【摘要】 目的克隆人呼吸道合胞病毒(human respiratory syncytial virus,HRSV)F基因,构建F基因的真核表达载体pcDNA3.1(+)/F,并进行表达和鉴定。方法根据编码F蛋白的基因序列设计引物,通过RT-PCR从感染HRSV的HEp-2细胞中扩增获得F蛋白的基因,然后克隆到pGEM-3zf载体中,序列测定正确后,将其亚克隆到真核表达载体pcDNA3.1(+),行酶切鉴定,脂质体法转染COS-7细胞,应用Western blotting方法分析F基因表达情况。结果测序证实得到HRSV F基因序列,序列分析显示没有发生无义突变。转染COS-7细胞后,利用Western blotting方法检测到了F蛋白的特异性条带。结论成功克隆HRSV F基因,并在真核细胞中获得表达。更多还原

【Abstract】 Objective To clone F gene of human respiratory syncytial virus(HRSV) subgroup A,construct eukaryotic expression vector containing F gene,and identify the expression of F protein using Western blotting assay.Methods According to the sequence of F gene,a pair of primers were designed and synthesized.F gene was amplified from HRSV-infected HEp-2 cells by using RT-PCR,and then cloned into pGEM-3zf vector.After sequence analysis,F gene was subcloned into eukaryotic expression vector pcDNA3.1(+).The resulting recombinant plasmid pcDNA3.1(+)/F was confirmed by restriction endonuclease assay,and then transfected into COS-7 cells by using Lipfectamin 2000.The expression of F protein was identified with Western blotting assay.Results DNA sequencing displayed no nonsense mutation in F gene.The specific expression of F protein in COS-7 cells was confirmed by Western blotting assay.Conclusion The eukaryotic expression vector containing F gene of HRSV is successfully constructed and expressed in eukaryotic cells.更多还原