前列腺特异性膜抗原基因片段在酿酒酵母中的诱导表达及意义

Inducible Expressing and Identification of Prostate Specific Membrane Antigen in Saccharomyces cerevisiae

[目的]分析前列腺特异性膜抗原(PSMA)基因片段在酿酒酵母中的诱导表达情况,以便进一步探讨重组人PSMA的免疫活性并完成抗前列腺癌PSMA蛋白疫苗的制备.[方法]应用基因重组技术将人PSMAcDNA序列克隆入酿酒酵母穿梭诱导表达载体pYES2/NT,构建重组酵母真核表达质粒pYES2/NT-PSMA,转化酿酒酵母菌株INVSc1,尿嘧啶筛选出阳性克隆转化子,经半乳糖诱导表达后进行菌体全蛋白的Western blot检测.[结果]成功构建了PSMA的酵母真核表达重组子pYES2/NT-PSMA,并在酿酒酵母INVSc1中得到有效表达,Western blot显示两个特异性的人重组PSMA全蛋白片段,相对分子质量分别为Mr=100×103及Mr=85×103.[结论]人PSMA基因片段能够在酿酒酵母中表达并进行翻译后糖基化修饰,为下一步进行抗前列腺癌PSMA疫苗的研制奠定了良好基础.

[Objective] To observe the inducible expression of gene fragment from human prostate specific membrane antigen （PSMA） in Saccharomyces cerevisiae, to explore the immune activity of the acquired recombinant human PSMA and to develop PSMA vaccine for prostate cancer in stepwise. [Methods] The cDNA encoding human PSMA （hPSMA） sequence was subcloned into yeast shuttle inducible expression vector pYES2/Nt; the recombinant plasmid pYES2/NT-PSMA was constructed. Saccharomyces cereviseoe INVScl was transformed and the positive transformants were selected in the medium of drop-out uracil. The positive transformants were induced by galactose to express recombinant hPSMA （rhPSMA）. The whole extract proteins of the lysis ceils were analyzed using Western blot analysis. [Results] pYES2/NT-PSMA was established successfully. Two specific rhPSMA were identified by the Western blot analysis; Mr=100×10^3 and Mr=85×10^3, respectivley. [Conclusions] Gene fragment from human PSMA could be expressed and processed post-translational modification of glycosylation in Saccharomyces cereviseoe, which laid foundation to develop PSMA vaccine for prostate cancer substantially.