呼吸道合胞病毒M2-1蛋白Hep-2细胞稳定表达株的构建及其在呼吸道合胞病毒感染中的特点

Construction of Stable Expression Respiratory Syncytial Virus M2-1 Protein Hep-2 Cell and Characteristics of the Cell in RSV Infection

目的:构建稳定表达呼吸道合胞病毒(RSV)M2-1蛋白的Hep-2细胞,并分析稳定表达细胞株RSV感染的特点。方法:构建M2-1真核表达质粒,转染至Hep-2细胞。通过G418筛选阳性单克隆;RT-PCR、qRT-PCR以及Western blot鉴定,最终得到稳定表达株。通过MTT法分析细胞增殖特征;通过分析不同浓度RSV病毒感染Hep-2细胞与所构建的稳定表达株细胞后在状态、细胞病变(CPE)出现时间以及RSV基因组核酸产量分析比较RSV在两种细胞中的感染特点。结果:获得一株稳定表达M2-1蛋白的细胞Hep-2 2F5。该株细胞的生长速度高于原始细胞株。采用1000~10?2 TCID50 (E1~E6)的病毒滴度对Hep-2 2F5与Hep-2两种细胞进行感染发现,1000 TCID50 (E1)的滴度下,两种细胞均产生病变,但Hep-2 2F5细胞产生的病变更明显;100~10 TCID50 (E2~E3)滴度下RSV感染两种细胞,Hep-2 2F5细胞产生的病变更早且更明显;1~0.1 TCID50 (E4~E5)滴度下RSV感染两种细胞,Hep-2 2F5细胞产生少量病变,Hep-2则无明显病变。E1~E4滴度下攻毒12 h后,RSV在Hep-2 2F5细胞中的核酸定量平均值高于Hep-2细胞,E5~E6滴度下攻毒12 h两细胞均未检测到RSV病毒核酸;随着时间延长,E1~E2滴度感染后的Hep-2中RSV含量高于Hep-2 2F5,在E4滴度及以下,RSV在Hep-2 2F5中的增殖优于Hep-2。结论:成功构建稳定表达M2-1蛋白的Hep-2 2F5细胞株。该细胞株在RSV感染中更具敏感性,低滴度病毒感染的情况下,更早产生CPE效应,且病毒核酸的含量也较Hep-2高。

Objective:To construct stable expressing respiratory syncytial virus M2-1 protein Hep-2 cells and analyze the characteristics of the cell in RSV infection.Methods:M2-1 eukaryotic expression plasmid was constructed and transfected into Hep-2 cells.The stable expression cell was screened by G418,identified by RT-PCR,qRT-PCR and Western blot.Cell proliferation was analyzed using MTT assay.The characteristics of Hep-2 and the constructed cell in RSV infection were analyzed by observing the state of the cells,the emergence of cytopathic effect(CPE),and the yield of RSV genomic RNA.Results:Hep-2 2F5 with stable expression respiratory syncytial virus M2-1 protein was obtained in this study.The growth rate of the cell is higher than that of the original Hep-2 cell.Hep-2 2F5 and Hep-2 were both infected with RSV with titer of 1000-10?2 TCID50(E1-E6).Under the titer of 1000 TCID50(E1),both cells developed CPE,but more obvious in Hep-2 2F5 cell;under the titer of 100-10 TCID50(E2-E3),the CPE developed earlier and more obvious in Hep-2 2F5 than Hep-2;Under the titer of 1-0.1 TCID50(E4-E5),Hep-2 2F5 cells developed a small amount of CPE,however,no CPE found in Hep-2.After 12 h infection at E1-E4 titer,the mean value of RSV genome in Hep-2 2F5 cells was higher than that in Hep-2 cells,and no RSV virus nucleic acid was detected in both cells 12 h post infection with RSV at E5-E6 titer.With prolongation of infection time,RSV genome content in Hep-2 was higher than that in Hep-2 2F5 under the titer of E1-E2.Under the titer of E4 or higher,the proliferation of RSV in Hep-2 2F5 was better than in Hep-2.Conclusion:Hep-2 2F5 cell with stable expression respiratory syncytial virus M2-1 protein was successfully constructed.The cell is more sensitive in RSV infection,and in the case of low titer virus infection,the CPE developed earlier,and the viral nucleic acid content was higher comparing to Hep-2.