非小细胞肺癌生物标记物角蛋白19基因的克隆、表达及纯化  

Cloning,expression and purification of CK19 protein

作　　者：孙晓林[1] 李梅[1] 唐佳昕[2] 李淑珍[1] 唐晓波[1] 

机构地区：[1]哈尔滨医科大学药学院生物制药学教研室,150081 [2]上海交通大学医学院

出　　处：《国际免疫学杂志》2013年第4期325-328,共4页International Journal of Immunology

基　　金：哈尔滨市科技创新人才研究专项资金项目（2008RFXXS018）

摘　　要：目的克隆人角蛋白19（CK19）基因，原核表达重组蛋白并纯化，为建立一种新的非小细胞肺癌诊断方法奠定基础。方法从宫颈癌HeLa细胞株中提取总RNA，经RT—PCR合成eDNA，设计特异性的引物，用PCR的方法扩增目的片段，构建pET．32a—CK19和pGEX-4T1-CK19表达载体，在BL21大肠杆菌中表达CK19重组蛋白纯化，并进行SDS—PAGE及Westernblot鉴定。结果经PCR扩增后得到一条420bp的DNA片段，构建载体后DNA测序，结果与预期序列一致。初步结果鉴定表明，表达和纯化的融合蛋白，与预期结果一致。结论成功克隆CK19基因，并获得高纯度的CK19融合蛋白，为进一步制备体外诊断试剂打下基础。Objective To clone human keratin 19 （CK19）gene and carry out prokaryotic expression and purification of CK19 protein, in order to establish an early diagnostic method of non-smaU cell lung cancer. Methods Human CK19 gene was cloned through RT-PCR and PCR techniques from total RNA of a HeLa cell strain. The recombinant plasmids PET-32a-CK19 and PGEX-4T1-CK19 were constructed and transformed into E. coli BL21, and CK19 was expressed, purified and identified by SDS-PAGE and Western blotting. Results The 420 bp DNA fragment was confirmed as CK19 gene by DNA sequencing. After expression and purification of the cloned gene, the protein was identified as CK19 by SDS-PAGE and Western blotting. Conclusions The gene encoding CK19 is successfully cloned, expressed and purified. The study lays a foundation for further de- velopment of diagnostic reagent of non-small cell lung cancer.

关 键 词：非小细胞肺癌 人角蛋白19 克隆 表达 纯化 

分 类 号：R734.2[医药卫生—肿瘤]