摘要
目的:克隆人mag1-基因,在大肠杆菌中进行表达并制备其抗体。方法:利用PCR方法从含有mag1-基因全长序列的pcDNA3-mag1-质粒上扩增mag1-基因开放阅读框(ORF),将其插入表达载体pET-28 a(+)中,构建重组表达载体pET-28 a-mag1-,用IPTG诱导目的基因在E.coli中的表达,并通过W estern印迹方法对表达产物进行鉴定。表达产物经过初步纯化后作为抗原免疫兔制备多克隆抗体。结果:所获得的序列经测序分析与源序列一致;SDS-PAGE分析表明,经0.1 mmol/L IPTG 37℃诱导6 h后在E.coli中有大量的融合蛋白表达,相对分子质量约为18×103。W estern印迹分析提示,诱导表达产物能与H is单抗发生特异反应。ELISA结果表明获得了高效价的多克隆抗体。结论:获得了H is-mag1-融合蛋白在大肠杆菌中高效表达,并制备了其相应的多克隆抗体。
Objective：To further investigate the new metastasis associated gene, mag-1 expressed in E. coli and its antibody was prepared in rabbit. Methods： mag-1 was amplified by PCR from pcDNA3-mag-1 and directly cloned into pET- 28a vector. The fusion protein was expressed in BL21 and identified by Western blot using anti-His monoclonal antibody. Rabbit was immunized with partially purified fusion protein subcutaneously. Results：Sequence analysis revealed identity of the sequence obtained to the previous report. The recombinant His-mag-1 could be expressed in E. coli as a fusion protein of 18 × 10^3. The recombinant protein was mostly expressed in the inclusion bodies on the induction by 0.1 mmol/L IPTG at 37% for 6 hours. Westem blot analysis showed that the recombinant protein could be recognized by His monoclonal antibody. The titer of polyclonal antibody against mag-1 was 1：160 000. Conclusion： The mag-1 gene is expressed in E. coli highly and its antibody is prepared successfully.
出处
《军事医学科学院院刊》
CSCD
北大核心
2006年第1期48-50,共3页
Bulletin of the Academy of Military Medical Sciences
基金
国家自然科学基金(30470389)
国家"973"计划(2002CB513105)资助
