人锌转运体8强免疫原性片段的预测及原核表达鉴定

Prediction of highly immunogenic fragment of human zinc transporter 8 and identification of its expression in prokaryotic system

目的筛选人锌转运体8(Zinc transporter8,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.coliBL21(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Westernblot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32000,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-Thiogalactopy-ranoside（ IPTG）. Expressed products were analyzed using SDS-PAGE and identified by Western 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 × 103 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.