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

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。

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.