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

作　　者：董磊[1] 张晓鹏[2] 易绍琼[2] 任军[2] 侯利华[2] 付玲[2] 于长明[2] 陈薇[2] 

机构地区：[1]空军总医院临床检验中心,北京100142 [2]军事医学科学院生物工程研究所,北京100071

出　　处：《军医进修学院学报》2012年第12期1296-1298,1311,共4页Academic Journal of Pla Postgraduate Medical School

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。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.

关 键 词：前列腺干细胞抗原 热休克蛋白 重组融合蛋白类 

分 类 号：R34[医药卫生—基础医学]