抗乙肝病毒shRNA表达载体转染条件的优化  

The Optimization of Transfecting HBV Specific shRNA Expression Vector in Vitro

作　　者：张秉强[1] 吴莹[1] 黄英[1] 张君[1] 张建军[1] 唐霓[1] 黄爱龙[1] 

机构地区：[1]感染性疾病分子生物学教育部重点实验室重庆医科大学病毒性肝炎研究所,重庆400016

出　　处：《生物技术通报》2006年第6期85-89,共5页Biotechnology Bulletin

基　　金：国家杰出青年基金(30225026);国家青年科学基金(30300298)资助

摘　　要：目的优化抗乙肝病毒(HBV)短发夹样RN(A short hairpin RNA,shRNA)表达载体转染条件,从而克服过量转染所致细胞大批死亡的弊端,以便更客观地反映RNA干扰抗乙肝病毒的效率、方法,通过脂质体介导,将HBV真核表达载体pHBV1.3与我们以前研究确定为能有效抗乙肝病毒的shRNA表达载体pTZU6-C共同转染肝癌细胞HepG2,将共转染的两种质粒及所需的脂质体设立成不同浓度和比例,以pHBV1.3与不表达shRNA的空载体pTZU6的共转染作为未干扰阴性对照,转染36h后收集细胞,通过Southern杂交来观察乙肝病毒DNA明显复制和显著受抑时,所需pHBV1.3与pTZU6-C的最适剂量和比例,以及相应脂质体的用量;同时,通过Western杂交来观察HBV蛋白表达的变化。结果通过southern杂交,发现当将pHBV1.(3μg):pTZU6-C(μg)固定为6:1,脂质体(μl):质粒(μg)为1:1时,对于100ml培养瓶中90%以上融合的细胞,用3.5μl的脂质体共转染0.5μg pHBV1.3和3μg pTZU6-C,即可检测出HBV DNA的表达,此时,存活细胞总数不受任何影响;随着pHBV1.3量的增多,HBV DNA的表达亦增多;当pHBV1.3增至4μg时,虽然表达量增多,但存活的细胞总数却开始明显减少,存活细胞约为70%～80%;当用8μg时,存活的细胞不足40%～50%,检测水平反而下降。当pHBV1.(3μg):pTZU6-C(μg)为1:1时,即可观察到乙肝病毒DNA表达的明显抑制作用,其抑制率为71%;当两者的比例为1:2时,其抑制率为70%;比例为1:6时,抑制率为84%。Western杂交的结果亦证实,共转染0.5μgpHBV1.3和3μg pTZU6-C,即可检测出HBV蛋白的表达;但转染36h后,尚未能观察到HBV表面抗原(HBsAg)表达明显受抑的情况。结论抗乙肝病毒shRNA表达载体的转染条件需要优化,对于100ml培养瓶中90%以上融合的细胞,用6μl的脂质体共转染2μg pHBV1.3和4μg pTZU6-C,即可观察到明显的HBV表达和显著的RNA干扰抑制效果,本研究结果为下一步针对乙肝病毒不同靶区shRNA表达载体的转染奠定了�Objective To optimize the transfection of HBV specific short hairpin RNA （shRNA）expression vector into culture cells,so that the investigation of the RNA interference on inhibiting HBV expression can be performed objectively. Methods Co-transfecting HBV expression vector （pHBV1.3）and shRNA expression vector（pTZU6-C）into HepG2 cell by lipofectamine,and investigating the does of vectors and lipofectamine by which it can be got a high expression and deep inhibition of HBV DNA in Southern blotting. Results we found that HBV DNA-replicative intermediates could be detected in Southern blotting 36 hours post transfection when using 6μl of lipofectamine to co-transfect 0.5μg of pHBV1.3 and 3μg of pTZU6-C to 100ml of culture vessels,and the expression of HBV DNA will be increased with the amount of pHBV1.3 enhanced when their ratio fixed to 1：6,however,the alive cells was decreased to 70 to 80 percent when 41μg of pHBV1.3 was used,and would be only 40 to 50 percent left when 81μg of pHBV1.3 was performed,meanwhile,the expression of HBV DNA was hardly cut down. The effect of RNA interference can be found when pHBV1.3：pTZU6-C ratio was only 1 to l：l,the inhibition rates of HBV DNA are 71,70,and 84 percent when pHBV1.3：pTZU6-C ratio was 1：1,1：2 and 1：6,respectively. West blotting show that the protein expression of HBV can be detected when co-transfecting 0.5μg of pHBV1.3 and 3μg of pTZU6-C,however, the inhibition rate of HBV protein expression was not so high as that of HBV DNA. Conclusion When using 6 μl of lipofectamine to co-transfect 2μg of pHBVI.3 and 6μg of pTZU6-C to 100ml of culture vessels,there can be found a high expression of HBV and a deep inhibition of RNAi.

关 键 词：细胞转染 RNA干扰 乙肝病毒(HBV) 

分 类 号：Q78[生物学—分子生物学]