重组质粒pGEX-6P-1/Sj-FABPc的构建及在大肠杆菌中的表达  

CONSTRUCTION OF RECOMBINANT pGEX-6P-1/Sj-FABPc AND EXPRESSION IN E.coli

作　　者：赵巍[1] 苏川[2] 吴海玮[2] 胡雪梅[2] 沈蕾 王荣芝 马磊 陈淑贞[2] 张兆松[2] 

机构地区：[1]宁夏医学院生物学教研室,访问学者银川750004 [2]南京医科大学医学分子生物学研究所

出　　处：《中国血吸虫病防治杂志》2000年第5期261-264,共4页Chinese Journal of Schistosomiasis Control

基　　金：江苏省医学分子生物学重点实验室开放课题

摘　　要：目的　构建基因工程菌株 ,获得重组蛋白 Sj- FABPc(日本血吸虫脂肪酸结合蛋白 )。方法　用 PCR法从日本血吸虫 c DNA文库中扩增 Sj- FABPc基因片段 ,再将该片段重组于 p GEM- T中并进行 DNA测序鉴定 ,经酶切后将目的片段构建成重组质粒 p GEX- 6 P- 1/ Sj- FABPc,转化于大肠杆菌 BL2 1 ,IPTG诱导表达。用 Glutathione Sepharose TM 4B亲和层析柱对表达产物进行纯化 ,Pre Scission TMProtease酶对融合蛋白进行解离 ,获得纯化的 14k Da Sj- FABPc。SDS- PAGE和 West-ern Blot方法对表达产物进行鉴定。结果　获得 p GEX- 6 P- 1/ Sj- FABPc菌株 ,分离纯化出 14k Da Sj-FABPc,表达量为 10 .5 2 m g/ L。Western Blot显示 ,表达蛋白能被日本血吸虫免疫兔血清识别。结论　重组蛋白 Sj- FABPc可高效表达并有良好的抗原性。Objective [WT5”BZ]To obtain recombinant protein [WT5”BX]Sj-[WT5”BZ]FABPc by gene engineering. [WT5”HZ]Methods [WT5”BZ]The gene fragment of [WT5”BX]Sj-[WT5”BZ]FABPc was amplified by PCR from the adult worm cDNA library of [WT5”BX]Schistosoma japonicum [WT5”BZ]and then subcloned into pGEM-T vector.The gene sequence was identified and the target fragment was restrictedly digested and subcloned into expression vector pGEX-6P-1. The protein is expressed and characterized. The fusion protein was purified by chromatography using Glutathione Sepharose TM 4B and was digested by the PreScission TM Protease. The 14kDa [WT5”BX]Sj-[WT5”BZ]FABPc can be obtained. The antigenicity of r[WT5”BX]Sj-[WT5”BZ]FABPc has been demonstrated by Western blotting. [WT5”HZ]Results [WT5”BZ]The pGEX-6P-1/[WT5”BX]Sj-[WT5”BZ]FABPc was constructed and expressed as r[WT5”BX]Sj-[WT5”BZ]FABPc in [WT5”BX]E.coli[WT5”BZ] BL 21 , and the yield of which was 10 52 mg/L. The r[WT5”BX]Sj-[WT5”BZ]FABPc can be recognized by the sera from the rabbits immunized with adult worm antigen of [WT5”BX]Schistosoma japonicum [WT5”BZ] using Western blotting. [WT5”HZ]Conclusion [WT5”BZ]The recombinant fusion protein could be expressed efficiently and had good antigenicity. [WT5”HZ]

关 键 词：日本血吸虫 脂肪酸结合蛋白 基因克隆 重组抗原 

分 类 号：R383.24[医药卫生—医学寄生虫学] Q784[医药卫生—基础医学]