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Production of recombinant bovine leptin in Pichia pastoris yeast, evaluation of biological activity and effect on sexual maturation of zebu heifers

Abstract

Nutrition exerts a clear effect over reproductive development of bovines, being leptin a possible link between nutrition and reproduction, by signalizing body fatness to the hypothalamus the body and exerting permissive effect on the attainment of puberty. The objective of this study is to clone the bovine leptin coding region and to produce the recombinant protein in Pichia pastoris yeast, in order to evaluate the effect of recombinant leptin application over sexual maturation of zebu heifers. The bovine leptin coding region will be obtained by PCR amplification of cDNA transcribed from total RNA extracted from adipose tissue of Nellore heifers. The gene will be cloned in an expression vector and inserted by electroporation in Escherichia coli bacteria TOP10TM. After colony selection, the plasmids will be isolated and sequenced to confirm that the insert is aligned with the secretion signaling sequence. Lines of P. pastoris will be transformed be electroporation for secretion of leptin on the culture medium, and the leptin will be purified by three phases of chromatography, dialyzed and lyophilized. The obtained leptin will be applied at four different doses in 16 pre-pubertal heifers during two days for determining the proper dosage for long term experiments. Thirty six prepubertal heifers will be used to test the hypothesis that the elevation of plasmatic concentration of leptin is capable to induce the beginning of puberty in Nellore heifers. The animals will be randomly allocated in three groups: high energy diet (H), low energy diet (L) and low energy diet with leptin application (LL). The heifers will be monitored twice a week for BW, body condition score, diameter of dominant follicle, and presence of corpus luteum, until the detection of the first ovulation. The age at puberty will be considered as the age at the first detection of a CL, assessed as being functional by serum progesterone concentration above 1 ng/ml by two consecutive collections. (AU)

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