【摘要】 目的:CMTM1-V17是我室克隆的人类新基因,为进一步研究CMTM1-v17的功能,需制备高纯度的CMTM1v17的抗体。方法:根据生物信息学分析的结果,用PCR法扩增人CMTM1-v17分子C端的cDNA片段(编码118~149位氨基酸),亚克隆到GST融合蛋白表达载体pGEX-4T-2上。将重组质粒pGST-CMTM1-v17导入大肠杆菌BL21,诱导表达GSTCMTM1-v17C端融合蛋白,经GST亲合层析、凝血酶切割后获得CMTM1-v17C蛋白作为抗原免疫家兔制备抗血清。抗血清经抗原亲合层析后用Westernblot和免疫组化方法进行鉴定。结果:通过重组表达获得抗原蛋白,免疫家兔并纯化抗血清得到特异的抗CMTM1-v17抗体,该抗体能检测到CMTM1-v17超表达和细胞内源性的蛋白。结论:成功制备了抗CMTM1-v17的多克隆抗体,对进一步研究CMTM1-v17的功能提供了一个有用的工具。更多还原

【Abstract】 Objective:To analyze a novel antigenic determinant of Homo sapiens CMTM1-v17 protein and prepare polyclonal antibody.Methods:By means of DNA recombination method, the C-terminal(The coding sequences of 118-149 residues) of CMTM1-v17 gene was inserted into pGEX-4T-2 vector. The recombinant plasmid was transformed into E.coli XL-1 Blue to express the fusion protein of GST/CMTM1-v17. The GST tag of the purified fusion protein was removed by enzymatic cleavage with thrombin. The polyclonal anti-CMTM1-v17 antibody were prepared by immunizing rabbits with the purified protein. Highly-specific, polyclonal anti-CMTM1-v17 antibodies was purified by affinity chromatographic column matrix coupled with antigen. The specificity of the anti-CMTM1-v17 antibody was identified by Western blot and immunohistochemistry.Results:The polyclonal antibody showed specific response to CMTM1-v17 by Western blot analysis and immunohistochemistry. These results suggested that the polyclonal antibody can be used for further studies of CMTM1-v17 function.Conclusion:A polyclonal anti-CMTM1-v17 antibody has been synthesized successfully.更多还原