人endostatin、K5及endostatin-K5重组腺病毒载体的构建及其表达  

Construction and expression of recombinant adenovirus vector carrying human endostatin,K5 and endostatin-K5 gene

作　　者：李慧瑾[1] 张伟锋[1] 王东阳[1] 李荣华[1] 齐宗利[1] 赵俊丽[1] 夏海滨[1] 

机构地区：[1]陕西师范大学生命科学学院基因治疗研究室,陕西西安710062

出　　处：《细胞与分子免疫学杂志》2009年第7期577-580,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金资助项目(30872993);陕西省自然科学基金资助项目(2007C201);陕西师范大学研究生培养创新基金(2008CXS038)

摘　　要：目的:人endostatin、K5及endostatin-K5基因克隆,重组腺病毒载体的构建及其表达产物体外活性检测。方法:采用PCR方法扩增基因,通过酶切连接方法将3种基因片段克隆到腺病毒shuttle载体pAd5-CMV-H1H2-MCS-6His中;用磷酸钙沉淀法分别将经PacI酶切的表达人endostatin、K5及endostatin-K5的腺病毒shuttle载体与PacI酶切的腺病毒骨架共转染HEK293细胞;用CsCl密度梯度离心法纯化制备病毒;用重组腺病毒感染HeLa细胞并在不同时间点收获蛋白,Western blot检测24h、48h和72h的蛋白表达量;通过MTT方法检测蛋白对ECV-304生长抑制作用。结果:成功制备表达人endostatin、K5及endostatin-K5重组腺病毒载体;Westernblot可以检测到蛋白表达,蛋白表达量随病毒感染HeLa细胞的时间延长而逐渐增高;由重组腺病毒所表达的3种蛋白对ECV304细胞均有明显的生长抑制作用。结论:由含有人en-dostatin、K5及endostatin-K5基因的重组腺病毒载体所表达的蛋白对ECV-304生长具有明显的抑制作用。AIM： To construct the recombinant adenovi- ral vectors expressing human endostatin, K5 and endostatin-KS gene respectively, and study their bioactivity in vitro. METHODS： Human endostatin, K5 and endostatin-K5 gene were amplified by PCR, which were then subcloned into shuttle vector pAdS-CMV-H1H2-MCS-6His by enzyme and ligation respectively. The positive recombinant plasmids linearized by Pac I were cotransfected into HEK 293 cells with the Pac I linearized adenoviral backbone plasmid using calcium phosphate precipitation method. The recombinant viruses were purified by CsCI density gradient centrifugation. The protein expression at different time points （24 h, 48 h and 72 h） was determined by Western blot. The inhibitory effect of the protein on ECV-304 growth was detected by MTT. RESULTS： The recombinant adenoviral vectors expressing human endostatin, K5 and endostatin-K5 gene were successfully constructed in HEK293 cells. Protein expression was detected by Western blot. The level of protein expression was increased with the prolonged incubation of the infected HeLa cells. Three kinds of the protein expressed by the recombinant adenoviral vectors showed obvi- ous inhibitory effect on ECV-304 cell growth. CONCLUSION： The protein expressed by adenoviral vectors carrying endostatin, K5 and endostatin-K5 gene has an obvious inhibitory effect on ECV-304 cell growth.

关 键 词：ENDOSTATIN K5 腺病毒载体 MTT检测 肿瘤 

分 类 号：Q291[生物学—细胞生物学]