HcNPV半胱氨酸蛋白酶,几丁质酶基因失活分析

将含有美国白蛾核型多角体病毒（Ｈｙｐｈａｎｔｒｉａ ｃｕｍｅａ ｎｕｃｌｅａｒ ｐｏｌｙｈｅｄｒｏｓｉｓ ｖｉｒｕｓ，ＨｃＮＰＶ）半胱氨酸蛋白酶基因（ＣＰ）的自然和几丁质酶基因（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。通过重组转移载体与含有家蚕促

Inactivation Analysis of HcNPV Cysteine Protease Gene and Chitinase Gene

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.