苜蓿丫纹夜蛾核型多角体病毒(AcMNPV)大立方形多角体突变株的分子生物学研究  

MOLECULAR RESEARCH ON AN AUTOGRAPHA CALIFORNICA MULTICAPSID NUCLEAR POLYHEDROSIS VIRUS (AcMNPV) LARGE CUBIC POLYHEDRON MUTANT

作　　者：林广云[1] 龙綮新[1] 何代芬 曲士芮 庞义[1] 

机构地区：[1]中山大学生物防治国家重点实验室

出　　处：《病毒学报》1997年第2期151-158,共8页Chinese Journal of Virology

摘　　要：利用空斑技术，从既能形成多角体又含ＴＫ酶基因的苜蓿丫纹夜蛾重组核型多角体病毒ＡｃＭＮＰＶ－ＴＫ中，纯化了一株形成大立方形多角体的病毒突变株ＡｃＭＮＰＶ－ＴＫｍｔ５１３。用ＥｃｏＲＩ、ＰｓｔⅠ、ＢｇｌⅡ及ＫｐｎⅠ等限制性内切酶对它做了酶切分析，并克隆了多角体蛋白全基因及其侧翼部分的片段。对所克隆的部分全序列测定结果，发现在多角体蛋白基因读码框内只出现了一个碱基的突变，导致了第２５位的氨基酸密码子由ＧＧＴ变为ＧＡＴ，该位氨基酸由甘氨酸（Ｇｌｙ）变为天冬氨酸（Ａｓｐｑ）还利用ＰＣＲ技术扩增出突变了的多角体蛋白基因部分片段，并将它克隆入转移载体质粒ｐＥＶ５５，与不形成多角体的重组病毒ＴｎＮＰＶ－ＨＢｓＤ４ＤＮＡ共转染Ｓｆ９细胞，结果产生同样的大立方形多角体病毒。A recombinant virus AcMNPV-TKmt51 3 with the insertion of thymidine kinase that can produce large cubic polyhedra was plaque-purified.It was analyzed with BglⅡ,EcoR-Ⅰ,KpnⅠ,PstⅠrestriction endonucleases and found to show the expected patterns.The polyhedrin gene,its coupled flanking regions,TK gene and promoters were cloned and se-quenced.It was found that in the polyhedrin ORF,the codon of the 25th amino acid has mutated from GGT to GAT,causing an amino acid change from Gly to Asp.In order to identify this mutation is the main reason for the change of polyhedron shape and amount,we employed PCR technique to amplify the fragment of polyhedrin gene from the startcodon ATG to the restriction enzyme KpnⅠrecognition site including the mutationsite, and introduced it into transfer vector pEV55 which originally contains the partial polyhedrin encoding sequence from KpnⅠto TAA.Cotransfection of the resultant recombinant plasmid and TnNPV-HBsD4 DNA into Sf9 cells.resulted in the appearance of the large cubic polyhedra,thus confirming that the single amino acid change is responsible for this mutant phenotype.

关 键 词：昆虫病毒 苜蓿丫纹夜蛾 核型多角体病毒 多角体 

分 类 号：Q965.8[生物学—昆虫学]