半胱氨酸蛋白酶基因在家蚕杆状病毒表达系统中的表达  

Expression of Cysteine Protease Gene in Bm-BmNPV Expression System

作　　者：林旭瑷 李卫国[2] 陈寅[1] 易咏竹[1] 张志芳[1] 何家禄[1] 

机构地区：[1]农业部家蚕生物技术重点开放实验室,中国农业科学院蚕业研究所,镇江212018 [2]山东农业大学林学院蚕学系

出　　处：《蚕业科学》2003年第3期241-245,共5页ACTA SERICOLOGICA SINICA

摘　　要：为了探讨杆状病毒的致病机理、改善杆状病毒表达系统的表达效率 ,将来源于家蚕核型多角体病毒ZJ8株的半胱氨酸蛋白酶 (cystenineprotease,CP)基因克隆到转移载体pVL 1393上 ,获得重组转移载体pVL cp ,与线性化的Bm BacPAK6病毒DNA共转染家蚕Bm 5贴壁细胞。通过蓝白斑筛选、纯化后得到的重组病毒经PCR鉴定证明 ,cp基因已被正确导入 ,注射感染家蚕 5龄幼虫 12 0h后表达产物活性达到最高。对表达产物进行SDS PAGE及酶活力分析 ,检测到半胱氨酸蛋白酶在家蚕生物反应器中的表达 ,其表达量约为 16 0 0U/mL。To inquire into the mechanism of infection of baculovirus and improve its efficiency as a vector in the expression of foreign protein, the cysteine protease gene from BmNPV ZJ8 through PCR was incorporated into the transfer vector pVL 1393, then Bm 5 cells were co transfected with linerizated parental virus (Bm BacPAK6) DNA and the above mentioned recombinant plasmid with lipofectin. The screened recombinant virus was identified by PCR, which expressed in the newly molted 5 instar larvae of silkworm Bombyx mori. The result indicated that rBmNPV CP can expressed in the silkworm and achieved its peak at 120 hours p.i., and the product of CP had been found through SDS PAGE and protease activity analysis, the yield of CP is about 1 600 U/mL.

关 键 词：半胱氨酸蛋白酶 家蚕杆状病毒表达系统 基因表达 致病机理 生物反应器 生物杀虫剂 

分 类 号：Q78[生物学—分子生物学]