Shi Xin-xin, Bao Lang, Qiu Yun-qing, Guo Si
Department of Infection and Immunity, Preclinical and Forensic Medicine School of West China Medical Center, Sichuan University, Chengdu 610041, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2010 Jun;26(6):539-42.
To construct, express and identify a recombinant prokaryotic expression vector pET-3872 carrying Rv3872 of Mycobacterium tuberculosis H37Rv strain, and to construct the recombinant Bacillus Calmette-Guerin (rBCG) strain expressing Rv3872 (PE35)of Mycobacterium tuberculosis H37Rv strain.
The Rv3872 gene was amplified by PCR from Mycobacterium tuberculosis H37Rv strain and cloned into prokaryotic expression vector pET32a(+). The recombinant plasmid pET-3872 was sequenced and transformed into E.coli BL21(DE3), and was induced with IPTG to express a 35 kD fusion protein, which was confirmed as His-Rv3872 by Western blot. The expression product was purified and the new Zealand rabbits were immunized. Rv3872 was amplified by PCR and cloned into E.coli-Mycobacteria shuttle vector pMV361. The recombinant vector was named as pMV-3872, and then pMV-3872 was transformed the to BCG via electroporation, the recombinant BCG-3872 strain. The PE35 protein expression of BCG-3872 was induced with heat shock reaction. Then BCG-3872 culture supernatant and bacterial precipitation were collected respectively and analyzed by Western blot.
A recombinant fused expression vector pET-3872 was constructed and His-3872 protein was confirmed by Western blot. BCG-3872 strain was constructed and Western blot confirmed the presenle of the PE35 protein in the supernatant of BCG-3872 strain culture.
The prokaryotic expression vector pET-3872 was constructed, and the 35 kD fusion protein His-3872 was expressed and purified successfully, which provides a tool for further functional study of the Rv3872 . The recombinant BCG strain expressing PE35 protein, BCG-3872 strain, was constructed successfully, which facilitates further study on BCG-3872 immune function.
构建、表达并鉴定携带结核分枝杆菌H37Rv株Rv3872的重组原核表达载体pET - 3872，并构建表达结核分枝杆菌H37Rv株Rv3872（PE35）的重组卡介苗（rBCG）菌株。
采用PCR从结核分枝杆菌H37Rv株扩增Rv3872基因，并克隆至原核表达载体pET32a(+)。对重组质粒pET - 3872进行测序后转化至大肠杆菌BL21(DE3)，用异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达35 kD融合蛋白，经蛋白质免疫印迹法（Western blot）确认为His - Rv3872。对表达产物进行纯化并免疫新西兰兔。采用PCR扩增Rv3872并克隆至大肠杆菌-分枝杆菌穿梭载体pMV361。将重组载体命名为pMV - 3872，然后通过电穿孔法将pMV - 3872转化至卡介苗，获得重组卡介苗-3872菌株。用热休克反应诱导卡介苗-3872菌株表达PE35蛋白。分别收集卡介苗-3872菌株培养上清和菌沉淀，并用Western blot进行分析。
构建了重组融合表达载体pET - 3872，Western blot证实有His - 3872蛋白。构建了卡介苗-3872菌株，Western blot证实卡介苗-3872菌株培养上清中存在PE35蛋白。
构建了原核表达载体pET - 3872，成功表达并纯化了35 kD融合蛋白His - 3872，为进一步研究Rv3872的功能提供了工具。成功构建了表达PE35蛋白的重组卡介苗菌株卡介苗-3872，便于进一步研究卡介苗-3872的免疫功能。
