内皮抑素基因定点突变体的构建  

Construction of a site-directed mutant of endostatin

作　　者：殷秀丽[1] 张春梅[1] 张晶旭 付安安[1] 尚巍[1] 刘平[1] 葛红岩[1] 

机构地区：[1]哈尔滨医科大学附属第一临床医学院眼科医院,中国黑龙江省哈尔滨市150001 [2]中国黑龙江省大庆市龙南医院眼科,163001

出　　处：《国际眼科杂志》2009年第2期277-279,共3页International Eye Science

摘　　要：目的:内皮抑素是一种内源性的血管生成抑制剂。运用定点突变技术对人内皮抑素基因进行改造,将内皮抑素中的RGAD改为RGD序列。构建野生型和突变型RGD-en-dostatin基因的真核表达载体,为进一步探讨其对角膜新生血管的影响奠定基础。方法:以pMD18-T/endostatin载体为模板扩增野生型内皮抑素基因全长编码序列,克隆至真核表达载体pIRES2-EGFP上,采用快速体外定点诱变技术获得RGD突变体。经PCR,酶切和序列测定方法鉴定重组质粒。结果:PCR成功扩增了野生型和RGD突变型的内皮抑素基因,并成功构建了其真核表达载体。结论:改造的含有RGD结构的内皮抑素基因真核表达质粒的成功构建并鉴定。AIM： To construct the eukaryotic express vector of wild type and RGD mutated endostatin gene, and to determine the effects of overexpression of RGD mutated endostatin in corneal neovasculization. METHODS： The full coding domain sequence of endostatin gene was cloned from pMD18-T/endostatin vector and inserted into plRES2-EGFP vector. Mutagenesis of RGD-endostatin was performed in rapid site-directed mutagenesis technique in the expression vector plRES2- EGFP. Then, the reconstructed plasmid was identified with PCR, enzyme digestion and sequencing. RESULTS： Wild type and mutated endostatin gene were successfully amplified by PCR and its eukaryotic expression plasmid was constructed. CONCLUSION; The successive reconstruction and verifying of eukaryotic expressive plasmid containing human wild type and mutated endostatin gene establish the foundation of studying of the mechanisms of corneal neovasculization.

关 键 词：内皮抑素 定点诱变 角膜新生血管 

分 类 号：R772[医药卫生—眼科] R346[医药卫生—临床医学]