肿瘤相关抗原17-1A在毕赤酵母中的表达与鉴定

目的利用Pichia pastoris酵母表达系统表达糖基化的肿瘤相关抗原17-1A，为进一步设计肿瘤蛋白疫苗提供研究基础。方法通过RT-PCR从小鼠肾组织中扩增17-1A的cDNA，将其定向克隆到pPICZαA质粒上，获得重组质粒pPICZαA-17-1A，测序正确后，重组质粒电转化到Pichia pastoris酵母菌株GS115中，在甲醇诱导下，利用其AOX I基因的α信号肽，分泌表达17-1A抗原糖蛋白，并对获得的蛋白进行SDS-PAGE及Western blot分析鉴定。结果构建了pPICZαA-17-1A重组质粒，通过电转化将目的基因整合人酵母基因组中，重组毕赤酵母表达能够表达17-1A抗原并检测到抗原发生了糖基化。结论能够通过毕赤酵母表达系统稳定表达糖基化的肿瘤相关抗原17-1A。

Expression and Characterization of Mouse 17-1A Antigen from Pichia pastoris

OBJECTIVE: This is a ground work in preparation for the development of a vaccine against tumor, in which 17-1A glucoprotein, a tumor associated antigen (TAA), was cloned and expressed in Pichia pastoris. METHODS: The cDNA of 17-1A gene was cloned from nephridial tissue of mouse embryo by RT-PCR and subsequently inserted into pPICZaA plasmid after digestion with Xho I and Xba I. The linearized recombinant plasmid was transformed into Pichia pastoris strain GS115. 17-1A glucoproteins were secreted into culture supernatant through alpha-signal peptide of AOX I after inducation with methanol. The protein products were analyzed and characterized by SDS-PAGE and Western-blot. RESULTS: The plasmid pPICZalphaA-17-1A was constructed and verified by sequenceing. 17-1A glucoproteins were expressed stably and glycosylated in Pichia pastoris. CONCLUSION: Glycosylated tumor associated antigen 17-1A can be expressed in Pichia pastoris.