Cloning And Eukaryotic Expression Of Cryptosporidium CaNBD Gene

Cryptosporidiosis is a zoonotic parasitic disease caused by Cryptosporidium infection.It was a worldwide distribution. In recent years, Cryptosporidium was highly valued because of its high infection rateserious and serious prejudice in infants and young children, young animals, the immune defective, especially in patients of AIDS. Domestic and overseas scholars took extensive and in-depth studies in cryptosporidiosis, there is no any effective drug for treatmenting cryptosporidiosis.Because of the new simple metabolic pathway, regulating the transporting of nutrients relying ATP-binding cassette transporter protein(CpABC) transporter in membrane and the character of evading the immune and multi-resistance in Cryptosporidium, many vaccines and drugs are difficult to prevent and treatment cryptosporidiosis. Therefore, further study at CpABC of Cryptosporidium has Important Significance in revealing the pathogenesis of Cryptosporidium and in screening the drug of treatment cryptosporidiosis.Firstly the cows source Cryptosporidium andersoni ABC protein gene nucleotide binding site-CaNBD was amplified by PCR in this topic,with cloning, sequencing and then got Cryptosporidium andersoni CaNBD gene; then4kinds of methods were used to isolate and cultivate small rat intestinal epithelial cells (IEC).A method of primary culture of mouse IEC cell was established. The rat intestinal epithelial cells where Cryptosporidium live were got. The CaNBD gene was inserted into pEGFP-C1vector to build the eukaryotic expression vector of pEGFP-C1-CaNBD.D. The recombinant plasmid of pEGFP-Cl-CaNBD was lead into IECs by means of lipofectamine media method to express. The protein expression of EGFP was observed. Finally,4concentrations of ions were tested after transfection. The significant differences were analysised by the SPSS16.0software to clear the transport function of CaNBD.Firstly, Cryptosporidium andersoni genomic DNA were extracted as template by using fecal DNA extraction kit. A pair of primers of plasmodium ABC protein gene sequencethe degenerate (with restriction sites EcoR I and Bgl II, the initial sub-ATG and stop codon of TAA) was synthed. The411bp nucleic acid of CaNBD gene was obtained after amplification, cloning and sequencing.It is137aa of the amino acid sequence.The ABC family characteristic sequences of "LSGGQ", Cryptosporidium ABC protein motif of Walker A-GETGSGKST" and Walker B-" DEATSSLD " were contained in the protein sequence of CaNBD. Therefore, the product of411bp is the Cryptosporidium andersoni ABC section of the ATP binding protein gene sequences, named CaNBD.Cloned recombinant plasmids with CaNBD gene and eukaryotic express plasmid pEGFP-C1were digested by restriction enzymes of EcoRâ…  and Bglâ…¡.The products of digestion were connected by T4ligase to construct the recombinant eukaryotic expression plasmid pEGFP-C1-CaNBD.pEGFP-C1-CaNBD was successfully constructed by the identification of PCR, restriction enzyme digestion and sequencing.Secondly,4methods of tissue culture, thermophilic bacteria protease digestion method, collagenaseXI/proteaseâ…  and collagenase â… /dispase VI were usde to isolated primary mouse intestinal epithelial cellsand culture.The cells were identified by two methods of immunohistochemistry and immunofluorescence. The results of4kinds of separation methods can get rat IECs. The IECs obtained by using method of tissue culture were polluted seriously by fibroblasts.The numcer of IECs obtained by using method of collagenase â… /protease VI is few. The numcer of IECs obtained by using method of thermolysin and collagenaseXI/dispase I is more and the IECs are fit to transfection.Finally, the recombinant eukaryotic expression plasmid of pEGFP-C1-CaNBD were transfected in IECs by means of lipofectamine media method.Negative control group were IECs transfected with empty plasmid of pEGFP-C1. Blank control group were IECs without any plasmid.EGFP were expressed in IECs with pEGFP-C1-CaNBD and with pEGFP-C1observed under a fluorescence microscope.No EGFP expressed in IECs without any plasmid. The IECs were broken by Ultrasonic Processor to be intracellular fluid.Cell supernatant was collected to be extracellular fluid.4kinds of ion concentration were detected by the Solution ion detection kit. The results showed that4kinds of ion were transported into IECs from extracellular. Therefore, the CaNBD was the gene of Cryptosporidium andersoni ABC protein of the nucleotide binding region gene and CaNBD was the protein of transporter which can transport nutritious.In conclusion,CaNBD-the Cryptosporidium andersoni ABC protein in the nucleotide binding gene was cloned the first time in this topic. Recombinant expressing plasmid of pEGFP-C1-CaNBD was structured. Primary rat IECs were obtained through establishment of cultured primary rat IECs method. Eukaryotic expression of CaNBD gene through importing into rat IECs.The CaNBD protein was able to import4kinds of ion through detecting the concentration of4kinds of ion. The successful cloning and expression of Cryptosporidium adersoni CaNBD gene establish a basis for drug-resistant mechanism of Cryptosporidium, and thus solve the drug resistance problem of Cryptosporidium and ultimately provide an important basis for the use of drugs to treat cryptosporidiosis.