SDS-PAGE and Western bolt analysis

Tachyzoites from three genotypic strains（RH，ME49-PLK and VEG）were lysed using M-PER Mammalian Protein Extraction Reagent(Pierce,Rockford,IL)containing 10 µg/ml each of antipain,leupeptin,chymostatin,pepstatin A,phenylmethylsulfonylfluoride and phenanthroline (Sigma,Dt.Louis MO).Aliquots equivalent to 15×106 tachyzoiteswere subjected to 12% SDS-PAGE under reducing conditions and transferred to nitrocellulose.Following transfer,theblots were blocked with 10% horse serum and then incubated overnight with anti-TgPCNA1 OR 2 mouse polyclonal sera(1:2500).Antigen detection was accomplished by incubating blots with alkaline phosphatase-conjugated anti-mouse IgG（Promega，Madison WIS）diluted 1:7,500,followed by development in nitroblue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate.

ResultsCloning and sequence comparison of the PCNA genes in T.gondii

Analysis of EST database entries along with conventional cDNA strategies identified two unique PCNA cDNAs in T.gondii,designated TgPCNA1 and TgPCNA2.The 951bp open reading frame of TgPCNA1 predicts a protein of 316 amino acids with a predicted molecular weight of 34,476 daltons.The TgPCNA2 clone contains a smaller open reading frame of 864 bp encoding a protein of 261 amino acids and a predicted molecular weight of 32,263 daltons.Consistent with all known eukaryotis PCNAs,the TgPCNA sequences contain basic helix-loop-helix DNA binding motifs(Fig.2-1)that demonstrate >50% similarity to the equivalent region in human PCNA[8].Other conserved motifs include the poltmerase-δ and p21 putative binding sites (D-SHV-,Fig.2-1)as well as the C-terminal sequences (-F/YLAP,Fig.2-1)that appear essential for proper folding. PfPCNA1 contains 10 and PfPCNA2 contains 9 additional C-terminal amino acdis while T.gondii PCNA1 and 2 contain C-terminal insertions of 53 and 28 amino acids.respectively.