人锌转运体8强免疫原性片段的预测及原核表达鉴定 - 第三军医大学学报.docVIP

人锌转运体8强免疫原性片段的预测及原核表达鉴定 吴 玥，刘宝英，徐 静，姜友昭，陈 兵(400038重庆，第三军医大学西南医院内分泌科) 【摘要】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，E. coli BL21(DE3)中，异丙基-β-D-硫代半乳糖苷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 筛选获得了人细胞来源的片段，并在表达；；Prediction of highly immunogenic fragment of human zinc transporter 8 and identification of its expression in prokaryotic system Wu Yue, Liu Baoying, Xu Jing, Jiang Youzhao, Chen Bing (Department of Endocrinology, Southwest Hospital, Third Military Medical University, Chongqing 400083, China) 【Abstract】 Objective To screen for 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）. lasmid 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）. xpressed products were analyzed using SDSand identified by Western blot. Results The ZnT8 (N) fragment was screened from 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 32×103 after transformed into E. coli, which was consistent with the expected value. Western blot 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. 【Key words】ZnT8；prokaryot expression；mmunogenicity Clinical Research Foundation of Third Military Medical University Corresponding author：Chen Bing；tel：86-23E-mail：chenbing3@ 【基金项目】第三军医大学临床科研基金（2008XG20） 【通