EHEC O157∶H7志贺毒素ⅡA1亚单位蛋白的构建表达与免疫原性鉴定

Construction,expression,and immunogenicity identification of STX2A1 subunit protein of EHEC O157∶H7

目的构建表达GST-STX2A1融合蛋白并鉴定其免疫原性。方法通过PCR反应从EHEC O157∶H7EDL933标准株菌中扩增STX2A1基因,将此基因克隆到GST融合蛋白原核表达载体PGEX-6p-1,得到阳性克隆PGEX-6p-1/STX2A1,转化大肠杆菌BL21,IPTG诱导GST-STX2A1融合蛋白的表达,采用亲和层析纯化该蛋白,SDS-PAGE分析其表达量、表达形式和纯度;用纯化的融合蛋白免疫Balb/c小鼠,免疫双向扩散鉴定抗血清效价,Western blotting鉴定抗血清的特异性。结果成功构建重组质粒PGEX-6p-1/STX2A1,经DNA测序证实插入序列与设计完全一致,并在大肠杆菌中表达出Mr约为5.3×104大小的可溶性目的蛋白,经亲和层析纯化后纯度可达90%,免疫Balb/c小鼠产生的抗血清效价为1∶16,Western blotting表明可与天然STX2A蛋白反应。结论在大肠杆菌中成功表达了可溶性的GST-STX2A1融合蛋白,该蛋白具有较好的免疫原性,为O157∶H7亚单位疫苗及制备抗STX2A1的单克隆抗体提供了必要的物质基础。

Objective To express GST-SFX2A1 fusion protein and identify the immunogenicity of the protein. Methods The STX2A1 gene was amplified from ,the EHEC O157：H7 EDL933 type strain with specific primers. The PCR product was inserted into the prokaryotic expression plasmid PGEX-6p-1 and confirmed by DNA sequencing. The constructed prokaryotic expression vector PGEX-6p-1/SFX2Alwas transformed into the competent E. coli BL21. The supernatant of the lysates was collected after the resuspension and sonication of the bacteria which were collected after IPTG induction. Then the recombinant GST-STX2A1 fusion protein was purified by GSTrap FF affinity cohann, desalted by HiTrap desahing cohann, and analyzed by SDS-PAGE. The antiserum against GST-STX2A1 fusion proteins was prepared by immunizing Balb/ c mouse with purified GST-STX2A1 fusion protein. The specificity of the antiserum against GST-STX2A1 fusion proteins was identified by Western blotting. Results The recombinant plasmid PGEX-6p-1/STX2A1 was constructed successfully. The soluble fusion protein GST-STX2A1, whose Mr was about 5.3 × 10^4, was expressed in E. coli. After purification by affinity column, the purity of the recombinant protein was about 90%. The antiserum against GST-STX2A1 fusion proteins could specifically react with the A subunit of native shiga toxion Ⅱ. Conclusion The high purity fusion protein GST-STX2A1 has been produced successfully, which provides a necessary foundation for preparing O157：H7 subunit vaccine and monoclonal antibodies against STX2A1 subunit of O157： H7.