逆转录病毒滴度测定新方法的实验研究

Experimental Study on a New Method for Retrovirus Titer Determination

[目的]研究一种简便、快速的能获取较高病毒滴度的体外培养方法。[方法]用质脂体转移法将逆转录病毒载体pLXIn转染到包装细胞PA317中,用G418筛选出抗性克隆,扩大培养,再收集上清,并测定病毒滴度。比较不同方法产生病毒的多少,为提高病毒滴度而进一步感染靶细胞奠定良好的实验基础。[结果]改良后的新方法(低温32℃、无CO2条件以及反复冻融法)较常规方法(37℃、5%CO2)可提高病毒滴度至少10倍。PTs,PTas和PLXIn组病毒颗粒数分别提高到1.3×105CFU/mL、1.2×105CFU/mL、1.5×105CFU/mL。[结论]逆转录病毒滴度测定新方法简便、快捷,能获取较高病毒滴度。

[Objective] To investigate a convenient and effective in vitro cell culture method to acquire high retrovirus titer.[Methods] The retrovirus vector PLXIN were transfected to PA317 packaging cells by liposomal transfection reagent,and were selected by G418 and expanded culturing.Then the supernanant was collected and the Virus titers were detected.To compare the retrovirus quantity generated by different methods can establish better experimental basis for improving the titer and further infecting target cell.[Results] Compared with the normal metuods（37℃,5% CO2）,the new improved method（low temperature32℃,CO2 non-existence and by using frozen-melting method）can enhanced the retrovirus titer at lease 10 times.PTs,PTas and PLXIn group retrovirus vector are enhanced to 1.3×105CFU/mL,1.2×105CFU/mL and 1.5×105 CFU/mL respectively.[Conclusion] New method for retrovirus titer determination is convenient and effective,by which higher virus titers can be acquired.