ALR基因RNA干扰慢病毒表达载体的构建及其在肝癌细胞中对ALR表达的抑制

Construction of recombinant lentiviral vector of siRNA for augmenter of liver regeneration gene and its expression in HepG2 cells

目的:构建肝再生增强因子(augmenter of liver regeneration,ALR)基因RNA干扰(RNA interference,RNAi)慢病毒载体,并检测其对人肝癌细胞株HepG2和QGY中ALR基因表达的干扰效率。方法:针对ALR基因RNAi的有效靶序列,合成靶序列的OligoDNA,退火形成双链DNA,与经HpaⅠ和XhoⅠ双酶切线性化的GV118慢病毒载体连接产生LV-ALR-shRNA重组慢病毒载体,经转化感受态细胞后筛选出阳性克隆,并进行测序鉴定。用293T细胞包装产生慢病毒,逐孔稀释法测定病毒滴度。再将包装浓缩后的慢病毒转染人肝癌细胞株HepG2和QGY,应用Real-time PCR和Western blot方法检测HepG2和QGY细胞中ALR基因mRNA和蛋白的表达情况。结果:酶切鉴定证实成功构建了针对人ALR基因的RNAi慢病毒载体,包装并浓缩慢病毒,病毒滴度为8E+8TU/ml。将病毒感染人肝癌细胞株HepG2和QGY后,2种细胞的干扰组ALR的mRNA表达水平较阴性对照组及空白对照组均明显下降(HepG2细胞,P干扰组vs.阴性病毒对照组=0.004,P干扰组vs.空白对照组=0.001;QGY细胞,P干扰组vs.阴性病毒对照组=0.005,P干扰组vs.空白对照组=0.001),同样,干扰组ALR在蛋白水平的表达也明显减降低。结论:成功构建ALR基因RNAi慢病毒载体,该载体能有效干扰人肝癌细胞株HepG2和QGY中ALR基因的表达。

Objective ： To construct a lentiviral vector for RNA interference （RNAi） for augermenter of liver regeneration （ALR） gene and to detect its interference efficiency in human hepatoma cell line HepG2 and QGY. Methods：Effective sequence of siRNA target- ing ALR gene was confirmed. The complementary DNA containing both sense and antisense Oligo DNA of the targeting sequence was designed, synthesized and cloned into the Hpa I and Xho I resriction sites of GV118 lentiviral vector. The resulting lentivirus vector containing ALR shRNA was named LV-ALR-shRNA. The competent E.coli cells were transformed and positive clone were screened,identified and sequenced. The recombinant lentivirus was packaged into mature lentivirus by 293T cells and the virus titer was measured by hole dilution method. The packaged lentivirus was used to infect HepG2 and QGY cells. The expression of ALR in the cells was detected by real-time PCR and Western blot. Results：PCR and sequencing verified that the recombinant lentiviral RNAi vector of ALR was constructed successfully. The titer of concentrated virus was 8E＋STU/ml.After being transfected by virus,ALR mRNA and protein levels were obviously reduced in both human hepatoma cell line HepG2 and QGY compared with those of nega- tive control and bland control cells （P〈0.05）. Conclusion：The lentiviral RNAi expression vector targeting ALR gene is successfully constructed and it could effectively silence the expression of ALR in human hepatoma cell line HepG2 and QGY.