鸭疫里默氏杆菌RecA作为一种内参蛋白的评价  

The Assessment of Rec A Acted as an Internal Reference Protein in R. anatipestifer

作　　者：廖何斌 刘马峰[1,2,3] 程安春[1,2,3] 

机构地区：[1]四川农业大学预防兽医研究所,成都6111302 [2]四川农业大学动物医学院禽病防治中心,雅安625014 [3]四川农业大学动物疫病与人类健康四川省重点实验室,成都611130

出　　处：《中国生物工程杂志》2015年第6期26-31,共6页China Biotechnology

基　　金：国家自然科学基金(31302131);高等学校博士学科点专项科研基金(20135103120006);中国博士后基金(2014M552378)资助项目

摘　　要：由于缺少适合的内参蛋白,所以用Western blot的方法来研究鸭疫里默氏杆菌蛋白表达的调节尚未见报道。以鸭疫里默氏杆菌基因组为模板,PCR扩增含有组氨酸标签His-tag的rec A基因和截段的rec AP基因,并分别将其克隆于原核表达载体p ET32a。将重组质粒转化到表达菌Rosetta中,经1mmol/L的IPTG诱导进行蛋白质表达。用组氨酸标签亲和试剂镍-琼脂糖进行重组蛋白质的纯化。用纯化的重组蛋白对4周龄的昆明鼠进行免疫,制备多克隆抗体。Western blot实验表明,截段表达的Rec AP的多克隆抗体血清比全长Rec A蛋白的多克隆抗体血清与鸭疫里默氏杆菌总蛋白反应更特异;在铁离子限制性环境和血红素丰富环境中培养的鸭疫里默氏杆菌的Rec A蛋白表达量一致。因此,鸭疫里默氏杆菌的Rec A蛋白可以在铁离子和血红素代谢等研究中作为Western blot的内参蛋白。Using Western blot to investigate the gene expression in R. anatipestifer is impracticable because of lacking of internal reference protein. Complete rec A gene and partial rec A gene harboring His-tag were amplified from the genome of R. anatipestifer and then were cloned into expression plasmid p ET32 a,respectively. Recombinant proteins Rec A and Rec APwere expressed in E. coli Rosetta under induction with1 mmol / L IPTG. Then,recombinant proteins were purified with Ni-affinity chromatography. The polyclonal antibodies against the recombinant proteins were prepared by immunizing 4 weeks old Kun Ming mouse with about50 μg purified proteins. Western blot indicated that the interaction between R. anatipestifer total proteins and serum against Rec APis more specific than that between R. anatipestifer total proteins and serum against Rec A.The expression of Rec A protein of R. anatipestifer is stable under different condition including common medium LB and TSB,iron chelated medium TSB ＋ Dip or sufficient hemin medium LB ＋ Hemin. In conclusion,Rec A protein of R. anatipestifer can act as an internal reference protein in iron and hemin metabolism researches.

关 键 词：鸭疫里默氏杆菌 内参蛋白 RecA蛋白 多克隆抗体 蛋白免疫印迹 

分 类 号：Q819[生物学—生物工程]