禽分枝杆菌副结核亚种map0862基因与map2154c基因的串联重组表达

Fusion expression of map0862 gene with map2154c gene from Mycobacterium avium subsp. paratuberculosis

以禽分枝杆菌副结核亚种参考株P18的基因组DNA为模板,扩增出了map0862基因和map2154c基因。采用重叠延伸剪接PCR技术(PCR-SOE)获得了融合基因map0862-2154c。将融合基因与原核表达载体pET32a(+)连接,构建了重组质粒pET0862-2154c。将重组质粒转化到大肠杆菌感受态细胞BL21(DE3)中,以IPTG(终浓度1 mmol/L)诱导后对表达产物进行SDS-PAGE分析和Western-blot检测。结果表明,表达的重组蛋白map0862-2154c的分子质量为76 ku,可用于建立诊断副结核病的ELISA。

The map0862 and 2154c genes were amplified by PCR from Mycobacterium avium subsp. paratutuberculosis P18 strain. The fusion gene map0862-2154c was obtained by PCR SOE technique. Then the fusion gene was inserted into the pET32a（＋） to construct the recombinant plasmid pET0862-2154c. The recombinant plasmid was transformed into Escherichia coli BL21（DE3） to express the fusion protein which was induced with 1 mmol/L IPTG. The expressed fusion protein was insoluble in SDS PAGE analysis. It was purified by nickel-NTA chromatography. Western-blot detection showed that the purified pro tein was 76 ku in molecular weight and could be used in ELISA for immunodiagnosis of bovine paratuberculosis.