鹿结核分枝杆菌流行株Rv3874-Rv3875融合基因的克隆及原核表达

Cloning and Prokaryotic Expression of Rv3874-Rv3875 Fusion Gene of Deer Mycobacterium tuberculosis Epidemic Strains

以吉林农业大学经济动物疾病实验室分离并保存的鹿结核分枝杆菌流行株为模板,利用重叠延伸剪接技术(Splicing by overlap extension,SOE)设计引物,克隆Rv3874-Rv3875融合基因,构建重组表达质粒p GEX-4T-1-Rv3874-75,在大肠杆菌BL21(DE3)中经IPTG诱导表达,并对目的蛋白进行纯化,表达产物通过SDS-PAGE和Western blot进行分析。结果表明:测序后融合基因Rv3874-Rv3875的序列与Gen Bank中序列的符合率为100%,重组表达质粒p GEX-4T-1-Rv3874-75在大肠杆菌中成功诱导表达,获得可溶性表达的融合蛋白,经SDS-PAGE分析该蛋白的相对分子质量约49 ku,经Western blot鉴定纯化好的融合蛋白能与鹿结核病的阳性血清发生反应,具有良好的反应原性。

Fusion gene Rv3874-Rv3875 was cloned by Gene-SOE using the genomic DNA of deer mycobacterium tuberculosis wild strain that were separated and saved in laboratory of economic animal disease of Jilin Agricultural University as template as template. Then the PCR product was transformed into p GEX-4T-1 to gain prokaryotic expression. Constructed recombinant plasmid p GEX-4T-1-Rv3874-75 was induced with IPTG in E. coli BL21（ DE3）. The purpose protein was purified by GST tag affinity chromatography purification system. The result showed that the compliance rate of the sequence of fusion gene Rv3874-Rv3875 and that in Gen Bank was 100% by sequencing. The fusion protein was expressed successfully in E. coli in a soluble form. SDS-PAGE and Western blot analysis induced that Rv3874-Rv3875 was expressed with the size about 49 ku and the purified protein could interact with the positive serum of deer tuberculosis,which demonstrated that the fusion protein had excellent antigenicity.