Expression Of Sheep KISSPEPTIN-53 Gene And Bioactivity Detection Of Expression Products

Kisspeptin(Kp,for short)is a neuropeptide encoded by the KISS1 gene and plays an extremely important role in the development of reproductive organ,puberty onset,seasonal estrus,spermatogenesis,follicle maturation and ovulation and other reproductive endocrine regulation process in mammals.Kisspeptin has potential application value in animal reproduction,but commercial kisspeptin drugs are now very expensive.The aim of this study is to express Kp53 using Pichia pastoris and detect its bioactivity,laying foundation for its application in animal reproduction.The coding sequence of sheep Kp53 was optimized based on the preferred codons of Pichia pastoris and cloned into p PICZ?A plasmid to construct a recombinant expression vector p PICZ?A-Kp53.The correct recombinant plasmid and empty plasmid were linearized with Sac I and electrotransformed into Pichia pastoris GS115.Positive transformants were induced by methanol and expressed.The total protein concentration in the fermentation supernatant was detected by BCA method.The expression products were identified by Tricine-SDS-PAGE and Western Blot.In order to increase the amount of expression,the experimental conditions including induction time,induction temperature and methanol concentration were optimized.The target product was purified by Ni affinity column chromatography.Finally,Kunming mice were injected intraperitoneally with purified products and its bioactivity is assessed by its effects on the levels of LH and FSH.The Pichia pastoris gene engineered bacterium is capable of expressing the sheep Kp53,and the target product size is approximately 9 k Da,which was in accordance with the theoretical molecular weight.The better induction conditions for shake flask fermentation were induction time 72 h,temperature 26°C,and methanol concentration 1%.Before optimization of the inducing conditions,the expression amount of the target product in the fermentation supernatant at 72 h is 0.59g/L,and the optimized expression amount reached 0.72g/L.The higher quality target expression product was obtained by purification.Compared with the saline-treated control,one hour after intraperitoneal injection of the purified product,the concentration of LH in the serum of mice was significantly increased,and there was no significant difference in FSH concentrations.The bioactive sheep Kp53 can be expressed in Pichia pastoris GS115,which lays the foundation for its application in animal production and reproduction.