摘要
目的制备小鼠SAP多克隆抗体,为进一步研究其功能和探讨其与炎症、肿瘤等疾病的相关性提供素材。方法PCR扩增小鼠SAP基因编码区的DNA片段,将其重组入原核表达质粒PET30a(+),转化入大肠杆菌BL21中,异丙基-βD硫代办乳糖苷(IPTG)诱导产生His/mSAP融合蛋白,SDS-PAGE分析结果表明,蛋白主要以包涵体形式存在。采用割胶回收的方法纯化目的蛋白,免疫新西兰白兔,制备抗血清。通过ELISA、Western印迹来检测抗血清效价及特异性。结果成功的构建了PET30a(+)/mSAP重组质粒,表达融合蛋白,免疫家兔所获得的mSAP多克隆抗体。结论mSAP多克隆抗体特异性强,效价高。
Objective To prepare mSAP polyclonal antibody and make it use widely in mSAP detection. Methods The DNA fragment of coding region of the mouse SAP gene was amplied by PCR, then cloned it in to the prokaryofic expressed vector, PET30α（ ＋ ）. The recombinant plasmid was transierred into E. coil BL21. Fusion protein His/mSAP was expressed after IPTG was induced and isolated and purified from inclusion bodies. New Zealand rabbits were immunized with the purify fusion protein. The antiserum was detected by ELISA and Western blot. Resuits the prokaryotic expressed PET30α（ ＋ ）/mSAP vector was successfully constructed, fusion protein His/mSAP was expressed efficiently. The polyclonal antibody was raised in the rabbits. Conclusion The mSAP antiserum re, veal high titer and specificity and maybe applicable in SAP function research.
出处
《国际遗传学杂志》
CAS
2006年第2期98-100,141,共4页
International Journal of Genetics
基金
国家自然科学基金(No.39925015)
