CAJ | 학술논문

目的在毕赤酵母细胞中表达PSA,为临床的检测和治疗提供材料.方法经RT-PCR获得PSA全长cDNA并测序.将编码成熟PSA 237氨基酸的cDNA序列插入毕赤酵母表达载体pPICZα-C,该载体含有AOX1启动子和α-factor信号肽序列,构建表达载体体pPICZαC-m PSA.甲醇诱导X33细胞表达rPSA,ELASA试剂盒筛选高表达rPSA的细胞株.结果获得了人PSA全长序列,构建了表达载体pPICZα-m PSA.获得了高表达rPSA的酵母X33细胞株,表达产量为1.2 mg/L.结论建立了重组人前列腺特异性抗原的毕赤酵母表达体系,规模化的生产有助于PSA的生物学特性的研究和临床检测和治疗的应用.
목적재필적효모세포중표체PSA,위림상적검측화치료제공재료.방법경RT-PCR획득PSA전장cDNA병측서.장편마성숙PSA 237안기산적cDNA서렬삽입필적효모표체재체pPICZα-C,해재체함유AOX1계동자화α-factor신호태서렬,구건표체재체체pPICZαC-m PSA.갑순유도X33세포표체rPSA,ELASA시제합사선고표체rPSA적세포주.결과획득료인PSA전장서렬,구건료표체재체pPICZα-m PSA.획득료고표체rPSA적효모X33세포주,표체산량위1.2 mg/L.결론건립료중조인전렬선특이성항원적필적효모표체체계,규모화적생산유조우PSA적생물학특성적연구화림상검측화치료적응용.
[Objective] To produce biologically active recombinant human (rh) prostate-specific antigen in a Pichia pastoris for clinic detection and therapeutic use. [Methods] The cDNA encoding the full length human PSA was obtained by RT-PCR and was sequenced. By inserting cDNA encoding mature hPSA 237 amino acid residues into Pichia pastoris expression vector pPICZα-C consisting of AOX1 promoter and the yeast α-factor signal sequence, the recombinant expression plasmid pPICZα-mPSA was constructed and transformed to yeast host strain X33. The protein was induced to express by methanol and the one transformant which expressed rPSA in high level was screened out by ELISA Kit. [Results] The cDNA encoding the full human PSA was obtained and the expression plasmid pPICZα-mPSA was constructed and the X33 cell strain which expressed rPSA in high level was obtained, and the final yield of active rPSA from Pichia pastoris was 1.2mg/L. [Conclusion] A method of expression of mature PSA in Pichia pastoris has been established. Large-scale production of active rPSA will be useful in the study of PSA biology character, clinic detection and therapeutic use.