用结核分枝杆菌HSP70启动子构建分枝杆菌穿梭表达质粒的研究

目的：用结核分枝杆菌HSP70(Heat shock protein70，HSP70)启动子改建分枝杆菌穿梭质粒pJEM11为分枝杆菌穿梭表达质粒。方法：利用聚合酶链反应(Polymerase chain reaction，PCR)扩增H37Rv基因组419607～419835位的HSP70启动子及调控序列，末端引入一个多克隆位点(Multiple clone sites，MCS)，再定向克隆人pJEM11的ApaI位点与XbaI位点之间，构建pJCH02载体，并对载体进行酶切鉴定及序列测定。将lhp-esat6融合基因克隆人pJCH02载体，评价其在BCG中的表达情况。结果：HSP70启动子、调控序列及引入的多克隆位点完全正确，lhp-esat6基因在BCG中成功表达。结论：成功改建穿梭质粒pJEM11为穿梭表达质粒pJCH02。本研究为构建BCG多价疫苗奠定了基础。

Construction of the Mycobacterium shuttle expression plasmid with Mycobacterium tuberculosis heat shock protein 70 promoter

Objective:To reconstruct Mycobacterium shuttle expression plasmid with Mycobacterium tuberculosis HSP70 promoter based on shuttle plasmid pJEM11.Methods:The cDNA fragment of 229bp promoter and its correlative sequence was amplified by PCR.Multiple clone sites was added to the end of amplified fragment,and then it was cloned into the pJEM11 to construct the plasmid pJCH02.The pJCH02 was identified by digestion with restriction endonuclease and sequence analysis.The lhp-esat6 fusion gene was cloned into the pJCH02,then its expression in BCG was confirmed by SDS-PAGE.Results:The sequence of HSP70 promoter and its correlative sequence was in accordance with the predicted sequence.The lhp-esat6 fusion gene was expressed in BCG.Conclusion:The shuttle expression plasmid pJCH02 is constructed successfully.The study provides the basis for BCG polyvaccine.