含MYND结构域的锌指蛋白10的原核表达及其多克隆抗体的制备

Prokaryotic expression of Zinc finger MYND domain-containing protein 10 and preparating its polyclonal antibody

目的表达纯化含MYND结构域的锌指蛋白10(ZMYND10)的重组蛋白并制备其大鼠多克隆抗体。方法全基因合成ZMYND10基因的开放阅读框,构建原核表达载体p ET-28m-ZMYND10。在大肠杆菌中诱导表达HisZMYND10,利用His标签镍柱纯化His-ZMYND10重组蛋白,并用泛素样蛋白酶1除去重组蛋白的His标签。用纯化蛋白免疫大鼠,蛋白A/G混合琼脂糖纯化血清得到抗体。通过Western blot和免疫沉淀方法鉴定抗体的特异性。结果成功构建原核表达载体p ET-28m-ZMYND10,并获得可溶性表达。利用His柱得到高纯度的重组ZMYND10抗原,免疫大鼠后纯化得到的ZMYND10多克隆抗体可以检测到B16F10细胞中过表达及内源性表达的ZMYND10蛋白,并可用于免疫沉淀实验。结论本实验成功制备了ZMYND10的多克隆抗体,为后续探索ZMYND10在肿瘤细胞中的功能奠定了基础。

Objective To express and purify recombinant protein of Zinc finger MYND domain-containing protein 10（ ZMYND10）,and to make preparation of polyclonal antibody against mouse ZMYND10. Methods The open reading frame of mouse ZMYND10 gene was synthesized and subcloned into the prokaryotic expression vector p ET-28 m and transformed into E. coli BL21（ DE3）. His-ZMYND10 recombination protein was purified by His60 Ni gravity columns,and the His tab was removed by ubiquitin protease 1. The fusion protein His-ZMYND10 was used as antigen to immunize rats. The rat antiserum was purified by protein A / G mix agarose and its specificity was examined by Western blot and immunoprecipitation. Results Prokaryotic expression vector p ET-28m-ZMYND10 was built successful and was made solubility expression. The high purified restructuring ZMYND10 antigen was obtained by His60 Ni gravity columns and was used to immunize rats to produce polyclonal antibody against ZMYND10 which was used to detected the over-expression and endogenous expression of ZMYND10 protein in B16F10 cell used in immunoprecipitation. Conclusion Polyclonal antibody against ZMYND10 was successfully prepared,providing a firm foundation for future investigation on the function of ZMYND10 in tumor cells.