Apo-tPA融合蛋白在鸡胚肝细胞中的分泌表达及其活性检测  

Expression of Apo-tPA Fusion Gene in Chick Embryo Liver Cells in Vitro and A Test for Its Biological Activity

作　　者：冯运巩[1] 李银聚[1] 程相朝[1] 张春杰[1] 吴庭才[1] 郁川[1] 王学强[1] 张旭[1] 

机构地区：[1]河南科技大学动物科技学院,河南洛阳471003

出　　处：《药物生物技术》2009年第6期517-521,共5页Pharmaceutical Biotechnology

基　　金：河南省自然科学基金项目(No.031103100);洛阳市重大科技攻关项目(No.0801048A)

摘　　要：为了探讨apo—B100介导的t—PA融合蛋白在鸡胚肝细胞中分泌表达的可行性和生物学活性，将编码人组织型纤溶酶原激活剂功能区的基因片段△t—PA与鸡VLDLy组装前体apo—B100进行融合，定向克隆到pCDNA3表达载体CMV启动子下游，构建pCDNA3-apo—△tPA表达载体。将pCDNA3-apo-△tPA用脂质体包裹后转染体外培养的鸡胚肝细胞，ELISA检测融合蛋白表达水平，发色底物法检测表达产物的生物学活性。结果显示，融合基因在鸡胚肝细胞中能够表达并分泌到细胞外，48h表达水平最高达575．3ng／10^5（cells·24h），表达产物活性为135．8IU／10^6（cells·24h）。apo—B100介导的t—PA在鸡胚肝细胞中成功表达，为外源基因在鸡体内表达并实现在卵黄中沉积奠定了坚实的基础。To study the apo - tPA fusion gene expression in chicken embryo liver cells in vitro, and to test the content and the biological activity of t- PA, the expression plasmid pCDNA3 - apo- △tPA was constructed and transfected into the chicken embryo liver cells cultured in vitro by Liposome. Apo - tPA fusion protein expression was confirmed by SDS- PAGE, the content tested by ELISA and the biological activity determined by chromogenic substrate assay. It was found that the plasmids pCDNA3 - apo - AtPA could express apo - tPA fusion protein in chicken embryo liver cells cultured in vitro. SOS - PAGE confirmed that the expressed protein was about 82 kDa, and ELISA tested the expression level was 575.3 ng/10^6 （cells · 24 h） in 48 h. Chromogenie substrate assay showed that the activity of apo - tPA fusion protein was 135.8 IU/10^6 （cells ·24 h）. It indicated that apo - tPA could be expressed in vivo by transfer pCDNA3 - apo - △tPA into hen＇s liver, and the fusion protein could transport to hen＇s ovary and accumulate in yolk.

关 键 词：组装前体apo—B100 人组织型纤溶酶原激活剂 融合基因 鸡胚肝细胞 活性检测 

分 类 号：Q78[生物学—分子生物学]