Construction Of Transfection Vector And Transient Transfection Of Cryptosporidium Baileyi

Cryptosporidiosis is one of the most important zoonotic protozoiasis. Transfection refers to the techniques that transfer the foreign genes into eukaryotic cells and expressed intra-cellula. Construction of transfection vector of Cryptosporidium will provide an effective tool for studing the function of genes, and screening the drug target genes and protective antigens genes. In our study, Cryptosporidium transfection vector was constructed by the model of Cryptosporidium baileyi and the reporter molecule of yellow fluorescent protein, the Cryptosporidium transfection system in chicken was cunstructed using restriction enzyme-mediated integration (REMI) method.Firstly,120chikens were divided to5groups, respectively inoculating sporazoites or oocysts via rectum and sporazoites or oocysts via crop.To detect the oocyst situation after inoculation, and made tissue sections and scanning electron microscope slices of bursa of Fabricius and trachea tissue. The results showed that the experiment groups which inoculated sporozoties via rectum or inoculated oocysts via crop can get more oocysts in the crest-time compared with the groups which inoculated sporozoties via crop or inoculated oocysts via rectum. It is suggested that inoculated sporozoties via recta and inoculated oocysts via crop were both the best inoculaton methods. The tissue section and scanning electron microscope (SEM) section showed that there were numerous developmental stages of C. baileyi adhere to the surfaces of bursa of Fabricius after inoculated sporazoites or oocysts via rectum and inoculated oocysts via crop, while a few of C. baileyi were observed in the surface of tracheal mucous membrane only after inoculated oocysts via crop, the burse of Fabricius and tracheal mucous membrane all damaged in different level after inoculated oocysts, these results indicated that C. baileyi in different tissue by different inoculation methods. It is the first report that C. baileyi can infect chickens by inoculating sporozoites or oocysts via recal in our study.Secondly, Cryptosporidium parvum and Cryptosporidium hominis actin, histone H4, CP15protein regulatory sequences were selected as reference to clone the promoter and terminator sequence of Cryptosporidium baileyi.We successfully obtained the suquences of promoter and terminator of histone4(H4) gene by Polymerase Chain Reaction (PCR) amplification, transformed clone and sequencing technology. The promoter and terminator were1295bp and977bp，and81bp starting from the initiation codon was the coding regin in promoter sequence.In the current study, the transfection vector pCbHYH was constrcted with the vector of laboratory, which was inserted with promoter,3’-UTR (untranslated region) and yellow fluorescent protein sequence. Transfection vector was identified the correctness by colony PCR, enzyme digestion and sequenciong method. The transfection system in chicken was constructed by using restriction enzyme-mediated integration (REMI) method and inoculating sporazoites or oocysts via rectum. The luminescence oocysts were detected in faeces in the7th days post inoculation (PI) and the luminescence C. baileyi polypides were observed by the fluorescence microscopy in mucosa of bursa of Fabricius in the11-13th days PI. The result showed that transient transfection was developed successfully in chikens.In our study, the method of inoculation of sporozoites via recta was established and provided the inoculation route for the realization of the transient transfection. the transfection vector of C. baileyi was constructed, and the transient transfection was accomplished in chickens. The results will promote the study of gene function such as invasion and metabolism, accelerate the anti-parasites drugs comes out and expand the research of live vaccine vector of C. baileyi.