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| Prokaryotic Expression of Canine (Canis lupus) Prorelaxin Gene and Preparation of Its Polyclonal Antibody |
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Abstract Abstract Relaxin, as a polypeptide hormone of insulin superfamily, plays importantly regulatory roles in the reproductive and non-reproductive physiology processes. Besides, relaxin appeared in peripheral blood, which had been seen as a sign of pregnancy in canine (Canis lupus) and other some animal. To prepare a polyclonal antibody against canine prorelaxin, the cDNA fragment of canine prorelaxin gene was amplified from the recombined plasmid pMD19-T-Cpreprorelaxin1 containing cDNA fragment of canine preprorelaxin gene by PCR, and cloned into the prokaryotic expression plasmid pET28a. Secondly, the recombined expression plasmid of canine prorelaxin was transformed into Escherichia coli BL21 (DE3) and induced by isopropyl-β-d- thiogalactoside (IPTG), to harvest the fusion protein which were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Finally, the polyclonal antibodies were harvested after injecting the rabbits (Oryctolagus cuniculus) with the purified fusion protein of canine prorelaxin emulsified with the Freund's complete adjuvant as the immune antigens, and analyzed by enzyme linked immunosorbent assay (ELISA) and Western blot. The results demonstrated that the fusion protein with the consistent molecular weight of 22 kD indicated by SDS-PAGE, was highly expressed as the form of inclusion bodies and positively reacted with the His-labeled antibody by Western Blot. The harvested anti-serum with more than 1∶80 000 titered by ELISA, reacted specifically with the purified fusion protein of canine prorelaxin. This study would lay a potential foundation for further study and clinical application of canine prorelaxin.
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Received: 21 September 2016
Published: 31 March 2017
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