人呼吸道合胞病毒F基因的克隆、真核表达与鉴定

Clone,construction,and expression of eukaryotic expression vector containing F gene of human respiratory syncytial virus subgroup A

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。

Objective To clone F gene of human respiratory syncyfial virus （HBSV） subgroup A, construct eukartotic expression vector containing F gene, and identify the expression of F protein using Western blotting assay. Methods Acconting 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 eukaryofie expression vector pcDNA3.1 （ ＋ ）. The resulting recombinant plasmid pcDNA3.1 （ ＋ ）/F was confmned by restriction endonuclease assay, and then transfeeted into COS-7 cells by using Lipfectamin 2000. The expression of F protein was identified with Western blotting assay. Results DNA sequeneing displayed no nonsense mutation in F gene. The specific expression of F protein in COS-7 cells was confmned by Western blotting assay. Conclusion The eukaryotic expression vector containing F gene of HBSV is successfully constructed and expressed in eukaryotie cells.