鸭疫里默氏杆菌RecA作为一种内参蛋白的评价

doi:10.13523/j.cb.20150605

中国生物工程杂志

2015, Vol. 35

Issue (6): 26-31 DOI: 10.13523/j.cb.20150605

研究报告

鸭疫里默氏杆菌RecA作为一种内参蛋白的评价

廖何斌^1,2,3, 刘马峰^1,2,3, 程安春^1,2,3

1. 四川农业大学预防兽医研究所成都 611130;
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

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

LIAO He-bin^1,2,3, LIU Ma-feng^1,2,3, CHENG An-chun^1,2,3

1. Institute of Preventive Veterinary Medicine, College of Veterinary Medicine, Sichuan Agriculture University, Chengdu 611130, China;
2. Avian Disease Reserch Center, College of Veterinary Medicine, Sichuan Agriculture University, Ya'an 625014, China;
3. Key Laboratory of Animal Disease and Human Health of Sichuan Province, Chengdu 611130, China

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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recA基因和截段的recA_P基因,并分别将其克隆于原核表达载体pET32a。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RecA_P的多克隆抗体血清比全长RecA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RecA蛋白表达量一致。因此,鸭疫里默氏杆菌的RecA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。

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

Abstract:

Using Western blot to investigate the gene expression in R. anatipestifer is impracticable because of lacking of internal reference protein. Complete recA gene and partial recA gene harboring His-tag were amplified from the genome of R. anatipestifer and then were cloned into expression plasmid pET32a, respectively. Recombinant proteins RecA and RecA_P were expressed in E. coli Rosetta under induction with 1mmol/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 KunMing mouse with about 50 μg purified proteins. Western blot indicated that the interaction between R. anatipestifer total proteins and serum against RecA^P is more specific than that between R. anatipestifer total proteins and serum against RecA. The expression of RecA 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, RecA protein of R. anatipestifer can act as an internal reference protein in iron and hemin metabolism researches.

Key words: R. anatipestifer Internal reference protein RecA protein Polyclonal antibody Western blot

收稿日期: 2015-01-05 出版日期: 2015-06-25

ZTFLH:

Q819

基金资助:

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

通讯作者: 刘马峰, 程安春 E-mail: liumafengra@163.com;chenganchun@vip.163.com

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引用本文:

廖何斌, 刘马峰, 程安春. 鸭疫里默氏杆菌RecA作为一种内参蛋白的评价[J]. 中国生物工程杂志, 2015, 35(6): 26-31.

LIAO He-bin, LIU Ma-feng, CHENG An-chun. The Assessment of RecA Acted as an Internal Reference Protein in R. anatipestifer. China Biotechnology, 2015, 35(6): 26-31.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20150605 或 https://manu60.magtech.com.cn/biotech/CN/Y2015/V35/I6/26

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