【摘要】 目的构建小鼠睾丸特异表达基因Biot2的原核表达载体,表达pQE30-Biot2融合蛋白。方法提取小鼠睾丸组织总RNA,经RT-PCR扩增Biot2基因片段,并将其克隆入原核表达载体pQE30中,构建重组质粒pQE30-Biot2。经限制性内切酶BamHI、HindIII双酶切鉴定及序列测定后,转化E.coliXL-Blue,经IPTG诱导表达组氨酸融合蛋白,对表达产物进行SDS-PAGE电泳分析和Western blot检测。结果构建pQE30-Biot2重组原核表达质粒,表达的融合蛋白经SDS-PAGE分析,在相对分子质量(Mr)约17700处出现了1条蛋白条带,该表达蛋白具有与His-tag单克隆抗体(mAb)特异性的结合能力。结论成功地构建了Biot2基因的原核表达载体,并表达出pQE30-Biot2重组蛋白,为下一步制备多克隆抗体和蛋白功能的深入研究奠定了实验基础。更多还原

【Abstract】 AIM: To construct a prokaryotic expression vector of mice gene Biot2, and to express the gene in E.coli/XL-Blue. METHODS: The total RNA was extracted from mice testes tissues. Biot2 gene fragment was amplified by RT-PCR and was cloned into the pQE30 vector. The recombinant vector was identified by restriction endonuclease digestion analysis and DNA sequencing, and then it was transformed into E.coli/XL-Blue through IPTG induction to express the target protein bearing 6-His tag, which was analyzed by SDS-PAGE and Western blot. RESULTS: After E.coli/XL-Blue was transformed with recombinant vector pQE30-Biot2 and induced with IPTG, the recombinant protein with relative molecular mass about 17.7 kD was obtained. CONCLUSION: Recombinant expression vector pQE30-Biot2 is constructed and expressed successfully, which will be helpful to our further research.更多还原