利用柞蚕核型多角体病毒表达载体系统在柞蚕蛹中表达增强型绿色荧光蛋白  

Expression of Enhanced Green Fluorescent Protein in Antheraea pernyi Pupa by Using Antheraea pernyi Nucleopolyhedrovirus Expression Vector System

作　　者：王林美[1] 张波[1] 叶博[1] 赵振军[1] 李树英[1] 李文利[1] 岳冬梅[1] 范琦[1] 

机构地区：[1]辽宁省农业科学院大连生物技术研究所,辽宁省野蚕重点实验室,辽宁大连116024

出　　处：《蚕业科学》2010年第5期792-799,共8页ACTA SERICOLOGICA SINICA

基　　金：国家人事部留学人员科技活动项目(No.LXZZ2005005);辽宁省科技攻关项目(No.2008208001)

摘　　要：为了探讨外源蛋白在柞蚕蛹-柞蚕核型多角体病毒(ApNPV)表达系统的表达水平和稳定性,将增强型绿色荧光蛋白(EGFP)基因克隆到柞蚕核型多角体病毒转移表达载体pApM748BE中,获得重组质粒DNA,与ApNPV DNA共转染樗蚕(Phi-losamia cynthiam)培养细胞Pc-01后,用末端稀释法筛选获得重组病毒ApNPV-Δph/egfp+。将该重组病毒感染柞蚕蛹,采用SDS-PAGE和Western blot方法检测EGFP在柞蚕蛹中的表达,结果显示:在柞蚕蛹感染重组病毒ApNPV-Δph/egfp+后6 d,EG-FP就有明显的表达;感染后12～18 d,蛹体液中的EGFP含量保持较高水平,以EGFP标准样品定量分析EGFP在柞蚕蛹体液中的表达水平高于1 mg/mL;感染后30～39 d仍可以检测到蛹体液中EGFP的表达,而且蛋白稳定。研究结果表明,利用柞蚕核型多角体病毒作表达载体,可在柞蚕蛹中高效稳定地表达EGFP,并且EGFP在雌雄蛹间的表达水平无明显差异。In order to examine the expression level and stability of expressing foreign genes in Antheraea pernyi pupa-nucleopolyhedrovirus expression system,an enhanced green fluorescent protein （EGFP） gene was cloned into pApM748BE,a transfer vector of Antheraea pernyi nucleopolyhedrovirus （ApNPV） .The recombinant plasmid DNA was obtained and used to co-transfect cultured Pc-01 cells of Philosamia cynthia together with ApNPV DNA.The recombinant virus,ApNPV-Δph/egfp ＋ ,was selected with limited dilution method.Pupae of A.pernyi were infected by this recombinant virus and the expression of EGFP was analyzed using SDS-PAGE and Western blotting methods.The results showed that EGFP was obviously expressed in pupae at 6 dpi （days post-infection） by the recombinant virus ApNPV-Δph/egfp ＋ .Contents of recombinant EGFP in haemolymph of pupae were maintained at a high level at 12 to 18 dpi,being over 1 mg /mL as shown by quantitative analysis in comparison with EGFP standard sample.The expression of EGFP was still detectable at 30 to 39 dpi and the expressed proteins were stable.These results indicate that EGFP can be efficiently and stably expressed in A.perryi pupae by using A.perryi nucleopolyhedrovirus as expression vector.Nevertheless,there is no obvious difference between the ex-pression levels of female and male pupae.

关 键 词：柞蚕蛹 核型多角体病毒表达载体系统 增强型绿色荧光蛋白基因 樗蚕培养细胞 

分 类 号：Q78[生物学—分子生物学]