脂联素基因去信号肽区原核表达及酶免检测方法研究

发布时间：2018-04-20 12:13

  本文选题：人脂联素去信号肽区 + 原核表达　； 参考：《河南师范大学》2011年硕士论文

【摘要】：近年来,肥胖症、Ⅱ型糖尿病、动脉粥样硬化发病率不断升高,血浆中脂联素(adiponectin,ADPN)的含量与这类疾病的发生呈负相关,通过检测人血浆中脂联素的含量可反应这类疾病的发生状况。本研究通过基因工程获得了人脂联素去信号肽区域重组蛋白,并以此制备标准品,初步建立了人脂联素ELISA定量检测方法。 根据NCBI上公布的人脂联素基因序列NM_004797.2,利用primer5.0设计一对引物。从人脂肪组织中提取总RNA,通过RT-PCR获得678bp的人脂联素去信号肽区的基因片段,将目的片段和载体pET-41a分别进行双酶切,琼脂糖凝胶电泳,回收纯化目的片段和载体并进行连接,将连接产物转入DH5α,涂布LB固体培养基,挑取单菌落,扩大培养后用碱裂解法提取质粒,将双酶切鉴定为阳性的重组质粒命名为pET-41a/ΔSADPN。将该重组质粒转入E.coli BL21(DE)3中,用IPTG进行诱导表达,SDS-PAGE表明重组蛋白以可溶性形式表达,并具有较好的免疫反应性。 通过诱导表达条件的优化,确立了最佳的诱导条件为:25℃,IPTG浓度0.03mmol/L,诱导时间8h。融合蛋白含有组氨酸标签,用镍离子亲和层析纯化目的蛋白,得到的融合蛋白溶液,经检测其浓度为2.1 mg/mL,纯化后的蛋白制备成标准品。 以抗人脂联素单克隆抗体作为包被抗体,采用改良的过碘酸钠法,对人脂联素多克隆抗体进行辣根过氧化物酶标记,作为标记抗体。棋盘滴定法确立人脂联素单克隆抗体的最佳包被浓度为1.0μg/mL;酶标抗体的最佳稀释倍数为1:4000;以脂联素去信号肽区重组蛋白为标准品绘制出的标准曲线,线性范围为1ng～30ng;与同类试剂盒进行临床试验比较,其检测结果通过t检验,P0.05,没有显著性差异。 本研究通过基因工程方法得到了免疫反应性较好的重组人脂联素去信号肽蛋白,制备了检测脂联素的标准品,初步建立人脂联素双抗体夹心检测方法,为进一步研制人脂联素ELISA定量检测试剂盒奠定基础。
[Abstract]:In recent years, the incidence of obesity, type 2 diabetes and atherosclerosis has been increasing. The plasma adiponectin ADPNs are negatively correlated with the occurrence of these diseases. Adiponectin levels in human plasma can reflect the occurrence of these diseases. In this study, the recombinant protein of human adiponectin de-signalling peptide region was obtained by genetic engineering, and the standard product was prepared, and the quantitative detection method of human adiponectin ELISA was established. According to the human adiponectin gene sequence NM004797.2 published on NCBI, a pair of primers were designed by primer5.0. Total RNAs were extracted from human adipose tissue. The gene fragment of human adiponectin designalling peptide region of 678bp was obtained by RT-PCR. The target fragment and vector pET-41a were digested by double enzyme, agarose gel electrophoresis, and purified fragment and vector were recovered and ligated. The ligation product was transferred into DH5 伪, coated with LB solid medium, and a single colony was picked up. After expanded culture, the plasmid was extracted by alkaline lysis method. The recombinant plasmid identified by double enzyme digestion was named pET-41a/ 螖 SADPN. The recombinant plasmid was transferred into E.coli BL21(DE)3 and expressed by IPTG. SDS-PAGE showed that the recombinant protein was expressed in soluble form and had good immunoreactivity. Through the optimization of the induced expression conditions, the optimal induction conditions were established as follows: the concentration of IPTG at 25 鈩，

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