【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。更多还原

【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 was 1∶5.12×105 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.更多还原