Analysis And Tertiary Structure Prediction Of CP15Antigen From Cryptosporidium Parvum And Construction Of GST Fusion Expression Vector

Cryptosporidiosis is a worldwide found parasitic disease caused by Cryptosporidium, which infects humans and many animals. Cryptosporidium is the major protozoan parasite in the phylum Apicomplexa, it causes watery diarrhea in infected individuals and can be fatal to immunocompromised individuals, e. g, AIDS patients. All infected organism will excret Cryptosporidium oocyst in feces, which can contaminate water sources, such as drinking water, swimming pool, rivers and lakes etc, threatening the human public hygiene. With the development of molecular biology, people found seven kind of Cryptosporidium main founctional antigenic gene. Among them the study about surface antigen was most in-depth, including Cpl5, Cp23and Cp15/60. Though many researchers have done plenty of study about prevention and treatment of Cryptosporidiosis, at present, no prophylactic therapy is available to prevent this parasitic disease in humans or animals, therefore the development of the diagnostic reagent and the vaccine are in imminent.In this study, we cloned the Cpl5gene from constucted cDNA library of C ryptosporidium, cloned it into pMD19(T) Vector, named it pMD19—Cp15, positive clone was gene sequenced and analyzed, and Cp15amino acid sequence was analyzed with biology software, such as ExpasyProtparam (http://web.expasy.org/protparam/)、 Swiss-Model(http://swissmodel.expasy.org)SOPMA(http://npsa-pbil.ibcp.fr/cgi-bin/npsa_a utomat.pl?page=npsa_sopma.htmL) and Psipred (http://bioinf.cs.ucl.ac.uk/psipred).The three-dimensional structure of CP15protein was predicted by three kinds of software, such as Swiss-PdbViewer.The Result showed that:Cp15gene can encode a protein composed of126amino acid residues of peptide, with the predicted protein molecular weight of14.79Kda, theoretical isoelectric point of4.84, and the proportion of hydrophilic amino acid of59.5%. Analyses of the structure indicated that:it maybe have two helixes and the irregular curls and strand structure composed the essential conponent of secondary structure. The B-cell epitope is maybe between the peptides of50-58、76-89and110-115. This study provided a theoretical basis for obtain valid epitope antigenic gene. Besides, Cp15fragment was obtained from plasmid pMD19—Cp15with BamH I and Xho I digested, then subcloned into vector pGEX4T-3, constructed GST fusion expression recombinant vector pGEX-Cpl5in E.coli DH5a, then transformed into E.coli BL21(DE3), The recombinant protein GST-CP15was induced by IPTG and the expressed condition was identified by SDS-PAGE and Western blotting. Highly expressed CP15antigen was used for establishing recombinant-indirect ELISA method, and goat and bovin serum was used foe testing prevalent situation of Cryptosporidium at our distict. Our work laid out the solid foundation for the diagnosis of candidate antigen peptide of Cryptosporidiosis.