HSP70基因上游调控序列对GST基因在耻垢分枝杆菌中表达效率的影响

将外源基因———日本血吸虫２６ｋＤ抗原（Ｓｊ２６ＧＳＴ）基因克隆到大肠杆菌分枝杆菌穿梭质粒中，构建成四个不同的表达截体，研究它们在耻垢后分枝杆菌（Ｍｙｃｏｂａｃｔｅｒｉｕｍｓｍｅｇｍａｔｉｓ）中的表达效率。首先将含有结核杆菌热休克蛋白７０（ＨｅａｔＳｈｏｃｋＰｒｏｔｅｉｎ，ＨＳＰ７０）的启动子的质粒ｐＭＴ７０用ＮｃｏＩ切，进行两种不同的修饰后，得到不同的ＳＤ序列；将Ｓｊ２６ＧＳＴ基因克隆进去。再将含ＨＳＰ７０启动子和Ｓｊ２６ＧＳＴ基因的片段切下，克隆到分枝杆菌大肠杆菌穿梭质粒ｐＢＣＧ２０００中，筛选出不同ＳＤ序列、不同方向和不同拷贝数的分枝杆菌表达载体四个。所表达的重组天然Ｓｊ２６ＧＳＴ（ｒＳｊ２６ＧＳＴ）为可溶性蛋白，在ＳＤＳＰＡＧＥ上分子量为２６ｋＤ处可见明显的表达蛋白带。通过薄层扫描分析，发现表达质粒中双拷贝启动子外源基因组合，表达效率最高，是单拷贝组合的１６倍，占分枝杆菌菌体总蛋白的２８％。而不同的克隆方向和不同的ＳＤ序列（两者相差３个碱基）对表达效率的影响不明显。

STUDY OF HSP70 GENE UPSTREAM REGULATION ELEMENT ON EXPRESSION EFFICIENCY OF GST GENE IN M.SMEGMATIS

Four different experssion vectors were constructed by cloning foreign gene which encode Schistosoma japonicum 26kD antigen (Sj26GST)into Escherichia coli Mycobacteria shuttle plasmid pBCG-2000 and investigated their expression efficiency in mycobacteria smegmatis.The plamid which contains promoter of human mycobacterial tuberculosis heat shock protein 70 was firstly digested with Nco I and modified with two different ways to lead to two kinds of SD squences, and ligated with Sj26GST encoding gene.Then,the DNA fragment contained HSP70 promoter and Sj26GST gene was obtained and cloned into E.coil mycobacteria shuttle plasmid pBCG 2000,and finally four recombinant mycobacterial expression vectors that differienciated in SD seuqence, orientation and copy number were selected.The expressed native recombinant Sj26GST(rSj26GST)was solube and could be observed on SDS PAGE about at the molecular weight of 26kD obviously. Analysis with protein density scanning indicated:the expression efficiency that contaning double copy promoter foreign gene vector was the highest and the expresed protein which was about 1 6 times than others was 28% of total protein of Mycobacteria smegmatis. The cloned direction and SD sequence had no significant effect on expression efficiency.