人视黄醇结合蛋白4多克隆抗血清的制备及初步应用

发布时间：2018-06-16 02:23

  本文选题：人视黄醇结合蛋白4 + pET-28a(+)　； 参考：《安徽医科大学》2010年硕士论文

【摘要】： 目的制备抗人视黄醇结合蛋白4(Retinal binding protein4, RBP4)的多克隆抗血清,初步应用于2型糖尿病(T2DM)患者的血清RBP4检测。 方法从成人正常肝脏组织中提取总RNA,RT-PCR法扩增人RBP4 cDNA全长,与pMD-18T Vector相连,构建克隆载体pMD-hRBP4。设计分别带BamHⅠ和HindⅢ酶切位点的上下游引物,PCR后酶切,构建原核表达载体pET28a(+)-hRBP4,氯化钙法转化入BL21(DE3),提取质粒双酶切鉴定并测序,将IPTG诱导表达的蛋白质产物用western blotting鉴定。对诱导表达的条件如IPTG的浓度、时间和温度等进行优化,镍亲和层析法纯化rhRBP4,免疫新西兰大白兔,制备多克隆抗血清,Western blotting检测其特异性,ELISA法测定抗体效价。用多克隆抗血清检测T2DM患者血清RBP4水平。 结果重组质粒pET-28a(+)-hRBP4的双酶切结果与预期大小完全一致,测序结果证实所得的cDNA序列与GenBank中hRBP4的序列基本一致,Western blotting结果证实重组蛋白能够与hRBP4单克隆抗体特异性结合。IPTG诱导表达的最适浓度是0.04 mmol/L,最适时间为6 h,最适温度为37℃,表达的重组蛋白最多可达菌体总蛋白的44%,且以包涵体形式存在。SDS-PAGE电泳的结果表明亲和层析法得到的重组蛋白纯度为96%。ELISA法测定抗血清的效价为1:512000。Western blotting结果显示抗血清能够与T2DM患者血清中分子量21kDa的蛋白结合,与天然RBP4的大小一致。 结论成功构建和获得了hRBP4 cDNA全长的原核表达载体及rhRBP4蛋白,制备的多克隆抗血清可初步用于T2DM患者血清中RBP4的检测,为进一步的RBP4功能研究及临床大规模血清样本RBP4的检测奠定基础。
[Abstract]:Objective to prepare a polyclonal antiserum against human retinol binding protein 4 (RBP4) and apply it to the detection of serum RBP4 in patients with type 2 diabetes mellitus (T2DM). Methods the full length of human RBP4 cDNA was amplified by RT-PCR from adult normal liver tissue and linked to pMD-18T vector. The cloned vector pMD-hRBP4 was constructed. A prokaryotic expression vector pET28a (pET-hRBP4) was constructed and transformed into BL21DE-DE3 by calcium chloride method. The plasmid was digested and sequenced. The protein products induced by IPTG-induced expression were identified by western blotting. The induced expression conditions such as the concentration, time and temperature of IPTG were optimized. RhRBP4 was purified by nickel affinity chromatography and immunized with New Zealand white rabbits. The polyclonal antiserum was prepared by Western blotting to detect the titer of antibodies by Elisa. The level of serum RBP4 in patients with type 2 DM was detected by polyclonal antiserum. Results the double digestion results of the recombinant plasmid pET-28a (hRBP4) were in good agreement with the expected size. The result of sequencing confirmed that the sequence of hRBP4 was basically consistent with the sequence of hRBP4 in GenBank. The results of Western blotting showed that the optimal concentration of recombinant protein binding to hRBP4 monoclonal antibody was 0.04 mmol / L, the optimum time was 6 h and the optimum temperature was 37 鈩，

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