福氏志贺菌毒力蛋白IpaC的表达、纯化及免疫活性鉴定

目的表达、纯化福氏志贺菌毒力蛋白IpaC并对其免疫活性进行鉴定。方法将含有ipaC基因的pET32a．ipaC表达质粒载体转入大肠杆菌BL21（kDE3）中表达，表达产物进行SDS—PAGE鉴定，并采用QIA expressionist^TM蛋白纯化系统纯化IpaC蛋白；用纯化的蛋白免疫动物获得抗血清，Western-blot检测抗原的免疫活性。结果诱导后的表达产物经SDS—PAGE发现有一相对分子量约为63000的条带，其含量约占总蛋白量的11％，对融合蛋白进行纯化纯度可达90％以上，抗His的抗体Western-blot分析，发现特异性区带出现在63000u处，证明该蛋白是所要表达的融合蛋白，制备的免疫血清可与Ipac发生特异免疫反应。结论pET32a-ipaC重组表达质粒转入大肠杆菌后，可稳定、高效地表达毒力蛋白IpaC，且纯化后的蛋白具有免疫活性。

Expression,Purification and Immunoreactivity of IpaC from Shigella flexneri

Objective To express, purify, and analyze the immunoreactlvity of IpaC protein from Shigellaflexneri. Method The prokaryotic expression plasmid pET32a-ipaC was constructed and transformed into E.coli BL21（λDE3）. IpaC protein was purified and monitored by QIA expressionist^TM system and SDS-PAGE, respectively. New Zealand rabbits were then immunized with purified recombinant protein and the antiserum was collected. The immunoreactivity of IpaC was analyzed by Western-blot. Result SDS-PAGE analysis showed that the fusion protein had a molecular weight of about 63 000, and the yield of this fusion protein was 11% of the total cellular protein. The purity of this protein was 90%. Protein band with a size of 63 000 u was also detected by immunoblotting with anti-His antibody. The fusion protein could also be identified by anti-IpaC antiserum. Conclusion Recombinant plasmid pET32a-ipaC was constructed and expressed in E.coli BL21 （λDE3）. The fusion protein is immunogenic.