携带LMO3基因逆转录病毒载体的构建及其在NIH/3T3细胞中的表达  

Construction of Recombinant Retroviral Vector Carrying LMO3 Gene and Its Expression in NIH/3T3 Cells

作　　者：罗芸[1] 张新宇[1] 万东君[1] 刘晓花[1] 付学锋[1] 

机构地区：[1]兰州军区兰州总医院干部病房,兰州730050

出　　处：《解放军医药杂志》2014年第5期22-26,共5页Medical & Pharmaceutical Journal of Chinese People’s Liberation Army

基　　金：甘肃省自然科学基金资助项目(1107RJZA106)

摘　　要：目的构建单纯LIM蛋白3(LMO3)的逆转录表达载体,并观察其在NIH/3T3细胞中的表达情况。方法重组载体pLXSN-LMO3经酶切及测序鉴定后,脂质体法转染到pA317包装细胞,G418筛选稳定的病毒产生细胞株。重组逆转录病毒体外感染NIH/3T3,并对其进行病毒滴度检测,NIH/3T3细胞分为3组:以pLXSN-LMO3转染为实验组,以pLXSN转染为阴性对照组,正常细胞为空白对照组。采用免疫荧光组化染色和蛋白印迹(Western blot)法鉴定NIH/3T3细胞中LMO3的表达。结果重组逆转录病毒载体pLXSN-LMO3经酶切及测序鉴定构建正确,其病毒滴度平均可达4.04×106cfu/ml,各组NIH/3T3细胞免疫荧光组化染色及Western blot检测均有LMO3蛋白表达,其中实验组高表达LMO3,与阴性对照组、空白对照组比较差异具有统计学意义(P<0.05)。结论成功构建了携带LMO3基因逆转录病毒载体,并能在NIH/3T3细胞中高表达LMO3蛋白,为下一步开展基因治疗奠定了基础。Objective To construct recombinant retroviral vector carrying LIM domain only 3 （LMO3） gene and study its expression in NIH/3T3 cells. Methods The retroviral vector pLXSN-LMO3 was identified by restriction en-zyme analysis and DNA sequencing analysis, and then was transfected into retrovirus packaging cell line pA317, and G418 was used to select stable virus-producing cell lines. The recombinant retrovirus was used to infect NIH/3T3 cells in vitro, and the value of viral titer was determined. The NIH/3T3 cells were divided into three groups：experimental group （infected with the pLXSN-LMO3）, negative control group （infected with the pLXSN） and control group. The expression of LMO3 in NIH/3T3 cells was identified by the immunofluorescence histochemistry and Western blot methods. Results The pLXSN-LMO3 recombinant retroviral vector had been constructed correctly by restriction enzyme analysis and se-quencing analysis, and the value of titer assayed on NIH3T3 cells was up to an average of 4. 04 × 106 cfu/ml. LMO3 al-bumen expression was detected in NIH/3T3 cells using immunofluorescence histochemistry and Western blot methods, and LMO3 was highly expressed in experimental group, and the differences in LMO3 expression were statistically signifi-cant when compared with those in negative control group and control group （P〈0. 05）. Conclusion The recombinant retroviral vector carrying LMO3 gene has been constructed successfully, which can highly express LMO3 in NIH/3T3 cells and has potential utility in further gene therapy.

关 键 词：单纯LIM蛋白3 NIH 3T3细胞 逆转录病毒科感染 LIM domain only 3 

分 类 号：R512.99[医药卫生—内科学]