小鼠睾丸特异性基因Vad1.2重组蛋白的构建及多克隆抗体制备

Construction and Polyclonal Antibody Preparation of Recombinant Protein Based on Testis-specific Gene Vad1.2

目的:构建小鼠睾丸特异性基因Vad1.2重组蛋白,制备多克隆抗体。方法:将小鼠睾丸组织Vad1.2转录本行RT-PCR扩增,通过DNA重组技术插入克隆载体p ET15b,进行酶切及DNA序列分析。将表达载体转化入大肠杆菌BL21(DE3)RIL感受态细胞,10 mmol/L IPTG诱导表达重组蛋白,通过10%SDS-PAGE、Western blotting及质谱分析进行鉴定。随后对重组Vad1.2蛋白进行纯化和进一步鉴定。最后,制备兔抗小鼠Vad1.2多克隆抗体,并通过Vad1.2-EGFP质粒转染GC-2spd(ts)细胞对其特异性进行验证。结果:大肠杆菌BL21(DE3)RIL细胞中诱导表达并纯化的小鼠Vad1.2重组蛋白构建正确并经Western blotting和质谱分析得到证实。所制备的多克隆抗体可特异性识别GC-2spd(ts)细胞中过表达的Vad1.2-EGFP融合蛋白。结论:成功构建小鼠Vad1.2重组蛋白,并制备多克隆抗体,为Vad1.2基因的进一步研究奠定了实验基础。

Objective： To construct the recombinant mouse Vad1. 2 protein related to Spermatogenesis and prepare a polyclonal antibody. Methods： Vad1. 2 transcript was amplified from mouse testis by RT-PCR. The amplified product was inserted into p ET15 b vector,and identified by endonuclease digestion and DNA sequencing. The Vad1. 2 over-expression vector was transformed into E. coli BL21（ DE3） RIL competent cells. The recombinant protein was over-expressed by 10 mmol / L IPTG induction,and confirmed by 10% SDS-PAGE,Western blotting and Mass Spectrometry.Then,the recombinant was further purified with the Protein Refolding kit and verified again. Finally,a rabbit anti-mouse Vad1. 2 polyclonal antibody was generated. The specificity of rabbit anti-mouse Vad1. 2 polyclonal antibody was tested with GC-2spd（ ts） cells transfected with the Vad1. 2-EGFP fusion vector. Results： Vad1. 2 recombinant protein was successfully over-expressed in E. coli. The protein was purified and confirmed by Western blotting and Mass Spectrometry analysis. The anti-Vad1. 2 antibody specifically recognized the Vad1. 2-EGFP fusion protein over-expressed in GC-2spd（ ts） cells. Conclusion： The construction and polyclonal antibody preparation of recombinant mouse Vad1. 2 protein are of significance for the functional study of Vad1. 2 gene in spermatogenesis.