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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。
OBJECTIVE: To clone cDNA sequence of human cholesterol ester transporter (CETP) and express human CETP in E. coli to prepare rabbit polyclonal antiserum against human CETP. Methods: The human CETP gene was cloned into pET-30b (+) by RT-PCR. The prokaryotic expression vector of CETP was constructed and transformed into E. coli. The recombinant plasmid was induced by IPTG. The antiserum titer and specificity were identified by ELISA, Western blot and immunofluorescence. Results: SDS-PAGE analysis showed that CETP gene was highly expressed in inclusion bodies of E. coli BL21 (DE3) induced by IPTG, and the optimal induction time was 4h. Rabbit was immunized with gel-purified protein. The antiserum titer determined by ELISA was 1: 5.12 × 105. Western blot and immunofluorescence showed that the antiserum could specifically bind to the prokaryotic and eukaryotic proteins of CETP. CONCLUSION: Prokaryotic expression of CETP and high titer and specificity of rabbit anti-human CETP antiserum were successfully performed, which laid the foundation for further study on the structure and function of CETP.