广西眼镜王蛇毒酸性磷脂酶A_2-1在不同载体的表达  

Expression of Acidic Phospholipase A_2-1(APLA_2-1) from Guangxi Ophiophagus hannah (King Cantor) Venom in Different Vectors

作　　者：林文珍[1] 舒雨雁[1] 庄茂辛[1] 林政炯[2] 吴祥甫[3] 周元聪[3] 

机构地区：[1]广西医科大学生物化学与分子生物学教研室,广西南宁530021 [2]中国科学院生物物理研究所生物大分子国家重点实验室,北京100101 [3]中国科学院上海生命科学院生物化学与细胞生物学研究所,上海200031

出　　处：《蛇志》2007年第3期175-179,共5页Journal of Snake

基　　金：国家自然科学基金资助项目(No.39970174)

摘　　要：目的构建广西眼镜王蛇毒酸性磷脂酶A2-1(APLA2-1)在不同载体的重组表达质粒,在E.coli中表达APLA2-1并比较不同表达系统对APLA2-1的表达效果。方法将广西眼镜王蛇毒酸性磷脂酶A2-1(AP-LA2-1)基因克隆至表达载体pBLMVL2和pET28a(+),分别转化入大肠杆菌RR1和BL21,经过诱导表达,应用SDS-聚丙烯酰胺凝胶(SDS-PAGE)及Western blot观察重组蛋白表达情况。结果成功构建了重组质粒pBLMVL2-APLA2-1和pET28a-APLA2-1。pBLMVL2-APLA2-1在SDS-PAGE上没见明显表达带,在Western blot上可见一14 kD的表达带。pET28a-APLA2-1在SDS-PAGE上有一明显的18 kD表达条带,表达产物AP-LA2-1约占细菌总量30%,并以包涵体的形式存在。结论APLA2-1可在大肠杆菌中表达,pET28a(+)对APLA2-1的表达效果优于pBLMVL2。Objective To construct recombinant plasmids encoding acidic phospholipase A2-1 （APLA2-1） from Guangxi Ophiophagus hannah （King cantor） Venom and express in E. coli so as to compare the expression efficiency in different expression systems. Methods A cDNA encoding acidic phospholipase A2-1 （APLA2-1） from Guangxi Ophiophagus hannah （King can- tor） Venom was cloned into bacterial expression vector pBLMVL2 ,pET28a（q-） and expressed in E. coli RR1 ,BL21 respectively. The expressed recombinant proteins were analyzed by sodi- um docedyl sulfate polyacrylamide gel electrophoresis （SDS-PAGE） and Western blot. Results The recombinant plasmids pBLMVLz-APLA2-1 and pET28a-APLA2-1 were constructed sucessfully. No obviously expression band was observed in SDS-PAGE by using pBLMVL2 expression system, but one band with molecular weight 14 kDa was observed in Western blot. Moreover, a strong band with molecular weight 18 kDa was observed in SDS-PAGE and Western blot using pET28a（＋） expression system. The expression amount of APLA2-1 was about 30M of that of total cell proteins. In addition, the protein was produced as insoluble inclusion bodies. Conclusion The APLA2-1 could be expressed in E. coli. The results showed that there was better expression efficiency in pET28a（＋） than pBLMVL2.

关 键 词：广西眼镜王蛇毒 酸性磷脂酶A2 表达 

分 类 号：R996.3[医药卫生—毒理学]