoter pET-His, then transformed into E.coli TOP10F. Positive clone was screened using double digestion and polymerase chain reaction, and farther identified by nucleotide sequence. [Rusults] Mtb8.4gene was successfully inserted into the prokaryotic plasmid pET-His. The reading frame and gene sequence of recombinant plasmid were correct by sequencing reaction. [Conclusions] Recombinant plasmid pET-His-Mtb8.4 is successfully constructed, It will provide further study for the expression Mtb8.4 protein and its immunological effect,and a potential candidate for inclusion in a subunit vaccine against tuberCulosis(TB).