A亚型人呼吸道合胞病毒G基因的克隆、真核表达

Cloning, eukaryotic expression of G gene of subgroup A human respiratory syncytial virus

目的克隆A亚型人呼吸道合胞病毒(HRSV)G基因,构建G基因的真核表达载体,并进行表达和鉴定。方法自行设计引物,利用RTPCR技术,从感染HRSV的HEp2细胞中获得G基因片段,克隆到pGEMTeasy载体,经核酸序列分析,进一步亚克隆到真核表达载体pcDNA3.1(+),酶切鉴定后,脂质体法转染COS7细胞,RTPCR和Westernblotting鉴定基因转录和蛋白表达。结果真核表达载体pcDNA3.1(+)G的限制性内切酶分析结果与预期一致,基因序列分析显示没有发生无义突变。转染COS7细胞后,RTPCR检测到G基因有转录,Westernblotting分析观察到G蛋白特异性的表达条带。结论成功克隆A亚型HRSVG基因,并在真核细胞中获得表达。

Objective To clone G gene of human respiratory syncytial vires （ HRSV）, construct eukaryotic expressive vector containing G gene, and identify the transcription and expression of G protein by RT-PCR and Western blotting. Methods According to the sequence of G gene, a pair of specific primers was designed and synthesized. G gene was amplified from HRSV infected HEp-2 cells by RT-PCR and cloned into pGEM-T easy vector. After sequence analysis, G gene was subcloned into eukaryotic expressive vector pcDNA3.1 （ ＋ ）. The recombinant plasmid pcDNA3.1 （ ＋ ）G was confirmed by restriction endonuclease assay and transfected into COS-7 cells by Lipofectamin 2000. Finally, the transcription and expression of G protein identified by RT-PCR and Western blotting respectively. Results DNA sequencing displayed no nonsense mutation in G gene. The transcription and expression of G gene were confirmed by RT-PCR and Western blotting. Conclusion The eukaryoic expressive vector containing G gene of HRSV was constructed and expressed successfully in eukaryotic cells.