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.pdfVIP

668 中国人兽共患病学报 2010. 26 (7) Chinese Journal of Zoonoses 文章编号: 1002 - 2694(2010)07 - 0668 - 04 结核分枝忏菌RDl 区 rv3873 基因的原核表达 及其产物的细胞兔茂特性初步分析骨 周海霞，陈 祥，郑佳玉，牛中伟，潘志明，焦新安 摘 要z 目的 克隆表达结核分校杆菌 RDl 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 BL2lC 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 溃|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丁细胞免疫反应.ELISPOT 实验结果 表明多肤 pep3873 剌激特异性 IFN-y 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。 关键词:结核分枝杆菌;Rv38731 IFN-y 中图分类号:R378.91 文献标识码:A Prokaryotic expression of the rv3873 gene located in RDl of Mycobacterium tuberculosis and its ceIlular immunoproperties ZHOU Hai-xia , CHEN XÎang , ZHENG JÎa-yu.NIU Zhong-wei ,PAN Zhí-ming ,JIAO XÎn-an (J iungsu Key Luboratory 0/ Zοonosü •Yangzhou University.Yaηgzhou 225009 , China) ABSTRACT: The rv3873 gene located in RDl of Mycobacterium tu加rculosis was expressed in E. coli BL21 CDE3) and its celIular immunoproperties was analysed. The rv3873 gene encoding Rv3873 was amplified by PCR from pYUB: RDl and then subcloned into prokaryotic expression plasmid pET30a( +) after sequence analysis. Then the expression of Rv3873 with His tag in E. coli BL21CDE3) was detected by SD5and 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- y specific secreting ce lI 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-IO proteins of RDl could induce T cell immune responses. KEY WORDS: