血管紧张素Ⅱ基因重组杆状病毒的构建  

Construction of recombinant baculovirus with angiotnensin Ⅱ gene

作　　者：欧霞[1] Yuri Kusov 郭莉莉[1] 米锴[1] 吴晋元[1] 尹娜[1] 张光明[1] 李鸿钧[1] 孙茂盛[1] 

机构地区：[1]中国医学科学院北京协和医学院医学生物学研究所分子生物室云南省重大传染病疫苗工程技术研究中心云南省重大传染病疫苗研发重点实验室,云南昆明650118 [2]Department of Medical Molecular Biology University Lübeck

出　　处：《中国生物制品学杂志》2013年第5期590-594,602,共6页Chinese Journal of Biologicals

基　　金：北京协和医学院研究生创新基金(2011-1001-031)

摘　　要：目的构建携带血管紧张素I(IAngiotensin Ⅱ,AngⅡ)基因的重组杆状病毒,并利用Bac-to-Bac重组杆状病毒表达系统进行表达。方法将重组杆粒bacmid-P1-2A-4AngⅡs-3ABC(rB/HAngⅡ)转染昆虫细胞sf9,包装出重组杆状病毒rBac-P1-2A-4AngⅡs-3ABC(rBAV),通过光学显微镜和透射电镜观察细胞病变(CPE)及病毒形态,采用蚀斑试验检测病毒滴度,PCR及间接免疫荧光技术验证该病毒及感染细胞中AngⅡ和HAV蛋白的表达。结果重组杆粒rB/HAngⅡ转染sf9细胞96 h后,出现典型的CPE;透射电镜下观察可见典型的杆状病毒颗粒;PCR可特异性的扩增出约3 000和1 000 bp的P1-2A-4AngⅡs和3ABC基因片段;P3代重组杆状病毒滴度约为4×108pfu/ml;感染后的sf9细胞中可见特异性的绿色荧光(AngⅡ蛋白)和红色荧光(HAV结构蛋白)。结论已成功构建了携带AngⅡ基因的重组杆状病毒,并在sf9细胞中获得表达,为研制抗高血压疫苗提供了新的思路。Objective To construct the recombinant baculovirus vector containing angiotnensin（Ang）Ⅱ gene and express recombinant AngⅡ by using Bac-to-Bac expression system.Methods Insect sf9 cells were transfected with bacmid-P12A-4Ang Ⅱs-3ABC（rB/HAngⅡ）to obtain recombinant baculovirus rBac-P1-2A-4AngⅡs-3ABC（rBAV）.The cytopathic effect（CPE）and morphology of virus were observed by optical and transmission electron microscopy.The virus titer was determined by plaque assay.The expressions of AngⅡ and HAV in the virus and infected cells were determined by PCR and indirect IFA.Results Typical CPE was observed in sf9 cells 96 h after transfection with rB/HAngⅡ.Typical baculovirus particles were observed under transmission electron microscope.P1-2A-4Ang IIs and 3ABC gene fragments,at lengths of about 3 000 and 1 000 bp respectively,were amplified specifically by PCR respectively.The titer of recombinant baculovirus of passage 3（P3）was about 4×10 8 pfu/ml.IFA showed specific green（AngⅡ）and red（HAV structural protein）fluorescences in infected sf9 cells.Conclusion The recombinant baculovirus with AngⅡ gene was successfully constructed and expressed in sf9 cell,which provided a new route for development of anti-hypertension vaccine.

关 键 词：血管紧张素Ⅱ 杆状病毒表达系统 高血压疫苗 

分 类 号：R544.1[医药卫生—心血管疾病] R392.33[医药卫生—内科学]