关键词: 弓形虫, cDNA文库, 免疫筛选, 大鼠

Abstract: 　Objective To screen and identify the potential candidates for the development of toxoplasmosis vaccine. Methods Rats were infected with Taxoplasma gondii ( T. gortdii) RH strain and their sera were used as a probe to screen T. gondii tachyzoite cDNA expression libraries. The positive clones were analyzed by PCR amplification and DNA sequencing. Results Thirteen positive clones were obtained from about 4 × 105 phage plagues after three rounds of screening. The size of the inserts ranged from 0.45 kb to 2.4 kb. A BLAST search of all available sequence databases using the partial sequences from four positive clones (LI, L2, L4, L5) showed that the-sequence of L2 clone was identical with T. gondii P24 major antigen gene (TgP24). Clone L4 had a high homology with Saccharum officinarum pyruvate orthophosphate dikinase. There is no significant hit of any sequences to clone L1, suggesting that L1 could be a novel gene(GenBank accession number AY180109), named T. g-R1, which encodes a non-transmembrane protein with 134 amino acid open reading frame. PROSCAN analysis of the T. g-R1 amino acid sequence showed that this gene product contains two protein kinase C phosphorylation site, two casein kinase Ⅱ phosphorylation site, one N-myristoylation site and one microbodies C-terminal targeting signal. Clone L5 was a small partial fragment. Conclusion The identification of positive clones provides a possible way for the development of toxoplasmosis vaccine.

Key words: Toxoplasma gondii, cDNA library, immunoscreening, rat