结核分枝杆菌RD1区rv3873基因的原核表达及其产物的细胞免疫特性初步分析

Prokaryotic expression of the rv3873 gene located in RD1 of Mycobacterium tuberculosis and its cellular immunoproperties

目的克隆表达结核分枝杆菌RD1区Rv3873蛋白并初步分析其细胞免疫特性。方法应用PCR技术扩增rv3873基因,插入表达载体pET30a(+),构建原核表达重组质粒pET-Rv3873,重组质粒在宿主菌BL21(DE3)诱导表达,利用表达的蛋白腹腔注射免疫小鼠,以细胞增殖和ELISPOT试验测定融合蛋白的细胞免疫应答。结果克隆的rv3873测序与已发表序列100%同源,融合蛋白大小43ku,淋巴细胞增殖实验显示该蛋白能引起小鼠T细胞免疫反应,ELISPOT实验结果表明多肽pep3873刺激特异性IFN-γ分泌细胞多于IL-4分泌细胞。结论成功地克隆表达了Rv3873蛋白,并发现该蛋白具有良好的细胞免疫特性。

The rv3873 gene located in RD1 of Mycobacterium tuberculosis was expressed in E.coli BL21（DE3）and its cellular immunoproperties was analysed.The rv3873 gene encoding Rv3873 was amplified by PCR from pYUB：RD1 and then subcloned into prokaryotic expression plasmid pET30a（＋）after sequence analysis.Then the expression of Rv3873 with His tag in E.coli BL21（DE3）was detected by SDS-PAGE and Western blot assay.Significant T cell proliferation was observed by stimulation of splenocytes of His-Rv3873 immunized C57BL/6 mice with specific peptide pep3873 and PPD protein.More IFN-γ specific secreting cell spots rather than IL-4 were detected in His-Rv3873 group by ELISPOT assay.These results showed the Rv3873 rather than ESAT-6 or CFP-10 proteins of RD1 could induce T cell immune responses.