Construction Of Cryptosporidium Parvum CDNA Library

Abstract Construction of Cryptosporidium parvum cDNA Library Construction of Cryptosporidium parvum cDNA Library XU Weidong Directed by Prof ZHANG Xic hen Abstract: A eDNA library of Cryptosporidium parvum was prepared successfully for the first time by using pUCI8 vector in this country. Two genes encoding protective antigens were cloned. Total RNA and mRNA of C. parvum were isolated and purified. cDNAs were synthesized by reverse transcription. Fragments shorter than 400bp were removed by a gel chromatography prior to adding EcoR I adaptors to both ends of the cDNAs and phosphorating their 5?ends. The insert cDNAs were cloned into EcoR I-digested, BAP-treated pUC18 vectors and transformed into E. coil strain DH5-alpha to generate library. Two sets of primers for amplifying 23-kDa and 15-and 60-kDa sporozoite surface proteins were designed and synthesized. Special bands given by PCR test using recombinant plasmids from the library as templates were recovered and cloned into pMDI8-T vectors. Suspected positive clones were digested with EcoR I and Hind III. Positive clones were sequenced and homology of the sequences was compared with known genes in GenBank. The library size is 1.9X 106 recombinants. The inserted cDNA fragments of the library are ranged between 0.4X ~ bp and 6.5 X i0~ bp. No. I clone was 354bp, 98% identity to P23, while No.2 was 459bp, 98% identity to both CP15160 and CPI5. The library will helpful in study of local strains RNAs and DNAs, in gene screening for diagnosis and prevention of Cryptosporidiosis as well as gene subcloning.