CDNA Cloning And Polyclonal Antibody Preparation Of Schistosoma Japonicum Infection Resistance Associated Gene E77.43 In Microtus Fortis

Schistosomiasis japanica is a parasitic zoonosis which does great harm to human beings. Schistosoma japonicum Can infect about 46 species of mammalian include human being, but either domesticated or wild Microtus fortis have natural resistance which inherits stably to Schistosoma japonicum infection. This resistance shows a new direction on research of aganist Schistosoma japonicum.In previous study, we constructed bone marrow cDNA library of Microtus Fortis, and screened a gene fragment E77.43 with anti-schistosomula function in some extent by expression cloning, then we obtain the full-length cDNA of E77.43 by RACE. Bioinformatics analysis show the coding product of E77.43 could be integrin-linked kinase-associated serine/threonine phosphatase.On the foundation upon, first we extracted total RNA from tissue of Microtus Fortis and constructed a cDNA library. Then designed primers for ORF of E77.43, after generated the part of coding region by PCR, we ligated the PCR product into the pQE30 vector. The transformants were isolated and confirmed by enzyme digestion and sequence analysis. The recombinant was expressed efficiently in prokaryotic expression system. After interest protein was purified by Ni-NTA resin, dialyzed several times and cryodesiccatted, we determined the concentration of it. In the further research, we generated polyclonal anti-E77.43 antiserum by immunizing rabbits with purified interest protein. We detected that antibody titer achieve 1:6400 by ELISA method, and specificity of polyclonal antibody was identified by western-blotting analysis. Subsequently, we used the polyclonal anti-E77.43 to ivestigate the distribution of E77.43 in different tissues of Microtus Fortis by western blotting.Polyclonal antibody preparation of E77.43 laid a strong basis for the further research.