HcNPV半胱氨酸蛋白酶、几丁质酶基因失活分析  

Inactivation Analysis of HcNPV Cysteine Protease Gene and Chitinase Gene

作　　者：贡成良[1] 小林淳[2] 金伟[3] 吴祥甫[4] 

机构地区：[1]苏州大学蚕桑学院基因工程实验室,苏州215151 [2]日本三重大学工学部材料化学系 [3]浙江大学蚕学系,杭州310029 [4]中国科学院上海生物化学研究所,上海200031

出　　处：《生物化学与生物物理学报》2000年第2期187-191,共5页

摘　　要：将含有美国白蛾核型多角体病毒 (Hyphantriacuneanuclearpolyhedrosisvirus,HcNPV)半胱氨酸蛋白酶基因 (CP)的片段和几丁质酶基因 (ChiA)的片段克隆进PCRII,构建了转移载体pHcCVdel;将含有HcNPV多角体蛋白全基因 ( polh)序列的片段插入到 pHcCVdel的EcoRI位点 ,得到重组转移载体pHcCVpolh。通过该重组转移载体与含有家蚕促前胸激素基因 (PTTH)的重组病毒HcNPVPTTH+的DNA共转染SPIM细胞 ,使 polh基因取代ChiA基因起始密码下游 +76位到CP基因起始密码下游 +2 0位之间的区域 ,从而构建了CP、ChiA两基因同时失活的、能表达PTTH并能形成多角体的重组病毒HcNPVpolh+CP-ChiA-PTTH+。感染SPIM细胞表明 :CP、ChiA两基因为病毒复制非必需基因 ,它们的失活不影响病毒的复制与多角体的形成 ,但感染细胞的存活时间比HcNPV、HcNPVPTTH+感染的多 2天。推测CP、ChiA两基因失活后 ,可延长细胞持续表达外源基因的时间。The fragment of Hyphantria cunea nuclear polyhedrosis virus (HcNPV), which contains cysteine protease gene( CP ) and chitinase gene ( Chi A),was inserted into plasmid PCRII to construct transfer vector pHc CV del. Recombinant transfer vector pHc CVpolh was constructed by inserting the fragment of HcNPV polyhedrin gene ( polh ) into the Eco RI site of the pHc CV del. By cotransfection of the pHc CVpolh and HcNPV PTTH + DNA, which contained Bombyx mori prothoracicotropic hormone gene ( PTTH ) into SPIM cells, the region from +76 downstream of Chi A gene initiation codon to +20 downstream of CP gene initiation codon was substituted by the polh gene, and the recombinant virus HcNPV polh +CP -Chi A - PTTH + was produced. The recombinant virus, in which CP gene and Chi A gene were inactive, could express PTTH gene and form polyhedra. SPIM cells infected with the recombinant virus revealed that CP and Chi A gene were not essential for viral replication and had no significant effect on polyhedron formation, but the infected cells survived 2 days more than those infected with HcNPV and HcNPV PTTH +. Therefore, the expression time of foreign gene may be prolonged when CP gene and Chi A gene have been inactivated.

关 键 词：HCNPV 半胱氨酸蛋白酶 几丁质酶 基因失活 

分 类 号：Q782[生物学—分子生物学]