论文部分内容阅读
目的:制备高效价抗visfatin抗体,探测visfatin在前脂肪细胞成脂分化过程中在空间上表达情况。方法:克隆鼠内脂素visfatin基因的蛋白编码区全长序列,构建其原核表达载体pET28a-visfatin,然后在E.coli(BL21)中诱导大量以包涵体形式表达,提取包涵体并用Ni-NTA亲和层析柱纯化蛋白,并以此为抗原免疫新西兰大白兔制备多克隆抗体。以纯化后的抗血清为一抗,Western blot检测小鼠肝、肌肉等组织中visfatin的分布。结果:SDS-PAGE及Western blot显示原核表达载体pET28a-visfatin在大肠杆菌中诱导表达,融合蛋白能够被抗hismAb识别。免疫新西兰大白兔获得抗体效价在1∶25600以上。结论:用制备得到的抗体对小鼠心、肝、肺、脾、脂肪、肌肉组织样品做Western blot鉴定,结果表明visfatin在各组织中均有表达,其中脾和脂肪组织中表达量高于其他组织。
OBJECTIVE: To prepare high titer anti-visfatin antibody and to detect the spatial expression of visfatin during adipogenic differentiation of preadipocytes. Methods: The full-length cDNA sequence encoding murine visfatin gene was cloned. The prokaryotic expression vector pET28a-visfatin was cloned and expressed in E.coli (BL21) as a large number of inclusion bodies. The inclusion bodies were extracted and purified by Ni-NTA Affinity chromatography purification of protein, and use this as antigen to immunize New Zealand white rabbits to prepare polyclonal antibody. The purified antiserum was used as the primary antibody. The distribution of visfatin in the liver, muscle and other tissues was detected by Western blot. Results: The prokaryotic expression vector pET28a-visfatin was induced in E. coli by SDS-PAGE and Western blot, and the fusion protein was recognized by anti-hismAb. New Zealand white rabbits immunized antibody titers above 1: 25600. Conclusion: Western blotting of mouse heart, liver, lung, spleen, adipose and muscle tissue samples with the prepared antibody showed that visfatin was expressed in all tissues, and the expression of visfatin was higher in spleen and adipose tissue than in other tissues organization.