重组大鼠ΔNΔC/VEGF-C/Cys152Ser逆转录病毒载体的构建及表达  

Construction of recombinant rat ΔNΔC /VEGF-C /Cys152Ser retrovirus vector and its expression in RAW 264. 7 cells

作　　者：杨国红[1,2] 姬文婕[3] 周欣[2] 毕莹[2] 李玉明[2] 

机构地区：[1]天津医科大学研究生院,天津300070 [2]武警后勤学院附属医院心血管病研究所心脏中心,天津300162 [3]武警后勤学院附属医院呼吸与危重症医学科,天津300162

出　　处：《细胞与分子免疫学杂志》2012年第7期677-679,共3页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81070121;81170238);天津市卫生局重点项目(2010KZ122)

摘　　要：目的:构建重组大鼠ΔNΔC/VEGF-C/Cys152Ser(ddVEGF-C)逆转录病毒载体,建立稳定表达ddVEGF-C的PT67包装细胞株,并验证其表达。方法:利用PCR从pSecTag-ddVEGF-C中获取大鼠ddVEGF-C基因,克隆到pLPCX逆转录病毒载体,经PCR、双酶切和测序验证,转染PT67包装细胞,嘌呤霉素筛选出稳定表达目的基因的包装细胞株,用反转录PCR(RT-PCR)和Western blot方法验证其在RAW 264.7细胞中的表达。结果:构建的pLPCX-ddVEGF-C逆转录病毒载体经双酶切和PCR鉴定正确,测序结果与ddVEGF-C序列一致,病毒滴度为2×107CFU/mL,RT-PCR和Western blot结果提示重组病毒感染RAW264.7细胞能正确表达ddVEGF-C。结论:成功构建了pLPCX-ddVEGF-C逆转录病毒载体,并建立了稳定表达ddVEGF-C的PT67包装细胞株,为以VEGF-C基础的实验研究提供了新工具。AIM： To construct a retroviral vector containing rat △N△C/VEGF-C/Cys152Ser （ddVEGF-C） and verify its expression in RAW 264.7 cells. METHODS： The ddVEGF-C gene was amplified by polymerase chain reaction （PCR） from pSecTag-ddVEGF-C and cloned into pLPCX vector. After the procedure of PCR, double enzyme digestion analysis and DNA sequencing, the recombinant plasmid was transfected into packaging cells PT67 using LipofectamineTM 2000, and the positive clones were collect- ed by means of puromycin selection and detected for the viral titer. The expression of ddVEGF-C mRNA and protein in the RAW264.7 cells was determined by RT-PCR and West- em blotting, respectively. RESULTS： PCR and double enzyme digestion analysis demonstrated that the recombinant pLPCX-ddVEGF-C was successfully constructed by displa- ying two positive bands of 382 bp and 6 191 bp as expected. Viral titer was 2 x 107 CFU/mL. RT-PCR and Western blotting showed the expression of ddVEGF-C at the mRNA and protein levels in RAW 264. 7 cells. CONCLUSION： The recombinant vector pLPCX-ddVEGF-C has been successfully constructed as well as PT67 packaging cells expressing stably ddVEGF-C, which provides a potential tool for further VEGF-C related study.

关 键 词：ΔNΔC/VEGF-C/Cys152Ser 逆转录病毒载体PT67 pLPCX RAW264.7 

分 类 号：R373.9[医药卫生—病原生物学]