日本血吸虫26kD抗原基因在BCG中的表达

Study on Expression of Schistosoma Japonicum 26 kD Antigen Gene in BCG

研究了外源基因日本血吸虫２６ｋＤ抗原（Ｓｊ２６ＧＳＴ）在卡介苗（ｂａｃｉｌｕｓＣａｌｍｅｔｅ－Ｇｕｅｒｉｎ，ＢＣＧ）、耻垢分枝杆菌（Ｍ．ｓｍｅｇｍａｔｉｓ）和大肠杆菌（Ｅ．ｃｏｌｉ）中的表达．运用重组ＤＮＡ和聚合酶链反应（ＰＣＲ）等分子生物学技术，以表达Ｓｊ２６ＧＳＴ的Ｅ．ｃｏｌｉｐＧＥＸ衍生质粒为模板，经ＰＣＲ得到编码Ｓｊ２６ＧＳＴ的全长ｃＤＮＡ片段．将其按正确的阅读框顺序，克隆到人结核杆菌热休克蛋白（ｈｅａｔｓｈｏｃｋｐｒｏｔｅｉｎ，ＨＳＰ）７０的启动子下游，再将ＨＳＰ７０启动子和Ｓｊ２６ＧＳＴ基因一起亚克隆到Ｅ．ｃｏｌｉ－分枝杆菌穿梭质粒ｐＢＣＧ－２０００中，得到Ｅ．ｃｏｌｉ－分枝杆菌穿梭表达质粒ｐＢＣＧ－Ｓｊ２６．ｐＢＣＧ－Ｓｊ２６电转化入ＢＣＧ和Ｍ．ｓｍｅｇｍａｔｉｓｍｃ２１５５中表达Ｓｊ２６ＧＳＴ抗原，所表达的天然重组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．其表达量分别占ＢＣＧ和Ｍ．ｓｍｅｇｍａｔｉｓ菌体总蛋白的１５％和１０％．可见，Ｓｊ２６ＧＳＴ基因能在ＢＣＧ中高效表达．

The expression of foreign gene,Schistosoma japonicum 26 kD antigen (Sj26GST),in bacillus Calmette Guerin (BCG), Mycobacterium(M.) smegmatis and Escherichia coli(E.coli) was studied by using techniqes of molecular biology,such as recombinant DNA technology,polymerase chain reaction (PCR),etc.BCG,a live attenuated bovine tubercle bacillus,which has been used to immunized against tuberculosis for many years,is a highly effective adjuvant,and can engender long lasting immune responses with only a single dose.The cloning and expression of the foreign gene in BCG were investigated.The cDNA fragment encoding Sj26GST was amplified by PCR using plasmid pGEX which could express Sj26GST in E.coli as templet.The Sj26GST cDNA was cloned into the downstream of human M.tuberculosis heat shock protein (HSP) 70 promoter with correct reading frame.The HSP 70 was a conserved protein,which had high homogenity between different organisms,and the cDNA fragments which were cloned into the downstream of HSP70 promoter could be upregulated in stress conditions.The DNA fragments containing HSP70 promoter and Sj26GST gene were subcloned together into E.coli Mycobacteria shuttle plasmid pBCG 2000,which contained multiple cloning sites and was capable of conferring stably kanamycin resistance both in mycobacterium and E.coli ,to construct the expression shuttle plasmid pBCG Sj26.The recombinant BCG and M.smegmatis mc 2 155 which were electroporated with pBCG Sj26 could express Sj26GST when induced by heating, and the recombinant Sj26GST (rSj26GST) was soluable and could be observed on SDS PAGE with molecular weight of 26 kD.This indicated that the M.tuberculosis HSP70 promoter could be recognized by the RNA polymerase of BCG.The content of rSj26GST contained 15% and 10% of total bacterial protein of BCG and M.smegmatis ,respectively.These facts suggest that the forein gene Sj26GST encoding gene can be expressed in BCG,which may facilitate the development of recombinant BCG vaccine against Schistosoma japonicum.