Cloning,Expression,Purification And Antigenic Activities Against Invasion Gene IpaC Of Shigella Flexneri

Shigella spp is a kind of Gram-negative bacteria that belong to Enterobacteriaceae family. They cause shigellosis, or bacillary dysentery, an invasive infection of the human colon that affects a spectrum of clinical presentations, from short-lasting watery diarrhea to acute inflammatory bowel disease, the classic expression of shigellosis characterized by the triad of fever, intestinal cramps, and bloody diarrhea with mucopurulent feces. According to serological reaction, Shigella spp are divided into four groups (designated species), Shigella boydii, Shigella dysenteriae, Shigella flexneri and Shigella sonnei. Shigelia flexneri and Shigella sonnei (1 serotype) account for shigellosis, the former being prevalent in the developing world, the latter in the developed world. S. flexneri 2a is prevalent in our country, shigellosis is the second infectious disease and the most fargoing diarrhea. The incidence of shigellosis would become two or four times higher than usually in arm when military operations were perfomed. That the military affairs being affected by the prevalence of shigellosis have been reported many times in the world. With the use of its medications, the drug resistance of Shigella spp has become more and more severe, not only to a kind of antibiotic, but also many kinds of antibiotics. What's more, treatment is often delayed, so 10% acute shigellosis are translated into chronic one every year. These are important infectors for the prevalent of shigellosis. In this way, study on a rapid diagnostic method and an anti-shigella vaccine had become the emphases of prevention and treatment for shigellosis. Selecting and gaining antigens with high sensitiveness, specificity, and immunocompetenceis the base of the study. In the present study, invasion plasmid antigen C (IpaC), secreted by the invasion plasmid of Shigella spp and enteroinvasive Escherichia coli, is the primary effector protein for Shigella invasion of epithelial cells. Consisting of three distinctive domains, IpaC can direct its own invasiveness into macrophaget. It will provide us an insight into understanding the precise role of IpaC in Shigella spp pathogenesis. It is essential to strengthening our future approaches aimed at the study on a rapid diagnostic method and an anti-shigella vaccine.1 To construct and identify a recombinant plasmid containinginvasion plasmid antigen(ipaC) of Shigella flexneriThe ipaC gene obtained successfully by PCR amplification from plasmid DNA of S. flexneri 2457T was cloned into T vector, then subcloned into expression plasmid of pET32a. DNA sequencing showed that the DNA sequence of the cloned gene was almost the same as the published sequence, but one neocleotide different with stain isolated from China and two neocleotide different with foreign isolated strain.2 Expression purification and antigenic activities of thefusion proteinThe prokaryotic expression plasmid pET32a-ipaC was constructed and transformed into BL21(DE3). The fusion protein was about 63KDa, with six his on C-terminal, and its expression of fusion reached up to 13% of the total bacteria protein. After sonication, the fusion protein of pET32a-ipaC was found mostly in supernatant. The fusion protein was purified by QIAexpressionist?system in light of the peculiarity of pET plasmid, The content of fusion protein was up to a level of 90% of the total protein. The fusion protein exhibited a specific reaction to the antibody of anti-his, representing IpaC-his fusion protein was identified. The fusion protein wasused to immunize rabbit for the preparation of polyclonal antibodies. Polyclonal antibodies could specifically recognize a 63KDa protein from 2457T and EIEC. These data suggested that the fusion protein contains the essential epitopes and has high antigenic activities, those would pave the way for further studying monoclonal antibodies against against IpaC of shigella spp and EIEC and establish Gold-immunochromatography assay (GICA) for die diagnosis of shigellosis.