Abstract: Based on the published mRNA sequence of Gallus gallus gonadotropin releasing hormone 1 （GnRH1） gene, a pair of primers was designed to clone the GnRH1 gene coding sequence of Jinghai Yellow chicken by RT-PCR. GnRH1 gene was cloned in to pET32a vector for expression in E.coil BL21 and bioinformatics analysis. Finally, a 352 bp gene was cloned, which contained CDS region, promoter region and part of the 3′region. It was found that the GnRH1 gene nucleotide sequence shared 93%, 81%, 54%, 58%, 61%, 76%, 76%, 59%, 76% and 66% identities with Meleagris gallopavo, Columba livia, Homo sapiens, Bos taurus, Babirussa, Capra hircus, Ovis aries, Pantholops hodgsonii, Equus caballus and Rattus norvegicus, respectively. Restriction and sequencing analysis confirmed that prokaryotic expression vector pET32a-GnRH1 was successfully constructed. SDS-PAGE analysis showed that the recombinant plasmid was successfully expressed in E.coli BL21 and the molecular weight was approximate 25 to 28 ku. The expression quantity in supernatant was higher than that in the precipitant. Western blotting analysis confirmed that the protein expressed in E.coli BL21 was the interest protein and expressed in soluble form with high activity. The results showed that GnRH1 gene of Jinghai Yellow chicken was cloned and GnRH1 prokaryotic expression system was constructed successfully. The interest protein was expressed successfully, which laid the foundation for further studies.

Key words:

Jinghai Yellow chicken; gonadotropin releasing hormone 1; cloning; prokaryotic expression