人肿瘤抗原基因Delta-like4(DLL4)可溶性表达、纯化及鉴定  

Soluble Expression,Purification and Identification of Human Tumor Antigen Delta-l ike4 (DLL4) Recombinant Protein

作　　者：刘建军[1] 胡月新 焦扬[1] 赵瑜 杨静[1] 何保丽[4] 

机构地区：[1]昆明医科大学分子临床医学研究院,云南昆明650500 [2]昆明医科大学科研实验中心,云南昆明650500 [3]昆明医科大学生物医学工程研究中心,云南昆明650500 [4]昆明医科大学动物学部,云南昆明650500

出　　处：《昆明医科大学学报》2013年第6期26-29,共4页Journal of Kunming Medical University

基　　金：云南省高校药学重点实验室项目(2011YXZD01)

摘　　要：目的研究克隆肿瘤抗原人DLL4基因,与原核表达载体可溶性融合表达、分离纯化及其鉴定,并计划将其应用于DLL4特异性肺癌肿瘤疫苗研究过程中的抗血清滴度检测.方法采用PCR方法扩增DLL4多核苷酸序列,克隆入pMD-18T载体.测序正确后,将其亚克隆入pGEX-KG原核表达载体,获得pGEX-KG-hDLL4载体.以该载体转化E.coli菌株BL21(DE3),IPTG诱导其表达,裂解大肠杆菌,以GSTrap亲和层析柱纯化目的蛋白,采用SDS-PAGE电泳,Western Blot方法鉴定目的蛋白的表达.结果 PCR扩增获得了hDLL4基因片段,经测序证实与GenBank公布的序列一致,重组的质粒经过PCR、酶切鉴定,证明重组质粒中已成功的插入了目的基因hDLL4.成功构建了该蛋白的原核表达载体pGEX-KG-hDLL4.结论融合蛋白GST-hDLL4在25℃,IPTG终浓度0.5 mmol/L条件下诱导表达,以GSTrap亲和层析柱纯化,获得纯化融合蛋白.采用SDS-PAGE,Western Blot的方法可检测到目的可溶性融合蛋白GST-hDLL4的表达.Objective To clone human tumor antigen DLL4 gene, then induce its prokaryotic expression and separate, purify and identify the soluble fusion protein. The result is intended to apply in the DLL4-specific antiserum titer detection of lung cancer vaccine research process. Methods DLIA segment was amplified by RT-PCR and cloned into pMD-18T vector. After sequencing, the DLL4 segment was sub-cloned into expression vector pGEX-KG to construct the expression plasmid pGEX-KG-hDLIA. The recombinant vector was transfected to BL21 （DE3） and GST fusion protein was expressed by IPTG. The protein was purified by GST affinity chromatography and identified by SDS-PAGE electrophoresis and Western blot. Results hDLL4 gene segment were obtained by PCR amplification, and the sequence was the same with the sequence which published by GeneBank after tested. We have successfully constructed the pProkaryotic expression vector pGEX-KG-hDLL4 by proving that the target gene hDLL4 had been inserted into the recombinant plasmid after PCR and enzyme digestion. Conclusion The fusion protein GST-hDLL4 could be induced under the condition that the temperature was 25℃ and the concentration of the IPTG was 0.5 mmol/L. After purified by GST affinity chromatography column, theexpression of the soluble fusion protein could be tested by SDS-PAGE and Western blot.

关 键 词：DLL4 原核表达 亲和层析 融合蛋白 

分 类 号：S852.6[农业科学—基础兽医学]