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目的筛选出新型呼肠病毒S1基因和宿主相互作用的关键区段,为后续的宿主受体蛋白筛选工作提供可靠的基础。方法将编码全长S1基因,以及N端和C端阅读框分别克隆入真核表达载体pIRSE2-EGFP,转染敏感细胞株鼠成纤维细胞(L929)和人宫颈癌细胞(Hela)后,通过荧光报告基因EGFP的表达分析,筛选出S1基因与宿主细胞相互作用时起决定性影响的区段。结果同等量的3种真核表达质粒在单独转染L929时,pGSN质粒转染后荧光表达量最大,pGSC质粒转染后荧光表达量最小。不同比例混合的pGS与pGSN进行共转染时,pGSN在转染质粒中的比例越高,荧光表达量也越高。在Hela细胞中的转染结果与L929相同。结论新型呼肠病毒R4株的S1基因N端阅读框编码区(62—406 bp)在S1基因转染时起关键作用。
Objective To screen out the key segments of the interaction between novel reovirus S1 gene and host, and provide a reliable basis for subsequent screening of host receptor proteins. Methods The full-length S1 gene and the N-terminal and C-terminal reading frame were cloned into the eukaryotic expression vector pIRSE2-EGFP and transfected into the sensitive fibroblasts (L929) and human cervical cancer cells (Hela) Fluorescent reporter gene EGFP expression analysis, screening S1 gene and host cells play a decisive role in the section. Results The same amount of three eukaryotic expression plasmids showed the highest expression of pGSN plasmid after transfected with L929, and the lowest expression of pGSC plasmid. When co-transfection of pGS and pGSN with different proportions, the higher the proportion of pGSN in the transfection plasmid, the higher the fluorescence expression. Transfection results in Hela cells are identical to L929. Conclusion The N-terminal reading frame (62-406 bp) of the S1 gene of the novel reovirus R4 strain plays a key role in S1 gene transfection.