Construction, Expression And Purification Of The Recombinant Human Growth Hormone In P. Pastoris And Its Biological Activity

Human growth hormone(hGH) has important effect on the growth of human body. Its deficiency can lead to human disease. Drugs containing hGH can be used to treat growth hormone deficiency disease. At present, Growth hormone is mainly producted in E.coli., which was constructed by recombinant DNA technology. The yeast belongs to the eukaryotic, and has more advantages than E. coli in the expression and purification of eukaryotic proteins. We chose pichia pastoris as host, and obtained rhGH by the process of construction, espressin and purification, and studied its biological activity.In this paper, we download the cDNA sequence of hGH from genebank to design, optimize, and synthesis, and employed three kinds of plasmid(pPIC9, pPIC9 K, pPICZα-A) as expression vector of target gene, and integrated them into three kinds of pichia pastoris(X-33, GS115, SMD1168) by electroporation, and screened the different combinations. We applyed the highest combination to optimize fermentation conditions of 1 L flask and 3 L fermentation, and isolated and purified the fermentation of 3 L fermentor, and finally studyed the function of rhGH in vitro. The results were as follows:1. The vector host combinations had different effect on the expression of rhGH, the expression of rhGH in combination(X-33 and pPICZα, X-Zα) was the highest.2. We investagated the effect of some factors on the expression amount in 1 L shaking flask and 3 L fermentation tank. The condition of highest expression quantity was 23℃、p H 6.0 in 1 L shaking flask, and the expression of rhGH was the highest under the conditions of 23℃、pH 6.0 in 3 L fermentation tank when used basal salts medium(BSM). The highest expression of rhGH was higher(300.0 mg/L) when being compared with the expression of rhGH in E.coli.3. We finished the purification of rhGH. We estimated protein yield above 60% and the purity more than 90% by high-speed refrigerated centrifuge, and the technology of two aqueous phase extraction, and DEAE resin chromatography.4. We studied the functions of rhGH, and found that rhGH had a certain role in promoting growth and proliferation of Bel-7402 and QSG-7701.This engineer yeast expressed high amount of rh GH, and its purification step was also relatively simple. Our experiment laid the foundation of industrialization and function research for rhGH.