大口黑鲈抗缪勒氏管激素(AMH)原核表达及多克隆抗体制备

Prokaryotic Expression and Polyclonal Antibody Preparation of Anti-Müllerian Hormone(AMH) in Micropterus Salmoides

为深入研究抗缪勒氏管激素在大口黑鲈性腺发育中的功能,本课题组利用前期获得大口黑鲈Amh基因,其cDNA全长序列2064bp,开放阅读框为1632bp,编码543个氨基酸,通过分析该基因的结构特点设计引物,构建重组质粒pET32a-AMH,将其导入大肠杆菌(BL21),获得重组蛋白(52.3kDa),以重组AMH蛋白免疫小鼠,制备多克隆抗体。采用酶联免疫吸附法对抗体效价进行检测。抗体血清的效价检测为2.7×10~5,可为后续实验提供原材料。本研究成功制备了大口黑鲈Amh基因的重组蛋白和多克隆抗体,为后续研究Amh基因的功能及其在大口黑鲈性腺发育中的分子机制提供参考。

In order to further study the function of Anti-Müllerian hormone in the gonad development of Micropterus salmoides, our research group used the Amh gene obtained in the previous stage, the full-length cDNA sequence was 2064 bp, Open reading frame 1632 bp, encoding 543 amino acids. By analyzing the structural characteristics of the gene, primers were designed to construct a recombinant plasmid pET32a-AMH, which was introduced into E. coli prokaryotic expression strain(BL21) to obtain a recombinant protein with a size of 52.3kDa, and the recombinant AMH protein was used to immunize mice, preparation of polyclonal antibody. Antibody titers were detected by enzyme-linked immunosorbent assay. The titer of antibody serum was 2.7 ×10~5, which could provide raw materials for subsequent experiments. In this study, the recombinant protein and polyclonal antibody of Amh gene of Micropterus Salmoides were successfully prepared, which can provide reference for further study on the function of Amh gene and its molecular mechanism in the gonad development of Micropterus Salmoides.