摘要
目的建立毕赤酵母表达PSA系统,获高活性人前列腺特异性抗原,为临床相关疾病的监测,诊断和治疗提供关键材料。方法构建表达载体pPICZα-mPSA质粒,采用电转法,将pPICZα-mPSA质粒转染毕赤酵母X33细胞,Zeocin筛选转染细胞,甲醇诱导X33细胞表达rPSA,ELASA试剂盒筛选高表达rPSA的细胞株。结果构建了表达载体pPICZα-mPSA,获得了高表达rPSA的酵母X33细胞株,表达量为1.2mg/L。结论应用酵母表达体系成功表达人前列腺特异性抗原。
Objective To study the biology character of PSA, and to provide PSA for clinic detection and therapeutic use. Methods A 0.730-kb fragment was amplified by PCR using the primer and pGEM-PSA. This fragment was ligated into TA vector, the resultant plasmid pGEM-mPSA was constructed. The pGEM-mPSA and pPICZ α -C were disgested by Xho I and Xba I, resulted in an inframe fusion of the mature PSA sequence with the yeast a-factor signal sequence. The resultant plasmid-pPICZ α -mPSA was electroporated in integration into the 5＇AOX1 region of the chromosome of Pichia pastoris strain X33. Through Zeocin selection, the protein was induced to express by methanol, and one transformant X33 which express rPSA in high level was chosen by ELASA. Results The expression plasmid pGEM-mPSA was constructed and the X33 cell strain which expressed PSA was obtained. The final yield of active rPSA from Pichia pastoris was 1.2mg/L. Conclusion Prostate-specific antigen was expressed in Pichia pastoris, which facilitate the study of PSA.
出处
《中国男科学杂志》
CAS
CSCD
2005年第5期23-25,共3页
Chinese Journal of Andrology
关键词
前列腺特异性抗原
毕赤酵母
基因表达
prostate-specific antigen
Pichia pastoris
gene expression
