家猪Gasdermin D基因的序列分析、原核表达及重组蛋白纯化

Sequence Analysis of Porcine Gasdermin D Gene and Prokaryotic Expression and Purifi cation of Recombinant Protein

本研究旨在对家猪Gasdermin D(GSDMD)基因进行序列分析,并采用原核表达系统表达、获得重组蛋白GSDMD。首先提取猪肺泡巨噬细胞(PAMs)的总RNA,通过RT-PCR扩增GSDMD基因序列并利用生物信息学软件进行序列分析;然后将该序列克隆至原核表达载体获得重组载体pET-28a-GSDMD;重组质粒转化BL21(DE3)感受态细胞后,经IPTG诱导表达并纯化该重组蛋白。结果表明,家猪GSDMD基因序列与牛源GSDMD基因序列核苷酸同源性最高,为81.7%;重组融合蛋白GSDMD约为58 kDa,以包涵体形式存在;纯化后的蛋白经Western blot检测可与抗His标签单克隆抗体发生特异性反应。本研究为探究猪源GSDMD蛋白的结构与功能,以及进一步探讨猪源疾病与细胞焦亡之间的关系及相关机制奠定基础。

The aim of this study was to analyze the sequence of the porcine Gasdermin D(GSDMD)gene and obtain the recombinant GSDMD protein by the prokaryotic expression system.The total RNA of porcine alveolar macrophages(PAMs)was extracted for RT-PCR amplifi cation and sequencing.Subsequently,the amplifi ed fragment was cloned into prokaryotic vector pET-28a to obtain the recombinant plasmid pET-28a-GSDMD.Prokaryotic expression was induced by IPTG and the recombinant protein was purifi ed.The results indicated that the porcine GSDMD sequence shared the highest level of nucleotide sequence identity with the bovine GSDMD(81.7%).The recombinant GSDMD protein was mainly in the form of inclusion bodies and the molecular weight was about 58 kDa.The purifi ed recombinant protein reacted specifi cally with anti-His monoclonal antibodies in Western blotting.This study laid the foundation for the research of structure and function of porcine GSDMD and contributed to further exploring the interaction and action mechanisms between the porcine diseases-related viruses and pyroptosis.