马尾松毛虫质型多角体病毒多角体蛋白基因的表达与定位

Expression and Localization of the Dendrolimus punctatus Cypovirus Polyhedrin gene'

为了表达马尾松毛虫质型多角体病毒(DpCPV)多角体蛋白基因(S10片段)并探讨多角体蛋白在真核细胞中的定位,从DpCPV中分离出S10,与pET-28a载体连接成重组表达质粒pET28-S10;将S10克隆到杆状病毒转座载体pFASTBACHTb中,依次筛选出重组转座质粒pFASTBACS10,重组穿梭质粒BacmidS10,重组杆状病毒AcS10。多角体蛋白基因表达后,用SDS-PAGE、Western-blot和免疫荧光技术对表达产物进行了检测。结果表明:S10原核表达质粒、重组杆状病毒成功获得;在昆虫细胞中表达的质型多角体蛋白主要定位于细胞质,同时有少量产物定位于细胞核。

To express the polyhedrin （the tenth segment, S10） of Dendrolimus punctatus Cypovirus （DpCPV） and investigate the localization of the polyhedrin in eukaryote, The polyhedrin gene was amplified from DpCPV by RT-PCR and cloned into pET-28a vector. The polyhedrin was then expressed in bacteria. The purified protein was used to immunized rabbit to produce the antibody, The S10 ORF was also cloned into the baculovirus vector pFASTBAC-HTb. The recombinantplasmid pFASTBACS 10 was transformed to E, coli containing AcMNPV bacmid to generate the recombinant Bacmid AcS10. AcSI0 bacmid DNA transefect Sf9 cell and the recombinant baculoviruse vAcS 10 was produced. After S10 was expressed, the products were detected and analyzed by the SDS-PAGE, Western-blot and Immunofluorescence technique. The polyhedrin expressed in SF-9 cells was mainly located in cytoplasm.