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纸质出版:2010
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吴玥,刘宝英,徐 静,姜友昭,陈兵.人锌转运体8强免疫原性片段的预测及原核表达鉴定[J].陆军军医大学学报(原第三军医大学学报),2010,32(11):1162-1164.
Wu Yue,Liu Baoying,Xu Jing,Jiang Youzhao,Chen Bing.Prediction of highly immunogenic fragment of human zinc transporter 8 and identification of its expression in prokaryotic system[J].J Amry Med Univ (J Third Mil Med Univ),2010,32(11):1162-1164.
吴玥,刘宝英,徐 静,姜友昭,陈兵.人锌转运体8强免疫原性片段的预测及原核表达鉴定[J].陆军军医大学学报(原第三军医大学学报),2010,32(11):1162-1164. DOI:
Wu Yue,Liu Baoying,Xu Jing,Jiang Youzhao,Chen Bing.Prediction of highly immunogenic fragment of human zinc transporter 8 and identification of its expression in prokaryotic system[J].J Amry Med Univ (J Third Mil Med Univ),2010,32(11):1162-1164. DOI:
目的 筛选人锌转运体8(Zinc transporter 8,ZnT8)中强免疫原性片段,并对其进行克隆表达及鉴定。 方法 采用生物信息学软件预测ZnT8结构特征,筛选出具有强免疫原性的N末端片段ZnT8(N),经PCR(Polymerase Chain Reaction)技术扩增,构建表达质粒pET32a-ZnT8(N),转化入E.coli BL21(DE3)中,异丙基-β-D-硫代半乳糖苷(IPTG)诱导目的蛋白的表达,并对表达产物进行SDS-PAGE分析及Western blot鉴定。 结果 从ZnT8分子中筛选出具有140个氨基酸的肽段ZnT8(N),重组质粒pET32a-ZnT8(N)经酶切和测序鉴定证实构建成功。转入大肠杆菌进行表达,表达产物相对分子质量(Mr)约32 000,与预期值相符,并经免疫印迹检测鉴定为阳性,融合蛋白的表达量约占菌体蛋白总量45%。 结论 筛选获得了人胰岛细胞来源的ZnT8强免疫原性片段,并在原核系统中成功表达。
Objective To screen highly immunogenic fragment of human zinc transporter 8 (ZnT8) and to identify its expression in prokaryotic system. Methods Characteristics of ZnT8 structure were detected using bioinformatics software. A highly immunogenic fragment of ZnT8 was screened at the N-terminal and amplified by polymerase chain reaction (PCR). Expression plasmid pET32a-ZnT8(N)was constructed and transformed to E.coli BL21(DE3). Expression of target protein was induced by isopropyl β-D-1-Thiogalactopyranoside(IPTG). Expressed products were analyzed using SDS-PAGE and identified by Weste
rn blotting. Results The screened ZnT8 (N) fragment had 140 amino acids. Recombinant pET32a-ZnT8 (N) was successfully constructed
which was confirmed by restriction analysis. The relative molecular weight of expressed products was about 32×10
3
after transformed into E.coli
which was consistent with the expected value. Western blot analysis showed that the positively expressed products contained over 45% of the total fused protein. Conclusion A highly immunogenic fragment of ZnT8 can be screened from human islet cells
which is expressed in the prokaryotic system.
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