鼻咽癌人源抗独特型基因工程抗体的原核表达及鉴定  

Expression and identification of human anti-idiotypic single chain antibody of nasopharyngeal carcinoma in E.coli

作　　者：汪静[1] 李官成[1] 童永清[1] 张志杰[1] 

机构地区：[1]中南大学肿瘤研究所,湖南长沙410078

出　　处：《中国现代医学杂志》2008年第8期1056-1059,共4页China Journal of Modern Medicine

基　　金：国家自然科学基金资助(No:30270512)

摘　　要：目的构建鼻咽癌人源抗独特型单链抗体原核表达载体,对其产物做初步鉴定,为鼻咽癌疫苗制备奠定基础。方法采用分子克隆技术,PCR扩增G22ScFv基因,并将其克隆到原核表达载体pET25b(+)上,将重组子转化大肠杆菌BL21(DE3),经IPTG诱导表达后,表达的蛋白经His-tag纯化试剂盒纯化后复性。SDS-PAGE及Western blot分析融合蛋白。结果构建的原核表达载体pET25b(+)-G22经测序检测,序列正确。在大肠杆菌中G22ScFv以包涵体形式获高效表达,SDS-PAGE及Western blot鉴定表达的目的蛋白Mr为25kD,同时证实载体构建及表达均正确。目的蛋白经试剂盒纯化后电泳分析纯度达95%以上。结论利用基因重组技术,在原核细胞中表达出重组G22ScFv并得到纯化,为研究鼻咽癌疫苗打下了基础。[Objective] To construct a new recombinant expression vector of nasopharyngeal carcinoma anti-idiotypic single chain antibody, and identify the product for the possible clinical use in active immunity therapy of nasopharyngeal carcinoma. [Methods] The G22 gene was emplified by PCR and digest by restrictive enzyme before cloned into pET25b （＋）. It was then transformed into E.Coll DtLSctand the positive recombinant plasmid named pET25b（＋）-G22 was sequenced. The target protein was expressed after transformed into E.Coll BL21 （DE3）, and induced with IPTG. The cell extracts were purified by His-tag fusion protein purification kit under denaturing condition. Western blot analysis was performed after solubility analysis. [Results] The sequence of target gene in recombinant plasmid pET25b（＋）-G22 was the same as G22 and could be efficiently expressed in E.coli. Protein production mainly was the inclusion body,but could be purified by His-tag fusion protein purification kit. The western blot analysis was proved that the protein had immunologic specificity and bioactivity. [Conclusion] The Ab2βScFv G22 is successfully cloned and expressed in this study, The purified product has biological activity. These results pave the way for further studies.

关 键 词：抗独特型抗体 鼻咽癌 原核表达 单链抗体 

分 类 号：R739.63[医药卫生—肿瘤]