| In recently, it had been got much focus on producing functional proteins by biotechnology method. Thus it was much more important to select appropriate expression system which can efficiently express gene coding valuable protein such as medical protein etc. The yeast had more advantages than other expression systems. Many scientists expressed heterogeneous gene effectively in yeast using DNA recombinant technology and yeast expression system. Pichia pastoris expression system was one of the widely used systems in research and application fields.The purpose of this study is to express heterologous proteins in our lab in Pichia pastoris system and to investigate the activity of these proteins. In the expression of HPV16 XJ E6 experiment, the 5' primer and 3' primer were designed according to HPV16 XJ E6 gene cloned in our lab, in which two restriction endonucleases (EcoR I & Xba I) were added respectively, meanwhile, the terminal condon of HPV16 XJ E6 gene was mutated so that the product of HPV16 XJ E6 protein could take 6×His tag that made purification of HPV16 XJ E6 protein easily. After amplified with PCR, HPV16 XJ E6 gene was cloned into pMD18-T vector. Then the HPV16 XJ E6 gene from positive clone after identified with restriction endonucleases digestion were retrieved. Following, HPV16 XJ E6 gene was cloned into shuttle plasmid pGAPZαA. The recombinant plasmid were linearized and transformed into Pichia pastoris and screened positive clone with zeocin+. At last, the supernatant of medium was analyzed by SDS-PAGE after fermentation. The results indicated that the HPV16 XJ E6 protein has been expressed successfully in Pichia pastoris eukaryotic expression system, and the MW of HPV16 XJ E6 protein is 20 kDa. In the expression of HPV16-E7 experiment, the 5' primer and 3' primer were designed according to HPV16-E7 gene sequence cloned from the cervical carcinoma of Xinjinag Uygur women, two restriction endonucleases' site (EcoR I & Xba I ) were added into those two primers respectively. After amplified with PCR, HPV16-E7 gene was cloned into pMD18-T vector, then the identified HPV16-E7 gene was cloned into shuttle plasmid pGAPZαA. The recombinant plasmid were linearized and transformed into Pichia pastoris. Before fermentation the positive clone was selected with zeocin+. At last, the supernatant of medium was analyzed by SDS-PAGE and western blotting after fermentation. The results indicated that the HPV16-E7 protein has been expressed successfully in Pichia pastoris expression system, and the MW of HPV16-E7 protein is about 22 kDa and 88 kDa . This could lead to further investigation about function of HPV16-E7 protein development.As for Cecropin-XJ, the cDNA of Cecropin-XJ was obtained from RT-PCR reaction with total RNA isolated from silkworm and cloned into pMD18-T vector, then Cecropin-XJ gene was subcloned into pGEX-4T-1 with EcoR I & Sal I. After that, the Cecropin-XJ was chiped off from pGEX-4T-1-Cecropin-XJ with EcoR I & Not I and constructed into pGAPZαA. As following the recombinant pGAPZαA-Cecropin-XJ was linearized by AvrII and transformed into Pichia pastoris SMD1168 strain with LiCl method transformation. Screening with Zeocin we got positive clones and those clones were fermented in flask. It was found that the Cecropin-XJ protein could be secreted into the culture leading by α-factor came from pGAPZαA for SDS-PAGE analysis. The MW of Cecropin-XJ is about 14.4-20.1 kDa and 31.0-43.0 kDa. Antibacterial activities were also observed and heat-stability was characterized. By Agar Hole Pervasion experiment, this polypeptide of Cecropin-XJ exhibited strong anti-bacterial activity. It could kill S. aureus that could lead disease to human being or other creatures. On the other hand, the Cecropin-XJ polypeptide still keep anti-bacterial activity after boiled for 2 hours. Subsequently, the properties of recombinant Cecropin-XJ isolated from Xinjing silkworm and expressed in Pichia yeast was investigated. According to Agarose Diffusion Assay, this recombinant Cecropin-XJ has...
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