小鼠睾丸特异表达蛋白GM1142抗体制备与应用

Antibody preparation and application of mouse testis specifically-expressed protein GM1142

目的制备睾丸特异性表达蛋白GM1142的多克隆抗体,研究其小鼠睾丸组织的表达与定位情况,为进一步探究其在精子发生中的功能奠定基础。方法通过生物信息学分析选取小鼠GM1142 N端1-153个氨基酸为抗原肽,通过分子克隆技术构建重组原核表达载体pET42b-T7-GM1142N153-8×His,诱导表达后纯化并用以免疫新西兰大白兔和小鼠制备GM1142的多克隆抗体。蛋白免疫印迹和免疫荧光染色检测GM1142在小鼠各组织、不同发育时期的睾丸、以及睾丸中各细胞类型中的表达与定位特征。结果GM1142在睾丸中特异性高表达;免疫荧光染色结果显示GM1142在小鼠精原细胞和早期精母细胞的胞质中呈现强荧光信号。结论GM1142在睾丸中特异性高表达并可能在精原细胞发育和精母细胞减数分裂中具有重要作用。

Objective To prepare the polyclonal antibody of mouse testis-specific protein GM1142 for exploring its expression and localization characteristics in testis,laying foundation for further investigation of its function in spermatogenesis.Methods The 1-153 amino acid fragment at N-terminus of mouse GM1142 was selected as antigen peptide for antibody preparation after bioinformatics analysis,and the recombinant prokaryotic expression vector pET42b-T7-GM1142N153-8×His was constructed by molecular cloning technology,and the target protein was purified after induced expression,followed by inoculating New Zealand white rabbits or mice with the purified protein for preparing polyclonal serum against GM1142.The expression and localization characteristics of GM1142 in different tissues,testes at different developmental stages and different testicular cell types were detected by western blot and immunofluorescence staining.Results Gm1142 was specifically highly expressed in testis.Immunofluorescence staining showed a strong fluorescence signal of GM1142 in the cytoplasm of mouse spermatogonium and early spermatocyte.Conclusion Gm1142 is specifically highly expressed in testis and it may play an important role in spermatogonial development and meiosis of spermatocytes.