Survivin、TGFβ3和TIMP1基因重组慢病毒多表达载体构建及其表达活性检测  

CONSTRUCTION AND IDENTIFICATION OF RECOMBINANT LENTIVIRAL VECTOR CONTAINING SURVIVIN,TGFβ3 AND TIMP1

作　　者：彭文彬[1] 杨堃[2] 马学晓[3] 岳斌[3] 胡有谷[3] 陈伯华[3] 

机构地区：[1]即墨市人民医院骨科,山东青岛266200 [2]青岛大学医学院附属医院中心实验室 [3]青岛大学医学院附属医院脊柱外科

出　　处：《青岛大学医学院学报》2015年第2期127-130,共4页Acta Academiae Medicinae Qingdao Universitatis

基　　金：国家自然科学基金面上项目(81171758)

摘　　要：目的应用Gateway技术构建生存素(Survivin)、人转化生长因子β3(TGFβ3)和基质金属蛋白酶抑制剂1(TIMP1)慢病毒多表达质粒,并检测其表达活性。方法应用Gateway技术,以自剪切多肽2A串联Survivin、TGFβ3、TIMP1的3段外源基因,并克隆到慢病毒多表达质粒上,通过RT-PCR和Western-blot技术分别检测质粒转染293T细胞后目的基因mRNA和蛋白质表达水平。结果 RT-PCR和Western-blot检测结果显示,慢病毒多表达质粒成功表达了3种目的基因,且其mRNA和蛋白质表达水平较空载体对照组、PBS对照组明显升高,差异有显著性(F=17.87~69.11,q=6.94~15.47,P<0.05)。结论成功构建了慢病毒多表达质粒pLenti6.3-Survivin-P2A-TGFβ3-T2A-TIMP1,为下一步病毒的制备以及体内实验提供了基础。Objective To construct a recombinant lentiviral vector containing Survivin,human transforming growth factor β3 （TGFI]3） and tissue inhibitor of metalloproteinases] （TIMP1） genes using Gateway technology, and identify its expression activity. Methods The Survivin, TGFβ3 and TIMP1 genes were amplified by RT-PCR, and ligated using 2A self-cleaving se- quences and cloned into lentiviral vector using Gateway technology. The recombinant lentiviral vector was then transfected into 293T cells. I^T-PCR and Western-blot analysis were employed to detect the expressions of Survivin, TGFβ3 and TIMP1 in 293T cells. Results RT-PCR and Western-blot analysis showed that the expressions of Survivin, TGFβ3 and TIMP1 genes were sig- nificantly increased in both mRNA and protein level in the transfected 293T cells as compared with non-transfected controls （F = 17.87-69.11,q=6.94-15.47,P〈0.05）. Conclusion The recombinant lentiviral vector carrying Survivin, TGFβ3 and TIMP1 genes is successfully constructed by Gateway technology, which provides a preclinical basis for further virus preparation and in vivo experiments.

关 键 词：生存素 转化生长因子Β3 基质金属蛋白酶抑制剂1 慢病毒属 椎间盘 退变 

分 类 号：R681.53[医药卫生—骨科学]