稳定表达甲型流感病毒血凝素蛋白的哺乳动物细胞系的建立

目的建立稳定表达甲型流感病毒血凝素(Hemagglutinin,HA)蛋白的哺乳动物细胞系。方法 PCR扩增流感病毒(A/PR/8/34)全长HA基因,并将其克隆入真核表达载体pcDNA5/FRT(pDF)中,构建重组表达质粒pDF-HA,将其与表达Flp重组酶的pOG44质粒共转染Flp-In-CHO细胞,通过体内同源重组使目的基因整合至宿主细胞染色体上。采用Hygromycin B持续压力筛选重组细胞系CHO-HA,通过间接免疫荧光法(IFA)和Western blot法检测HA蛋白的表达。重组细胞连续培养10代后,采用PCR和IFA法检测细胞中HA的基因遗传和蛋白表达的稳定性。结果重组表达质粒pDF-HA经双酶切及测序,证实构建正确;通过Hygromycin B抗性及IFA,共筛选出20株高表达HA蛋白的重组细胞株,Western blot结果进一步证实,HA蛋白在重组细胞中获得表达,并被切割成HA1和HA2蛋白;连续培养10代后,PCR与IFA方法分别检测到重组细胞HA基因和蛋白的表达。结论已成功建立了稳定表达甲型流感病毒HA蛋白的哺乳动物细胞系,为针对HA蛋白和流感病毒的免疫学检测以及HA蛋白的功能研究提供了靶细胞。

Establishment of Mammalian Cell Line for Stable Expression of Hemagglutinin Protein of Influenza Virus Type A

Objective To establish a mammalian cell line for stable expression of hemagglutinin(HA) of influenza virus type A.Methods Full-length HA gene of influenza virus(A/PR/8/34) was amplified by PCR and cloned into eukaryotic expression vector pcDNA5/FRT(pDF).Flp-In-CHO cells were co-transfected with the constructed recombinant plasmid pDF-HA and plasmid pOG44 expressing Flp recombinase,and the target gene was integrated into chromosome of CHO cells by homologous recombination in vivo.Recombinant CHO-HA cell line was screened by continuous pressure screening with hygromycin B,and the expression of HA protein was determined by IFA and Western blot.The recombinant cell line was subcultured for 10 passages and tested for the stability of genetic and expression of HA by PCR and IFA respectively.Results Both restriction analysis and sequencing proved that recombinant plasmid pDF-HA was constructed correctly.Twenty recombinant cell strains highly expressing HA protein were screened with hygromycin B.Western blot proved that HA protein was expressed in the recombinant cells and were cleaved into HA1 and HA2 proteins.The expressions of HA at gene and protein levels were proved by PCR and IFA in the recombinant cells after subculture for 10 passages.Conclusion The mammalian cell line for stable expression of HA of influenza virus type A was successfully established,which provided target cells for immunological determination of HA protein and influenza virus as well as study on function of HA protein.