H3、H5、H7、H9亚型流感病毒通用多表位重组鸡痘病毒构建  

Construction of mulfiple-epitope rFPV against H3,H5,H7,H9 Subogpe in fluenza A Vinus

作　　者：陈义锋[1,2] 金宁一[1] 贾雷立[3] 鲁会军[1] 南文龙[1,2] 田明尧[1,2] 陈晓月[1,2] 马海利[1,2] 马鸣潇[1,2] 刘成宏[1,2] 

机构地区：[1]吉林大学畜牧兽医学院,长春130062 [2]中国人民解放军军事医学科学院11所全军基因工程重点实验室,长春130062 [3]军事医学科学院疾病预防所,北京100071

出　　处：《免疫学杂志》2007年第5期524-527,共4页Immunological Journal

基　　金：教育部重点项目(306006);吉林省科技发展计划重大项目(20065020)资助

摘　　要：目的构建表达H3、H5、H7、H9亚型流感病毒通用多表位重组鸡痘病毒活载体疫苗候选株。方法筛选流感病毒主要抗原(HANANPM)优势抗原表位基因,以生物信息学软件优化其排列结构合成流感多表位基因(Epi),以H5、H7HA融合表达基因为骨架,将多表位基因插入构建H7HA-Epi-H5HA阅读盒,定向克隆至鸡痘病毒表达载体pUTA-2复合启动子(ATI-P7.5×20)下游,构建重组鸡痘病毒中间转移载体pUTA-2-H7HA-Epi-H5HA。应用脂质体介导的转染法,将该重组鸡痘病毒中间转移载体与282E4株鸡痘病毒共转染鸡胚成纤维细胞(CEF),经BrdU进行3次加压筛选挑斑纯化后传代,并对重组以鸡痘病毒进行PCR和IFA检测和Western blot分析。结果成功构建了重组鸡痘病毒中间转移载体pUTA-2-H7HA-Epi-H5HA,PCR、IFA和Western blot检测阳性。结论筛选出稳定共表达H5H7HA基因和通用多表位基因的重组鸡痘病毒vUTA2-H7HA-Epi-H5HA株,该重组鸡痘病毒的构建为跨种通用型流感病毒活载体疫苗的研究奠定了物质基础。Objective To construct recombinant fowlpox virus （rFPV） co-expressing multi-epitopes of influenza/A subtype H3, HS, HS, H7, and H9 virus. Methods The epitope gene of dominant antigen （HA NA NP M） d of influenza A viruses was selected. The multiepitopes gene was synthesized and optimized by bioinformatics methods, and then inserted into the HSH7HA gene to construct an ORF, which should be cloned into downstream of the complex prompter （ATI-P7.5×20） of pUTA-2 vector to construct the recombinant fowlpox virus middle transfer vector pUTA-2-H7HA-Epi-HSHA. The CEF was then co-transfected with the reconstructed vector and FPV 282E4 strain under the pressures of Brdu. PCR, IFA, and Western blotting were also utilized to identify the recombinant virus and detect the bioacfivity of the expression products in CEF, respectively. Results The middle transfer vector pUTA-2-H7HA-Epi-HSHA for rFPV was constructed successfully. The antibody specificity of the rFPV expressional product was detected by IFA and Western blotting. Conclusion The rFPV obtained by three times of pressure of Brdu could express efficiently, furthermore the expression product can specially recognized by the corresponding antibodies.

关 键 词：重组鸡痘病毒 多表位基因(Epi) 构建 

分 类 号：S852.65[农业科学—基础兽医学]