采用杆状病毒表达系统表达EGFP标记的HPV-16L1病毒样颗粒  

Expression of EGFP-HPV-16L1 fusion protein by bac-to-bac and insect cell system

作　　者：杨军[1,2] 王一理[2] 司履生[2] 

机构地区：[1]西安交通大学医学院第二附属医院病理科,710004 [2]西安交通大学医学院生物医学信息工程教育部重点实验室,生命科学与技术学院癌症研究所,710061

出　　处：《中华微生物学和免疫学杂志》2007年第3期207-212,共6页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金（No.30300305）

摘　　要：目的制备增强型绿色荧光蛋白（EGFP）标记的人乳头状瘤病毒16型（HPV-16）病毒样颗粒，为研究HPV-16L1的生物学活性提供一种方法。方法采用Xbal＋HindⅢ双酶切pGEM-T-HPV-16质粒，获得HPV-16L1基因片段，用PCR法从pEGFP质粒中扩增EGFP基因片段，NcoⅠ＋SalⅠ双酶切，依次克隆入杆状病毒转移载体，获得重组pFB-EGFP-HPV-16L1转移载体；在DHIOBac内通过转座子Tn7的介导，获得重组Ac-EGFP-HPV-16L1杆状病毒；感染Sf-9昆虫细胞；荧光显微镜、激光共聚焦显微镜和免疫组织化学法确定融合蛋白的荧光特性及细胞学定位；利用Western blot分析融合蛋白的表达；电子显微镜观察病毒样颗粒（VLP）的形成；利用小鼠红细胞凝集试验及凝集抑制试验分析生物学活性。结果重组pFB-EGFP-HPV-16L1辅助表达载体及重组杆状病毒中EGFP-HPV-16L1融合基因构建正确；荧光显微镜和激光共聚焦显微镜证实感染重组杆状病毒AC-EGFP-HPV-16L1的Sf-9细胞核内可见耀眼的绿色荧光，免疫组织化学证实EGFP-HPV-16L1融合蛋白主要位于Sf-9细胞核内；Western blot证实EGFP-HPV-16L1融合蛋白相对分子质量（Mr）为83×10^3，与理论值一致；透射电子显微镜观察发现感染重组Ac-EGFP-HPV-16L1杆状病毒的Sf-9细胞核内和裂解上清内均可发现大量形态均一的vIJP形成；小鼠红细胞凝集试验证实EGFP-HPV-16L1融合蛋白可引起小鼠红细胞凝集，且可被抗HPV-16L1单克隆抗体抑制。结论杆状病毒表达系统表达的EGFP标记的HPV-16L1蛋白既能自组装成病毒样颗粒、具有构象依赖性生物学活性，又能发射易于检测的绿色荧光，有可能用于HPV-16L1蛋白的生物行为研究，并实时可视化追踪其在宿主细胞内的转运过程。Objective To produce EGFP tagged HPV-16 VLP, study its biological activity, and to create an experiment model for investigation of HPV-16. Methods HPV-16LI gene was gained from pGEM-T-HPV-16 plasmid by Xbal ＋ HindⅢ restriction enzyme, EGFP gene was amplified from pEGFP plasmid by PCR. Mter the genes were recombined into baculovirus transfer vector pFastbac-Hb, a recombinant transfer vector pFB-EGFP-HPV-16L1 was created. When it was used to transform E. coli DHIOBac, the recombinant baculovirus Ac-EGFP-HPV-16L1 encampashag EGFP-HPV-16L1 fusion gene with the action of Tn7 transposon was obtained. Mter the insect Sf-9 cells were infected with the recombinant baculovirus, the fluorescence of EGFP-HPV-16L1 fusion protein was observed in fluorescent microscopy and laser confocal microscopy. While the expression and the intracellular localization of EGFP-HPV- 16L fusion protein in Sf-9 cells was identified by Immunohistochemical method with anfi-HPV-16L1 antibody. The expression of EGFP-HPV-16L1 fusion protein was analyzed by Western blot. The infected Sf-9 cells and the supernatant of Sf-9 cell lysate were examined in tranmission electron microscopy （TEM） respectively. Meanwhile, the biological activity of the interested protein was analyzed by hemagglufination assay （HA） and hemagglutination inhibition assay （HAI）. Results PCR and restriction enzymes digestion confirmed the identity of recombinant transfer vector pFB-EG- FP-HPV-16L1 and recombinant baculovirus containing EGFP-HPV-16L1 fusion gene. After transfected with the indicated expression vectors 4 hours, the green fluorescence can be finding from the Sf-9 cells. And from then on, the green fluorescence congregated in the nuclear of Sf-9 cells transfected with the recombinant Ac-EGFP-HPV-16L1 virus. Finally glare green fluorescence was demonstrated mainly in the nuclei of infected Sf- 9 cells . But the green fluorescence spreaded through the nuclear and cytoplasm of Sf-9 cells tmnsfected with the recombinant Ac-EGFP virus. The molecular mass of E

关 键 词：人乳头状瘤病毒16型 重组杆状病毒 增强型绿色荧光蛋白 病毒样颗粒 融合蛋白 

分 类 号：R686[医药卫生—骨科学]