Expression Of Pig Growth Hormone In Pichiapastoris And Fermentation

Porcine Growth Hormone(pGH)is a kind of polypeptide hormones,synthesized and secreted by the porcine anterior pituitary acidophil,which can promote adipose decompose,improve muscle weight and protein deposition.It can also improve the daily gain of pig,the percentage of carcass lean meat,the area of eye muscle and can largely improve the quality of the pig carcass.If used in the pig breeding industry,the pGH will bring great social and economic value.In this study,total RNA was extracted from the porcine pituitary,the cDNA sequence of porcine somatotropin was obtained by reverse transcription amplification,the gene sequence was cloned into vector pPICZA,pPICZαA,constructed the Pichia pastoris expression vector pPIC ZA-pGH,pPICZαA-pGH.After induced expression,detected the expression of p GH in Pichia pastoris by SDS-PAGE and Western-blot,the results is that pGH protein could insoluble in the cells,p GH protein in the low expression of extracellular,only 78 mg/L.In order to improve pGH expression levels,the pgh gene of porcine growth hormone was optimized by software,the optimized gene sequence was constructed as a recombinant pGH Pichia pastoris.The results showed that the pGH protein was still in the form of insoluble protein in Pichia pastoris,and the soluble pGH protein was few.To optimize the content of intracellular soluble pGH protein,pGH expression vector pPIC ZA-pGH(optimized)-HDEL located in endoplasmic reticulum was constructed and co-expressed with molecular chaperone Bip and PDI X33-pGH(optimized)-Bip-PDI,pGH protein was detected in the intracellular precipitated samples of co-expressed strains,but the content of intracellular soluble p GH protein was still low,indicating that the chaperones Bip and PDI could lead to the degradation of insoluble pGH protein without improving the solubility of p GH protein.The co-expression pGH and the molecular chaperone protein Ssa1,Sis1 was detected by SDS-PAGE and Western-blot.The results showed that the expression of pGH protein was significantly higher than that of the control group.The expression of pGH protein was detected by SDS-PAGE and Western-blot.And the soluble p GH protein was 70 mg / L.Indicating that the molecular chaperone protein Ssa1,Sis1 can improve the solubility of p GH protein in Pichia pastoris cells.In addition,the expression of p GH protein in Pichia pastoris was studied.The expression level of p GH protein was the highest at 20 ℃,48 h and p H 6.0,and the expression level was 340 mg / L.Using pGH protein to stimulate porcine intestinal epithelial cell proliferation,MTT assay was performed at 48 hours,the proliferation rate of 125 μg / L soluble pGH protein treated group was 249% compared with the control group,and the proliferation rate of the pGH protein treated group was 211% compared with the control group.The soluble pGH protein and the purified pGH protein a ll have the role of promoting porcine intestinal epithelial cell proliferation.In summary,codon optimization is necessary for the intracellular expression of pGH protein in Pichia pastoris.The co-expression of the chaperones Ssa1 and Sis1 can improve the solubility of p GH protein in Pichia pastoris cells,and to lay the foundation for the subsequent study on the soluble expression of p GH protein in Pichia pastoris.