霍乱弧菌O139 mshA基因的克隆、表达及免疫反应性鉴定

Cloning,expression and immunoreactivity of mshA gene of Vibrio cholerea O139

目的克隆霍乱弧菌O139临床菌株的甘露糖敏感血凝素(Mannose-Sensitive Hemagglutinin,MSHA)基因,构建原核表达系统,鉴定重组表达产物的免疫反应性。方法高保真PCR扩增mshA基因,T-A克隆后测定核苷酸序列并构建原核表达系统pET-28a(+)-mshA-E.coliBL21(DE3),IPTG诱导目的重组蛋白(rMSHA)表达、Ni-NTA亲和层析法收集蛋白;Western blot鉴定其免疫反应性。结果所克隆的mshA基因与GenBank报道的(AE004128)序列完全一致。构建的mshA基因原核表达系统pET-28a(+)-mshA-E.coliBL21(DE3)表达量占细菌总蛋白的20%以上。表达的蛋白(约20kD)可与His单克隆抗体发生特异性免疫结合。结论成功构建了霍乱弧菌O139mshA基因的高效原核表达系统,表达的rMSHA蛋白有良好的免疫反应性,为进一步研制MSHA及其抗体检测试剂盒奠定了基础。

To clone mshA gene and construct its prokaryotic expression system, and to identify immunoreactivity of the target recombinant protein（rMSHA）. The mshA gene was amplified by high fidelity PCR and then T-A cloned into the new type of prokaryotic expression system pET-28a（＋）-mshA-E, coli BL21DE3. The output of rMSHA was measured by SDSPAGE and Ni-NTA affinity chromatograthy was performed to collect rMSHA. The immunity of rMSHA was determined by Western blot. DNA sequence analysis showed that the nucleic acid sequence of the cloned mshA gene was identical to that published in GenBank. The output of rMSHA expressed was as high as 20% or above of the total bacterial proteins. A 20kD fusion protein was detected by SDS-PAGE and its his tag was recognized by its MAb in Western blot. It is evident that the fusion protein rMSHA of Vibrio cholerea O139 is well expressed in prokaryotic expression system,with fine immunoreactivity.