Studying Of Expression And Transfer Function On Cryptosporidium Andersoni ABC1 And ABC2 Gene

Cryptosporidiosis is a worldwide zoonosis, this disease is caused by Cryptosporidium. In the immune normal crowd, cryptosporidiosis will only cause self-limiting diarrhea, for immune suppression or immunodeficiency may cause fatal diarrhea. The nutrients of Cryptosporidium in transportation regulation is to rely on the membrane ATP binding cassette transporter (CpABC, at the same time also has the multi drug resistance and avoid the characteristics of immune function, which is the reason why many drugs and vaccines against cryptosporidiosis is invalid. Therefore, in-depth studying cryptosporidium CpABC protein, to reveal the pathogenic mechanism of Cryptosporidium parvum, it is the vital significance for researching and developing the effective anti Cryptosporidium drugs.Firstly, According to ATPase gene sequences in the Genbank synthesize the ATPase gene primers, while Cryptosporidium andersoni genomic DNA as the template. Translating the 876bp gene fragments which is amplificated, and getting 292aa.Secondly, According to CpABCl gene sequences in the Genbank synthesize the CaABCl gene primers (plus restriction enzyme sites BglⅡ and EcoRI, initialor ATG and terminator TAA), while Cryptosporidium andersoni genomic DNA as the template. Translating the 522bp gene fragments which is amplificated, and getting 174aa. Connecting the recombinant plasmid CaABCl and eukaryotic expression plasmid pEGFP-C1 after double enzyme digestion, in order to construct recombinant eukaryoticexpressionplasmidpEGFP-C1-CaABC1, then transfecting into IEC for expression. Detecting the concentration of K+, Ca2+, Na+ and Mg2+ in the fluid inside cells and extracellular fluid of the blank group, the contrast group, transfection group and add inhibitors, which have been dealed, and finding that 4 kinds of ions are tansported from outside to inside of the cell, while the transport effect of Ca2+ is the most obvious.Third, According to CpABC2 gene sequences in the Genbank synthesize the CaABC2 gene primers (plus restriction enzyme sites XhoI and XbaI, initialor ATG and terminator TGA), while Cryptosporidium andersoni genomic DNA as the template. Translating the 756bp gene fragments which is amplificated, and getting 252aa. Connecting the recombinant plasmid CaABC2 and eukaryotic expression plasmid pEGFP-C1 after double enzyme digestion, in order to construct recombinant eukaryoticexpressionplasmid PEGFP-C1-CaABC2, then transfecting into IEC for expression. Detecting the concentration of Cholesterol, glucose, phosphatase and glutathione in the fluid inside cells and extracellular fluid of the blank group, the contrast group, transfection group and add inhibitors, which have been dealed, and finding that 4 kinds of metrial are tansported from outside to inside of the cell, while the transport effect of Cholesterol is the most obvious.