小鼠肿瘤/睾丸抗原Biot2的原核表达载体的构建及表达  

Construction of the prokaryotic expression vector and expression of murine testis-specific gene Biot2

作　　者：王春婷[1] 张鹏[1] 彭枫[2] 杨寒朔[1] 

机构地区：[1]四川大学华西医院肿瘤生物治疗国家重点实验室,四川成都610041 [2]四川大学华西医院肿瘤中心生物治疗科,四川成都610041

出　　处：《细胞与分子免疫学杂志》2007年第5期406-408,共3页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家高技术研究发展计划(863)资助项目(2004CB518800);国家自然科学基金资助项目(30130260;30572148)

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。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.

关 键 词：睾丸 组织特异表达 重组融合蛋白/表达 

分 类 号：Q78[生物学—分子生物学]