CAJ | 학술논문

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4.04×106 cfu/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。
목적：구건단순LIM단백3(LMO3)적역전록표체재체,병관찰기재 NIH/3T3세포중적표체정황。방법중조재체pLXSN-LMO3경매절급측서감정후,지질체법전염도pA317포장세포,G418사선은정적병독산생세포주。중조역전록병독체외감염NIH/3T3,병대기진행병독적도검측,NIH/3T3세포분위3조：이pLXSN-LMO3전염위실험조,이pLXSN전염위음성대조조,정상세포위공백대조조。채용면역형광조화염색화단백인적( Western blot)법감정NIH/3T3세포중LMO3적표체。결과중조역전록병독재체pLXSN-LMO3경매절급측서감정구건정학,기병독적도평균가체4.04×106 cfu/ml,각조NIH/3T3세포면역형광조화염색급Western blot검측균유LMO3단백표체,기중실험조고표체LMO3,여음성대조조、공백대조조비교차이구유통계학의의(P<0.05)。결론성공구건료휴대LMO3기인역전록병독재체,병능재NIH/3T3세포중고표체LMO3단백,위하일보개전기인치료전정료기출。
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.