BJ46a基因在昆虫细胞的转染、蛋白表达及超微结构观察  

BJ46A GENE TRANSFECTION,PROTEIN EXPRESSION IN BACULOVIRUS EXPRESSION SYSTEM AND ULTRASTRUCTURAL CHANGES OF HOST CELLS

作　　者：徐剑文[1,2] 林建银[1] 林旭[1] 张晓艳[1] 

机构地区：[1]福建医科大学分子医学研究中心 [2]福建医科大学基础医学院人体解剖学与组织胚胎学系,福州350004

出　　处：《中国组织化学与细胞化学杂志》2009年第3期249-253,共5页Chinese Journal of Histochemistry and Cytochemistry

基　　金：福建省产业技术研究开发项目(闽发改高技[2004]-686);福建省科技重点项目(2005D092);福建医科大学重大科技项目(XZ04002);福建省高校创新团队培养计划(闽教科[2006]36号)

摘　　要：目的研究蛇毒金属蛋白酶抑制剂BJ46a在杆状病毒表达系统的表达及宿主细胞Sf9超微结构的变化。方法将构建的杆状病毒重组穿梭载体Bacmid BJ46a,经Cellfectin脂质体介导转染Sf9昆虫细胞,获重组杆状病毒颗粒;以高滴度病毒感染Sf9昆虫细胞,行Western blot观察BJ46a融合蛋白的表达。在病毒扩增、蛋白表达过程应用透射电镜和超薄切片技术观察Sf9细胞超微结构的变化。结果Western blot分析表明:在转染病毒的Sf9细胞出现BJ46a融合蛋白表达条带。电镜观察表明:Sf9细胞在病毒扩增过程,细胞核增大,病毒发生基质形成,杆状病毒在其周围装配。蛋白表达期间,杆状病毒量剧增,细胞核内外存在大量丝状纤维。结论BJ46a基因可在杆状病毒表达系统成功表达,并伴随着宿主细胞超微结构的显著变化,其结果为杆状病毒表达系统的研究奠定坚实的基础。Objective To study the ultrastructural changes in host cells and the baculovirus expres sion system during virus amplification and protein expression. Methods Cellfectin reagent complexes containing BJ46a （ snake venom metalloproteinase inhibitor） gene were used to transfect insect cells Sf9 to produce recombinant baculovirus. After the baculoviral stock was amplified and titered, the high-titer stock was used to infect insect cells for large-scale expression of the BJ46a recombinant protein. Western blot was used to detect the protein expression, and the ultrastructural changes of host cells were observed by trans- mission electron microscopy and ultramicrotomy. Results Western blot analysis confirmed that BJ46a recombinant protein was produced by Sf9 cells infected with high-titer viral stock. Transmission electron mi- croscopy showed that virogenic stroma was located centrally in the nucleus after infection of viral stock during virus amplification, and there were many nucleocapsids and filament bundles in the nuclear periphery during protein expression. Conclusion BJ46a recombinant protein was expressed successfully in Sf9 cells with ultrastructural changes dramatically. The study of Sf9 offers the theoretical foundation and prerequisite for development and application of the baculovirus expression system.

关 键 词：蛇毒金属蛋白酶抑制剂(BJ46a) 杆状病毒表达系统 Sf9 超微结构 

分 类 号：R349.6[医药卫生—基础医学]