| Cryptosporidium is a worldwide zoonotic human parasitic protozoa, which can cause lethal diarrhea in immunosuppressed populations, and has important public health implications. By now we have not yet found the treatment to prevention this disease.ABC transporters have fews of transmembrane protein families, the family members can take advantage of the energy of ATP hydrolysis to transport substrate across membrane. ABC proteins exist in a wide variety of biological bodies, and are related with the transporting of nutrition, medicines, the absorption of toxin excretion. Therefore, we need to do some preliminary reseach about Cryptosporidium transport protein to confirm that's a potential drug target.10 transporter proteins of Cryptosporidium parvum and other related membrane protein sequences are referred by professor Guan Zhu. And we obtain the corresponding gene and gene description in the NCBI Blast database. Besides,we obtain the sites which small peptides seat in the integrity protein by Clastal X1.83.we did not have got homology of these peptides, thus these peptides are specific.9 of these peptides are synthesized by GL Biochem (Shanghai) Co., Ltd,and their purity and composition analysis were determined by HPLC and mass spectrometry. the map in the following appendix.Each hapten (peptide EC et.al, 6 of 9 peptides) is conjugated with BSA, and evaluated by SDS-PAGE . The result is that peptide is coupled to BSA successly. We use these 6 complete antigen to immune 6 Japanese white rabbits respectively, and each rabbit weight 2 kg, first,we mix the complete antigen with the same volume FCA,and each rabbit is immuned 1 mg complete antigen. Then Immune once every 14 days with mixture which Contains complete antigen and FIA. The rabbits are immuned 6 times totally. The last time we immune the each rabbit each complete antigen 2mg. After the third time immuning, we did an ELISA test once every 2 week. The ELISA condition is each antigen is diluted 100 times, coating plate in 4℃over night, block for 1 hour with 1% BSA-PBST, serum is diluted 100 times and double dilution. At last, each serum is diluted 1000 times parallelied each other. P/N≥2 is positive, and results are positive.We infected gerbils with C. parvum oocysts and C. muris oocyst which saved in lab successfully. histopathological and ultrastructural studies indicate that C. parvum parasites in gerbils mainly in the duodenum, jejunum, ileum, Causing loss of intestinal microvilli became shorter, and C. muris parasites in gerbils is in cardia, pylorus, fundus.several staining methods which contain HE, PAS, toluidine blue staining are used to observe parasite, infected tissueand inflammatory cells.In this experiment, we have speculated peptides of Cryptosporidium parvum transporters and related proteins, and rabbit antiserum was prepared for further study of Cryptosporidium parvum.these preliminary works lay the foundation for the location of transporters of Cryptosporidium and the metabolism mechanism of Cryptosporidium parvum... |
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