前列腺干细胞抗原与热休克蛋白70羧基端结构域的融合表达及纯化
Expression and purification of prostate stem cell antigen and heat shock protein 70 carboxyl-terminus
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摘要: 目的 构建前列腺干细胞抗原(prostate stem cell antigen,PSCA)及热休克蛋白70(heat-shock protein,HSP70)羧基端结构域的重组表达质粒,对重组融合蛋白进行诱导、表达及纯化,为肿瘤疫苗的研究奠定基础。 方法 克隆人HSP70羧基端基因及PSCA基因,构建重组表达载体pET21-PSCA-HSPC,重组融合蛋白经(isopropyl-β-D-thiogalactoside,IPTG)进行诱导表达,利用单克隆抗体进行Western blot鉴定,最后进行蛋白纯化。 结果 成功构建了重组表达质粒pET21-PSCA-HSPC;SDS-PAGE结果显示目的蛋白以可溶性形式存在,经镍柱亲和层析、苯基柱疏水及凝胶过滤柱纯化后,重组融合蛋白纯度在95%以上。 结论 该研究成功实现了PSCA与HSP70羧基端结构域的可溶性表达。Abstract: Objective To lay the foundation for the development of tumor vaccine by constructing,expressing and purifying recombinant plasmid of prostate stem cell antigen（PSCA） and heat shock protein 70 carboxyl-terminus（HSPC70）. Methods Recombinant plasmid pET21-PSCA-HSPC was constructed by cloning PSCA gene and carboxyl-terminus gene of HSP70.Expression of recombinant plasmid pET21-PSCA-HSPC was induced with IPTG and identified by Western blot analysis and then recombinant plasmid pET21-PSCA-HSPC was purified. Results The recombinant plasmid pET21-PSCA-HSPC was successfully constructed.SDS-PAGE showed PSCA-HSPC in a soluble form.The purity of recombinant protein PSCA-HSPC was over 95% after purification with Ni-ATA,phenyl sephrose FF and Superdex 75. Conclusion Recombinant fusion protein PSCA-HSPC is expressed in soluble form.
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