江浙蝮蛇毒酸性磷脂酶A_2基因的表达  

Expression of the APLA2 Gene from Agkistrodon halys Pallas

作　　者：刘小龙[1] 潘华[1] 杨冠珍[1] 吴祥甫[1] 周元聪[1] 蔡明德[2] 

机构地区：[1]中国科学院上海生物化学研究所 [2]上海市高血压研究所

出　　处：《生物化学与生物物理学报》1998年第4期397-401,共5页

基　　金：国家自然科学基金;中国科学院重大项目基金

摘　　要：将江浙蝮蛇毒酸性磷脂酶Ａ２基因克隆至表达载体ｐＢＬＭＶＬ２，转化入大肠杆菌ＲＲ１，经过温敏诱导，ＳＤＳ－ＰＡＧＥ检测，在约１４ｋＤ处有一表达条带。表达产物酸性磷脂酶Ａ２约占细菌蛋白总量的３０％，并以包涵体的形式存在。纯化包涵体后，将产物变性、复性，然后用ＦＰＬＣＳｕｐｅｒｏｓｅＴＭ１２纯化，产物经过ＳＤＳ－ＰＡＧＥ检测只有单一条带。通过Ｗｅｓｔｅｒｎ检测，证实纯化产物即是酸性磷脂酶Ａ２。最后对表达的酸性磷脂酶Ａ２进行了酶活性和生物活性的测定。至此我们成功地在大肠杆菌中表达蝮蛇毒酸性磷脂酶Ａ２基因。The APLA2 gene from Agkistrodon halys Pallas has been cloned into the expression plasmid pBLMVL2 and expressed in E.coli RR1. The molecular weight of the expressed product is approximately 14 kD as shown by SDSPAGE, its expression level is about 30% of the total cellular proteins. The protein was produced as insoluble inclusion bodies. After partially purified by washing the inclusion bodies, the product was denatured and refolded into active form. Then, the expressed APLA2 was purified by FPLC Superose TM 12 and was a single band as shown by SDSPAGE. The purified expressed protein had specific activity as the native enzyme and crossreacted with antisera prepared against the native enzyme. The successful expression of the APLA2 gene from Agkistrodon halys Pallas provides a good basis for further structurefunction studies.

关 键 词：江浙地区 蝮蛇毒 酸性磷脂酶A2 表达 活性 蛇毒 

分 类 号：Q78[生物学—分子生物学] Q55