摘要
目的克隆并在原核表达载体pET-28a中表达小鼠IL-13受体α2(sIL-13Rα2)胞外区基因。方法利用小鼠3T3细胞总RNA,逆转录为cDNA,设计引物,常规PCR法扩增出小鼠sIL-13Rα2基因,将PCR产物克隆入pMD18-T载体,经双酶切及测序鉴定后,再亚克隆入pET-28a,构建重组质粒pET-28a/sIL-13Rα2,并转化到大肠杆菌BL21感受态细胞。IPTG诱导表达后表达产物经Western blot鉴定。结果PCR扩增产物与预期大小相符合,重组质粒经双酶切鉴定及基因序列测定表明构建正确;sIL-13Rα2蛋白表达以包涵体形式存在,可与山羊抗小鼠sIL-13Rα2单克隆抗体发生特异性反应,相对分子质量约为36 ku。结论成功原核表达sIL-13Rα2基因,为进一步探讨sIL-13Rα2在血吸虫病肝纤维化过程中的调节作用奠定了基础。
Objective To clone murine IL-13 receptor α2 extracellular domain gene（sIL-13Rα2） and express in prokaryotic expression vector pET-28a.Methods Total RNA was extracted from murine 3T3 cells for amplification of sIL-13Rα2 gene by RT-PCR.The amplified gene fragment was cloned into pMD18-T simple vector and identified by double endonuclease digestion and gene sequencing.The fragment was subcloned into a prokaryotic expression vector pET-28a again and recombinant pET-28a/sIL-13Rα2 was constructed and transformed into E.coli BL21 for expression under induction of IPTG.The expressed product was identified by western blot.Results The size of PCR product was consistent with the expected size,and pMD-18T/sIL-13Rα2 and pET28a/sIL-13Rα2 were constructed correctly by double endonuclease digestion and gene sequencing.The expressed sIL-13Rα2 protein existed in a form of inclusion body,and showed specific reaction with goat anti-mouse sIL-13Rα2 McAb.The relative molecular weight was approximately 36 ku.Conclusion Murine sIL-13Rα2 gene is successfully cloned and expressed in pET-28a,which lay a foundation of further study sIL-13Rα2 on regulation of liver fibrosis in schistosomiasis.
出处
《安徽医科大学学报》
CAS
北大核心
2012年第2期109-112,共4页
Acta Universitatis Medicinalis Anhui
基金
国家自然科学基金(编号:30872209)
安徽省高等学校省级自然科学研究项目(编号:KJ2008B282)
安徽医科大学博士科研资助项目(编号:XJ200708)
教育部博士点基金(编号:200803660003)
关键词
基因表达
小鼠sIL-13Rα2基因
原核表达载体
gene expression
murine soluble interleukin-13 receptor α2 gene
prokaryotic expression vector
