猪水肿病毒素B亚单位的表面表达

Cell-Surface Display of the B Subunit Gene of the Shiga-Like Toxin Type Ⅱ Variant

用聚合酶链式反应 (PCR)技术 ,从大肠杆菌PPSLT_ⅡeB中扩增出猪水肿病素 (SLT_Ⅱe)的B亚单位基因 (slt_ⅡeB)的 2 0 4bp的编码序列。将此PCR扩增产物按预定的阅读框架克隆进质粒表达载体PZSPAX ,并转化到大肠杆菌HB10 1中 ,筛选到含有slt_ⅡeB的重组质粒PZBSPAX。将重组质粒PZBSPAX进行序列分析 ,结果表明 :重组质粒PZBSPAX中的插入序列与发表的slt_ⅡeB是一致的 ,证明PZBSPAX就是含有slt_ⅡeB的重组表达质粒。含有重组质粒PZBSPAX的大肠杆菌HB10 1命名为重组大肠杆菌PZBSPAX。通过在不同温度条件下、不同培养时间情况下、不同IPTG诱导条件下 ,利用荧光抗体染色证明重组大肠PZBSPAX可表面表达预期的融合蛋白PP_SLT_ⅡeB_SPAX ;利用SDS_PAGE、Western_blot对重组大肠杆菌PZBSPAX的表达进行了分析 ,结果表明重组大肠杆菌PZBSPAX表达的融合蛋白PP_SLT_lleB_SPAX与预期大小一致 。

The B subunit gene(slt_ⅡeB,204bp)of the Shiga_like toxin H variant (SLT_Ⅱe) was amplificated from the Escherichia coli PPSLT_ⅡeB by polymerase chain reaction (PCR).The PCR product of slt_ⅡeB was choned into PZSPAX plasmid between the EcoRⅠ and BamHⅠ sites.Restriction endonucleases analysis were used to identify the recombinant plasmids,and the recombinant plasmids,PZBSPAX were obtained.The PZBSPAX sequences were analyzed (with the MicroGenic computer program),and compared with the published sequences of slt_Ⅱe.The data of sequences for the slt_ⅡeB genes were shown that they were in accord with the sequences of slt_Ⅱe,and demonstrated that the recombinant plasmid PZBSPAX was slt_ⅡeB clone.The PZBSPAX was transferred into E.coli .HB101 to express fusion protein.The effects of selected culture conditions were examined.lncubation time,incubation temperature,and IPTG conditions were investigated.The data of Fluorescent Antibody technique demonstrated that SLT_ⅡeB were display on cell surface of E.coli.HB101.The data of SDS_PAGE analysis and Westem_blot conformed the fusion protein PP_SLT_ⅡeB_SPAX is we interested,and the fusion protein PP_SLT_ⅡeB_SPAX have the same antigenicity of SLT_ⅡeB.