人胆固醇酯转运蛋白的cDNA克隆、原核表达及抗血清制备  

cDNA cloning and prokaryotic expression of human cholesteryl ester transfer protein and preparation of its antiserum

作　　者：刘惠荣[1] 曾武威[2] 周冰[3] 申乐[2] 吴刚[2] 薛红[2] 陈保生[2] 

机构地区：[1]内蒙古农业大学生物工程学院,内蒙古呼和浩特010018 [2]中国医学科学院基础医学研究所医学分子生物学国家重点实验室,北京100005 [3]北京市药品不良反应监测中心,北京100024

出　　处：《细胞与分子免疫学杂志》2008年第5期471-474,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金资助项目(39770168);国家重点基础研究发展计划(973)资助项目(2006CB503801)

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4h。将切胶纯化的蛋白免疫家兔,ELISA法测定的抗血清效价为1∶5.12×105。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。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 SDSPAGE was I：5.12 × 10^5 and the antiserum reacted specifically with CETP expressed in BL21 （DE3） and COS7 cells. CONCLUSION： The preparation of the specific rabbit antiserum against CETP will be valuable for the study on the structure and function of human CETP.

关 键 词：胆固醇酯转运蛋白 原核表达 抗血清 

分 类 号：R392.11[医药卫生—免疫学]