Cloning, Prokaryotic Expression And Characteristics Analysis Of Yak Growth Hormone Gene

Growth hormone(GH)secretion by single-chain polypeptide hormone, a wide exist in vertebrates. GH can promote growth and development of animals with increased milk yield, wool production and improve the feed efficiency and lean meat percent- age.Yak is one of a special breed of the Qinghai-Tibet Plateau. It is main production and means of subsistence of the local herdsmen.Yak is a breed by a long-term natural selection and artificial selection. Low levels of yak production, milk annual output about 450 kg, adult body weight 210-270kg. Based on growth-promoting role of growth hormone, it is necessary to conduct a study of yak growth hormone (YGH) gene.In this article, specific primers were designed and 707bp DNA sequence was cloned by RT-PCR from yak pituitary, in which included 654bp the total coding sequence (CDS) of Yak growth hormone gene and it encoded 217 amino acids(GeneBank Accession No. EF154193). The sequence analysis of YGH showed that the amino acids sequences shared 100%, 99.1%, 98.6%, 88.9%, 88.5%, 88.9% and 89.4% sequence similarity with cattle, goat, sheep, horse, pig, cat and dog respectively, the result showed the growth hormone was very conservative; The protein have two strong hydrophobic regions and two strong transmembrane helixs, it is a secreted protein; The protein second structure main isα-helix and irregular curly. The result of the protein three-dimensionalsolution structure prediction is composed of 28-215 amino acids, which had the highest similar with second structure ; YGH protein had a good antigen reactivity.A pair of specific primers contain enzyme sites were designed according to the YGH gene sequence. The YGH gene was amplified by PCR and linked PET-28a (+) together by ligase, and then transformed into E. coli BL21 (DE3) plysS and induced by IPTG. The expression product was ananlyzed by SDS-PAGE and the expression condition was optimized. The recombinant protein was purified and identified by Western blotting. The results indicated that fusion protein was expressed in E.coli mainly as the inclusion bodies form.The optimum expression of YGH fusion protein was obtained with 1.0mM IPTG for 5 hours at 40℃. Western blotting analysis showed the fusion protein has a good antigen reactivity.