Construction Of Stable Transfected Cell Line Of IL-6 Overexpression Lentiviral And Its Effect On The Expression Of HBsAg,HBeAg,HBV-DNA

Objective:To construct stable transfect cell line of IL-6 overexpression lentiviral and explore its effect on the expression of HBsAg,HBeAg and HBV-DNA.Methods: IL-6 fragment was amplified by PCR using Huh7 cell gDNA as template and cloned into pMIGR1 vector.After sequencing analysis sucessfully,pMIGR1 plasmid and pMIGR1-IL-6 plasmid was used respectively with lentiviral packaging plasmid pCL-10A1 to co-transfected 293 T cells to obtain recombinant lentiviral particles,and then transfected Huh7 cell.The cells named Huh7/con cell line and Huh7/IL-6 cell line respectively were screened by puromycin,expanded the cells obtained.IL-6 expression was confirmed by Electrochemiluminescence and qRT-PCR;HBsAg,HBeAg expression was detected by Chemiluminescence microparticle immunoassay;HBV-DNA expression was detected by PCR-fluorescence probe.Results:The MIGR1-IL-6 plasmid was confirmed successfully by sequencing analysis,which indicated that IL-6 lentiviral expression vetor was successfully constructed.IL-6 expression in the Huh7/IL-6 cell lines increased significantly compared with Huh7/con cell lines.The expression of IL-6 in Huh7,HepG2 and Huh7/IL-6 cells transfected with pcDNA1.3 plasmid after 6 h,12 h,24 h and 48 h of transfection were significantly increased;The expression of HBsAg,HBeAg and HBV-DNA in Huh7 and HepG2 cells treatment with pcDNA1.3 plasmid and dealed with 20 ng/ml IL-6 after 6 h,12 h,24 h and 48 h of transfection were significantly decreased;The expression of HBsAg,HBeAg and HBV-DNA in Huh7/IL-6 cells transfected with pcDNA1.3 plasmid after 6h,12 h,24h and 48 h of transfection were significantly decreased.Conclusion:The IL-6 lentiviral expression vector is successfully constructed,and the Huh7 cell line stably expressing IL-6 is established;Transfection of pcDNA1.3 plasmid up-regulates IL-6 expression;IL-6 decreases the expression of HBsAg,HBeAg and HBV-DNA.