hVEGF_(121)与βhCG融合蛋白的克隆、分离纯化与初步鉴定  

Cloning,Purification and Preliminary Identification of the Fusion Protein Including hVEGF_(121) and βhCG

作　　者：曹荣月[1] 宦晓军 孙翁 李盼[1] 郑梅[1] 赵鹤[1] 杨梦琪[1] 龙军[2] 张晓红[3] 

机构地区：[1]中国药科大学生命科学与技术学院微基因药物实验室,江苏南京210009 [2]南京中医药大学,江苏南京210023 [3]南京医科大学南京市妇幼保健院妇科,生殖医学国家重点实验室,江苏南京210004

出　　处：《药物生物技术》2014年第1期1-6,共6页Pharmaceutical Biotechnology

基　　金：中国国家自然科学基金委员会资助项目(No.31270985);国家基础科学人才培养基金资助项目(No.J1030830);国家自然科学基金资助项目(No.81373232);江苏高校优势学科建设工程资助项目

摘　　要：构建一种新型抗肿瘤融合蛋白疫苗,包含hVEGF121和βhCG抗原表位,并探索融合蛋白的纯化条件和方法。采用PCR方法,克隆VEGF和βhCG基因,并通过限制性内切酶NcoⅠ、EcoRⅠ和HindⅢ将两个基因逐个插入pET-28a(+)质粒中,利用质粒自带的卡那霉素抗性筛选出阳性克隆,获得重组基因的表达载体。对重组菌进行乳糖诱导表达后,通过超声破碎,硫酸铵分级沉淀,阴离子交换层析等方法进行融合蛋白的分离纯化,Western-blot鉴定。测序结果表明成功构建重组载体pET-28a-VEGF-M2-X10-βhCG-CTP37,重组基因长993 bp,编码331个氨基酸残基。SDS-PAGE显示,经乳糖诱导,Escherichia coli BL21(DE3)重组菌表达hVEGF121-M2-X10-βhCG-CTP37多肽,约占菌体总蛋白量的35%。融合蛋白经分离纯化后纯度可达到电泳纯,约占蛋白冻干粉的80%。结论:克隆、测定了融合蛋白基因,并在Escherichia coli BL21(DE3)中诱导表达hVEGF121-M2-X10-βhCG多肽,同时成功分离纯化此多肽。To construct a new type of antitumor fusion protein vaccine containing two antigen epitopes（hVEGF121andβhCG）,meanwhile to explore purified process of the fusion protein,VEGF and βhCG gene cloned by polymerase chain reaction（PCR） were inserted into a pET-28a expression vector through the restriction enzymes Nco Ⅰ,EcoR Ⅰ and Hind Ⅲ and then the recombinant vector was acquired after screening positive clones.After being expressed in recombinant strains induced by lactose,the fusion protein was purified through ultrasonic broken,ammonium sulfate fractional precipitation and ion exchange chromatography,and identified by Western blot.We successfully constructed the recombinant vector,pET-28a-hVEGF121-M2-X10-βhCG-CTP37.The longth of the recombination gene was 993 bp,encoding 331 amino acid residues.SDS-PAGE showed that the fusion protein expressed in recombinant strains achieved up to 35% of the total protein content.The fusion protein was purified at electrophoresis level.The recombination gene was cloned,identified successfully and the fusion protein,hVEGF121-M2-X10-βhCG-CTP37,was expressed in Escherichia coli BL21（DE3）.The purified fusion protein was achieved after utilizing several methods for purification.

关 键 词：hVEGF121 ΒHCG 克隆 表达载体 分离纯化 融合蛋白 

分 类 号：Q51[生物学—生物化学] Q785