| 柞蚕NPV转移载体组建及外源基因在Sf9细胞、柞蚕卵巢原代细胞和蛹体中的表达 |
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| 引用本文： | 张春发,刘淑珊,范琦,王林美,李文利,李广泽.柞蚕NPV转移载体组建及外源基因在Sf9细胞、柞蚕卵巢原代细胞和蛹体中的表达[J].蚕业科学,1992(3). |
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| 作者姓名： | 张春发 刘淑珊 范琦 王林美 李文利 李广泽 |
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| 作者单位： | 辽宁省农科院大连生物技术研究所
(张春发，刘淑珊，范琦，王林美，李文利)，辽宁省农科院大连生物技术研究所(李广泽) |
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| 摘 要： | 根据对载有ApNPV核多角体蛋白基因的片段的酶谱和核苷酸序列分析结果,采用人工合成寡核苷酸引物引导的点突变方法,去掉了该基因的起始密码子(ATG突变为ATT),经系列克隆组建了pApM740转移载体,其外源基因插入位点为nt+141(BamHI)。系用多聚酶链反应(Polymerase Chain Rea-ction简称PCR)技术,分别将nt+2～+140,nt+9～+140、和nt+37～+140切掉,组建了pApM740、741、748和736转移载体,其外源基因克隆位点分别为nt+1、+8和+36(均为BamHI切点)。将IL-4、DE基因分别克隆到pApM740、741、748和736载体中,并进行了转染表达实验:(1)将载有DE基因的上述四种载体质粒DNA分别与AcNPV_(WT)DNA共转染Sf9细胞、经免疫抗体荧光检测,供试4个载体所载DE基因均得到表达;(2)将pApM741-IL_4和pApM748-IL_4重组载体质粒DNA分别与ApNPV_(WT)DNA共转染柞蚕卵巢原代细胞,待多角体出现后,用PCR法检测病毒基因,证实IL-4基因已整合到ApNPV DNA基因组中;(3)将pApM748-DE重组载体质粒DNA与ApNPV _(WT)DNA共转染柞蚕蛹获得成功。对病蛹病毒DNA进行PCR检测,证实DE基因已整合到病毒基因组中;取病蛹体液,用免疫沉淀及Western blotting方法检测DE蛋白,证实DE蛋白确已被表达。从而初步建立了柞蚕NPV载体—昆虫细胞宿主和柞蚕NPV载体—柞蚕蛹活
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| 关 键 词： | 柞蚕 核型多角体病毒 转移载体 基因突变 载体表达系统 |
CONSTRUCTION OF GENE TRANSFER VECTORS OF CHINESE OAK SILKWORM, A. PERNYI NUCLEAR POLYHEDROSIS VIRUS AND THEIR USE FOR EXPRESSION OF FOREIGN GENES IN INSECT CELLS AND THE HOST PUPAE OF A. PERNYI |
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| Zhang Chun-fa Liu Shu-han Fan Qi Wang Lin-mei Li Wen-li Li Guang ze.CONSTRUCTION OF GENE TRANSFER VECTORS OF CHINESE OAK SILKWORM, A. PERNYI NUCLEAR POLYHEDROSIS VIRUS AND THEIR USE FOR EXPRESSION OF FOREIGN GENES IN INSECT CELLS AND THE HOST PUPAE OF A. PERNYI[J].Acta Sericologica Sinica,1992(3). |
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| Authors: | Zhang Chun-fa Liu Shu-han Fan Qi Wang Lin-mei Li Wen-li Li Guang ze |
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| Abstract: | Four gene transfer vectors of ApNPV,pAp M 740,741 ,748 and 736,were constructed using the polyhedrin gene promoter which the translation initiation codon was removed (ATG to ATT) by site-derected mutagenesis and the different length of the N-terminal protein coding sequence (nt+ ,+8,. + 35 and nt+140) were remained in these vectors for it's important in the regulation of translation. For detectin of the expression function,IL-4 and DE genes were cloned to these vectors: ( 1) according to the high homology of polyhedrin gene sequences between AcN-PV and ApNPV,these recombinant plasmid DNA containing DE gene were cotransfected with AcNPV (WT) DNA to Sf9 cells,and DE protein was expressed by all these four vectors detected by immunof luorescence: (2) The 1L-4 gene was inserted to the vectors of pAp M 741 and 748,cotransfected with ApNPV (WT) DNA to the primary cultural ovary cells of A. pernyi,the cells were infected and the recombinant IL-4 gene was detected by PCR using the specific primers to IL-4 gene; (3) The pAp M 748-DE recombinant plasmid DNA was cotransfected with ApNPV (WT) DNA directly to ApNPV host pupae of A. pernyi. and it was succeeded. The recombinant DE gene was detected in the infected pupae by PCR and the DE protein expressed in the pupae were idenfified by immunoprecipitation and western blot,the expression level (v/v) was much higher compared with AcNPV-Sf9 cell expression system. |
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| Keywords: | Antheraea pernyi Nuclear polyhedrosis virus Gene transfer vector Gene mntation Gene expression |
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