Construction Of CPV Transfer Vector And GFP Expressed In Cryptosporidium Parvum

Cryptosporidium is a widespread protozoan parasite that develops in epithelial cells lining the digestive and respiratory tracts of human and other vertebrates. Cryptosporidiosis caused by the Cryptosporidium has become a serious diarrheal disease of humans and other mammals throughout the world. Cryptosporidium has been classified to 15 species according to host specificity ,oocysts morphous, and gene characterization. C.parvum is generally considered to be the parasite species responsible for serious infection in human and domestic animals. Although progress has been made, control of cryptosporidiosis remains problematic due to the absence of approved preventive measures and lack of consistently effective parasite-specific pharmaceuticals.CPV is a kind of dsRNA virus obligate parasitized in C.parvum,the CPV were found to reside within the cytoplasm of sporozoites.there are two kinds of CPV L-dsRNA and S-dsRNA in the C.parvum,first reported by Nikolai in 1997,the discovery of CPV provide an important tool for the study of molecular biology of C.parvum.For the past few years, the vector system reconstructed with GLV has been used in several area ,for example:expression of exogenous gene, structure and function analysis of G.. lamblia gene.now,there was no report about CPV gene transfected vector, the full﹣length cDNA sequence of CPV L-dsRNA and S-dsRNA was analyzed CPV transfer vector was constructed and green fluorescent protein was successfully expressed in the C.parvum,and these provide a method for the study on gene expression and regulation of C.parvum.The full﹣length cDNA Sequence Analysis of Cryptosporidium parvum Virus The oocyst of C.parvum is purified with sucrose density gradients,then with the treatment of sodium hypochlorite ,thus purified oocyst without bacterium can be acquired, the oocyst was schizolysised with freeze and solution again and again to extract the total nucleic acids. According to the published nucleotide sequence of CPV L-dsRNA,two pairs of primers were designed and a pair of primer was designed for S-dsRNA total nucleic acids from the oocyst of C.parvum.were used as template for RT﹣PCR. After RT﹣PCR,the products were linked into pMD18﹣T vector,then cloned,sequenced and analyzed.as a result we get the Genome sequence of CPV L-dsRNA, the Genome sequence of CPV L-dsRNA was 1783bp(EU183403),which revealed the presence of an open reading frame.which encodes a polypeptide of 523 amino acid residues. without overlapping genes . which contains all consensus RNA﹣dependent RNA polymerase sequence motifs. The ratio of G+C in the Genome was 38.53%. The homology of CPV L-dsRNA virus to that of sequences (U95995)was 96% in the nucleotide,it lake three basic radical TTC which lay between 1305bp and 1307bp compared to the CPV L-dsRNA reported abroad.which lay in the coding region of RNA﹣dependent RNA polymerase sequence motifs. the Genome sequence of CPV S-dsRNA was 1375bp(EU183404),which also encode an open reading frame. It may encode capsid protein. The ratio of G+C in the Genome was 41.02%. compared to the C.parvum Virus S-dsRNA reported abroad.it has a G in 1311bp. The homology of CPV L-dsRNA virus to that of sequences (U95996) was 98% in the nucleotide. Construction of CPV Transfer Vector and GFP Expressed in C.parvum According to transcriptional start site,replication origin and packaging site of CPV genome L-dsRNA and S-dsRNA,a system was developed here for expression of a foreign gene in this organism by flanking the green fluorescent protein(GFP) gene with the fragments of L-dsRNA and S-dsRNA positive-strand RNA,transcript of the construct was synthesized in vitro with T7 polymerase and used one or mixture of two RNA transcript to transfect C.parvum oocyst by electroporation. centrifuged at 5000×g for 7min, the precipitation was resuspended in excysted liquid. Incubate at 37℃,5%CO2 incubator.After 90nin,they were washed with PBS buffer there times at 2000×g for 5 min, they were cultivated in 37℃5%CO2 incubator,After 18h inoculated, when observed with fluorescence microscope,some oocysts and sporozoites can also erupt green fluorescence after 18h incubated at 37℃,5%CO2 incubator, the ratio of oocysts erupt green fluorescence are about 10%. The GFP mRNA was detected by RT-PCR in transfected C.parvum which demonstrated that the RNA transfer vector may thus providing foundation for the study on gene expression and regulation of C.parvum...