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| The Assessment of RecA Acted as an Internal Reference Protein in R. anatipestifer |
| LIAO He-bin1,2,3, LIU Ma-feng1,2,3, CHENG An-chun1,2,3 |
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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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 RecAP 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 RecAP 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.
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Received: 05 January 2015
Published: 25 June 2015
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