人胆固醇酯转运蛋白的cDNA克隆,原核表达及抗血清制备.docVIP

人胆固醇酯转运蛋白的cDNA克隆、 原核表达及抗血清制备作者：刘惠荣????作者单位：内蒙古农业大学生物工程学院, 内蒙古 呼和浩特 010018; 【摘要】? 目的: 克隆人胆固醇酯转运蛋白（CETP） cDNA序列, 利用大肠杆菌表达人CETP, 制备兔抗人CETP的多克隆抗血清。方法: 采用RTPCR方法, 将人CETP基因克隆到pET30b(+)上, 构建CETP原核表达载体并转化大肠杆菌, IPTG诱导表达, 切胶纯化蛋白后免疫家兔, 制备CETP抗血清, 以ELISA、 Western blot、 细胞免疫荧光方法对抗血清效价及特异性进行鉴定。结果: SDSPAGE分析表明, 经IPTG诱导, CETP基因在大肠杆菌BL21(DE3)的包涵体中高效表达, 最佳诱导表达时间为4 h。将切胶纯化的蛋白免疫家兔, ELISA法测定的抗血清效价为15.12×105。Western blot及细胞免疫荧光检测结果显示, 抗血清可以与CETP原核及真核表达蛋白特异结合。结论: 成功地将CETP进行了原核表达并制备了高效价、 高特异性的兔抗人CETP抗血清, 为进一步研究CETP的结构与功能奠定了基础。 【关键词】? 胆固醇酯转运蛋白 原核表达 抗血清 　　Corresponding author, Email: bschen@ibms.pumc.edu.cn 　　cDNA cloning and prokaryotic expression of human cholesteryl ester transfer protein and preparation of its antiserum 　　LIU Huirong, ZENG Wuwei, ZHOU Bing, SHEN Le, WU Gang, XUE Hong, CHEN Baosheng 　　1Bioengineering College, Inner Mongolia Agricultural University, Hohhot 010018; 2National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100005; 3Beijing Center for ADR Monitoring, Beijing 100024, China 　　[Abstract]? AIM: To clone human cholesteryl ester transfer protein (CETP) cDNA, express and purify human CETP in E.coli and prepare CETPspecific rabbit antiserum. METHODS: by RTPCR method, the encoding sequence of human cholesteryl ester transfer protein (CETP) was cloned into pET30b(+) vector. Then BL21 (DE3) of E.coli transformed with recombinant vector pETCETP was induced to express CETP in high level by IPTG. The expressed protein was purified from SDSpolyacrylamide gel, and the antiserum against CETP was raised in rabbit. The titer and specificity of rabbit antiserum were evaluated by ELISA, Western blot and immunofluorescence assay. RESULTS: The results of SDSPAGE showed that CETP was expressed in the form of inclusion bodies in? BL21(DE3) and the best expression time was about 4 hours. The titer of the rabbit antiserum prepared with CETP purified from SDSPAGE wa