人脂联素acrp30基因的原核表达、纯化及活性鉴定 - 第三军医大学学报.doc

人脂联素基因的原核表达、纯化及ELISA检测方法的建立 （1郑州大学 郑州450002 2 河南省生物工程技术研究中心 郑州450002） 摘要：目的 克隆人（adiponectinAcrp30）基因，获得有活性的人脂联素蛋白，为进一步开发应用奠定基础。方法 以人新鲜皮下脂肪组织为材料，通过RT-PCR得到人Acrp30基因的全长片段。构建重组质粒pET-CKSAcrp30，转化表达菌Balgold，并对转化子进行诱导表达，利用Ni-NTA 亲和层析纯化目的产物，蛋白印记验证其活性。结果 获得了人Acrp30基因全长片段的正确序列，表达和纯化了人脂联素蛋白，表达量占菌体总蛋白的35%，纯度达到97%。方阵滴定法确定了抗血清最佳稀释度(1600倍)和包被抗原的最佳工作浓度(1mg/L)。结论基因在原核表达载体的表达产物具有免疫活性。初步优化了间接ELISA检测条件。 关键词：人脂联素；包涵体；基因表达；酶联免疫 Prokaryotic ExpressionPurification and immunological activity detection of Human Adiponectin Gene WANG Yun-long1,2,* LIANG Xiao-yan1 LIU Wei1 LI Yu-lin2 JIA Li-feng1 LI Zhao-xue3 LI Zhong-xin3 （1 Zhengzhou UniversityZhengzhou 450002 2 Henan Biotechnology Research Center Zhengzhou 450002 3 Henan University of Technology Zhengzhou 450002） Abstract：Objective: To clone the sequence of human Adiponectin(Acrp30) cDNA and obtain the protein of recombinant Acrp30 for the other development. METHODS: Fresh human fat tissure was used for the extraction of total RNA and amplification of the Adiponectin cDNA through RT-PCR method. After the recombinant plasmid pET-CKS-Adiponectin was constructed and confirmed,it was transduced into Balgold.Then the transformation was used for fermentation and induced expression. The protein was identified by Western blotting and purified by Ni-NTA affinity chromatography. RESULTS:The complete sequence of human Adiponectin was identical with GenBank. Human Adiponectin was expressed and purified. The target protein made up 35% of the total bacterial protein. The purity is above 97%. The optimal dilution of antiserum and the best packages concentration of antigen were defined by titration. CONCLUSION:Acrp30（Acrp30）with immune activity can be obtained using Prokaryoti Expression vectorand the indirect ELISA test conditions were initial optimized. Key words:Acrp30;inclusion body;gene expression;ELISA 脂联素是最近发现的由脂肪细胞分泌的一种脂肪细胞因子，具有抗动脉粥样硬化、调节脂肪酸氧化和糖代谢、增加胰岛素敏感性、抑制肝糖产生、抑制反馈TNF-α的生成与释放，并且脂联素具有一定的抗炎作用，对肝细胞损伤有重要的保护作用而备受关注[1-3]。 人脂联素adiponectin /Acrp3