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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 construct a rapid eukaryotic expression system of hemagglutinin (HA) gene of novel H7N9 strain by lentivirus vector and express in human embryonic kidney cell (HEK293T) for functional identification. Methods Genomic RNA of H7N9 bird flu virus was extracted. The full length of HA reading frame was amplified by reverse transcription polymerase chain reaction (RT - PCR). The HA gene was ligated into pMD18-T vector to construct pMD18-T-HA recombinant vector. Primers containing the Kozak sequence were designed to amplify the blunt-ended HA gene from the pMD18-T-HA plasmid and the expression vector pLenti-HA-V5 was constructed using Topo cloning. The expression vector was transfected into HEK293T cells. The expression of HA protein was identified by indirect immunofluorescence (IFA) and Western blot. The biological activity of the recombinant protein was identified by hemagglutination assay. Results The full length gene of 1 683 bp was obtained by reverse transcription PCR. The eukaryotic expression vector was constructed and the recombinant protein with relative molecular mass (Mr) of 70 000 was expressed. The result of IFA and Western blot showed that the protein had good immunoreactivity with the positive serum, meanwhile, the hemagglutination test confirmed that it has hemagglutination activity. Conclusion The rapid eukaryotic expression system of HA protein was established successfully using lentiviral vector, which laid the foundation for the development of H7N9 virus subunit vaccine, neutralizing epitopes and packaging of pseudovirus.