构建具有靶向治疗作用的工程化生长转化因子-β3蛋白的实验研究  

The in vitro construction of engineered transforming growth factor-beta 3 protein with potential of targeted therapy

作　　者：李进[1] 杨述华[1] 郑东[1] 杨操[1] 冯勇[1] 傅德皓[1] 梅荣成[1] 徐亮[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院骨科,武汉430022

出　　处：《中华创伤骨科杂志》2007年第9期842-846,共5页Chinese Journal of Orthopaedic Trauma

基　　金：国家自然基金资助项目（30471753）

摘　　要：目的构建前体相关蛋白（LAP）为潜伏作用，基质蛋白金属酶（MMP）酶切位点为导向作用，生长转化因子-β3（TGF-β3）活性部分为治疗作用的具有靶向治疗作用的工程化TGF-β3蛋白，并检验其特异性的靶向治疗作用。方法将人工合成的编码MMP酶切位点氨基酸的正、反义DNA序列经基因重组定向克隆插入真核表达载体pIRES-EGFP中，获得pIRES—EGFP—MMP重组体，用PCR从大鼠胚胎组织中TGF-β3的LAP段和TGF-β3的活性部分mTGF-β3，分别插入pIRES-EGFP-MMP中MMP的上游和下游，获得pIRES-EGFP-LAP-MMP-mTGF-β3重组体。然后将其转染入CHO细胞，最后用Westen Blot检测不同环境下细胞上清液中TGF-β3蛋白的表达。结果经过测序和酶切鉴定，确认成功构建了pIRES—EGFP—LAP—MMP-mTGF-β3质粒，转染CHO细胞后也成功表达了工程化TGF-β3蛋白，而且这种蛋白只在有MMP存在的条件下才能特异性地释放出有活性的mTGF-β3。结论通过基因工程技术，可成功构建工程化TGF-β3蛋白真核表达载体，其蛋白产物具有特异性的靶向治疗功能。Objective To investigate the targeted therapy function of engineered transforming growth factor-beta 3 （TGF-β3） gene transferred to CHO cells in vitro after construction of the engineered TGF-β3 protein in which latency associated peptide （LAP）, matrix metalloproteinase （MMP） enzyme and TGF-β3 play the roles of latency, targeting and therapy respectively. Methods The recombinant pIRES-EGFP-MMP was constructed by combination of DNA encoding MMP enzyme cutting sites and eukaryotic expression vectors plRES-EGFP. LAP and TGF-β3 fragments were obtained from rat embryos by RT-PCR and inserted into the upstream and downstream of MMP from pIRES-EGFP-MMP, respectively, so as to construct the recombinant plasmid of plRES-EGFP-LAP-MMP-mTGF-β3, which was then transferred to CHO cells. The superuatant was harvested after 36 hours of transfection and divided into 2 parts, one for detection of the protein expression by Western Blot directly and the other after addition of MMP enzyme or acidization. Results Enzyme cutting and sequencing analyses revealed that pIRES-EGFP-LAP-MMP-mTGF-β3 was successfully constructed. The expression of engineered TGF-β3 protein was detected after transfection to CHO cells. Meanwhile, only the presence of MMP enzyme could lead to release of mature TGF-β3 from the recombinant plasmid. Conclusion We have successfully obtained the engineered TGF-β3 protein with targeted therapy function by the technique of gene engineering.

关 键 词：工程化蛋白 靶向治疗 生长转化因子-β3 真核细胞 软骨分化 

分 类 号：R686[医药卫生—骨科学]