慢病毒介导的H7N9禽流感病毒血凝素基因快速真核表达系统的建立  

Establishment of a lentivirus-mediated rapid eukaryotic expression system of novel avian influenza H7N9 virus hemagglutinin gene

作　　者：张黎[1] 彭海燕[1] 周明浩[1] 余慧燕[1] 曾晓燕[1] 祁贤[1] 崔仑标[1] 焦永军[1] 史智扬[1] 

机构地区：[1]江苏省疾病预防控制中心卫生部肠道病原微生物重点实验室,江苏南京210009

出　　处：《细胞与分子免疫学杂志》2014年第6期630-634,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家科技重大专项(2012ZX10004-210-004)

摘　　要：目的利用慢病毒载体构建新型H7N9禽流感病毒血凝素(HA)基因的快速真核表达体系,在人胚肾细胞(HEK293T)中表达并进行功能鉴定。方法提取H7N9禽流感病毒基因组RNA,采用反转录PCR技术扩增HA读码框全长基因,将HA基因连接pMD18-T载体构建pMD18-T-HA重组载体。设计含Kozak序列的引物从pMD18-T-HA质粒中扩增出平末端HA基因,采用Topo克隆构建表达载体pLenti-HA-V5。将表达载体转染HEK293T细胞,通过间接免疫荧光法(IFA)和Western blot法鉴定HA蛋白的表达,通过血凝实验鉴定重组蛋白的生物学活性。结果经反转录PCR获得1 683 bp的HA全长基因,完成真核表达载体的构建并表达出相对分子质量(Mr)为70 000的重组蛋白。IFA和Western blot法结果显示该蛋白与阳性血清具有良好的免疫反应,同时血凝实验证实其具有血凝活性。结论成功利用慢病毒载体建立了HA蛋白的快速真核表达系统,为研制H7N9病毒亚单位疫苗、中和表位研究、假病毒包装奠定基础。Objective To establish a rapid eukaryotic expression system of hemagglutinin （HA） gene of novel avian influenza H？N9 using lentiviral vector, express the recombinant protein and study its functions in human embryonic kidney HEK？,93T cells. Methods The full-length HA gene was amplified from H7N9 genomic RNA by reverse transcription PCR （RT-PCR） and linked with pMDI8-T vector to generate pMDIS-T-HA plasmid. Blunt-end HA gene with Kozak sequence was amplified from pMD18-T-HA vector, and then pLenti-HA-V5 expression vector was constructed by Topo cloning for transient expression in HEK293T cells. Expression of HA-V5 recombinant protein was confirmed by immunofluorescence assay （IFA） and Western blotting. Hemagglutination test was performed to evaluate the biological activity of the recombinant protein. Results The full-length HA gene （1 683 bp） was obtained and eukaryotic expression plasmid was constructed successfully. A recombinant protein with relative molecular mass （Mr） 70 000 was expressed and the antigenicity and binding specificity to positive serum were demonstrated by IFA and Western blotting. The hemagglutination activity was proved by hemagglutination test. IFA and Western blotting showed that the Mr 70 000 recombinant protein had an immuoreactivity to positive serum. The hemagglutinaUon activity was confirmed by hemagglutination test. Conclusion The rapid eukaryotic expression system of HA gene was successfully constructed, which laid a solid foundation for further research on subunit vaccine development, neutralizing epitope mapping and packaging pseudovirus.

关 键 词：H7N9禽流感病毒 血凝素基因 慢病毒载体 真核表达系统 

分 类 号：R392.33[医药卫生—免疫学]