大鼠天然抗体相关的弓形虫抗原基因的免疫筛选与克隆

Immunological screening and cloning of genes encoding Toxoplasmaantigens related to the serum antibodies in rats

目的 研究大鼠对弓形虫感染的天然抗性分子 ,为弓形虫病疫苗的研制提供新的抗原分子。方法 用正常大鼠血清作为探针筛选弓形虫速殖子cDNA文库 ,对阳性克隆的插入片段分别进行PCR扩增及DNA序列测定。结果 从cDNA文库 2× 10 5个噬菌斑中筛选出 5个阳性克隆 ,其插入片段大小分别为 0 5 5～ 1 8kb。对P1、P4、P6和P8四个克隆进行测序 ,将所得序列查询基因序 ,结果显示 :P8与弓形虫棒状体蛋白 2 (ROP2 )基因相同。P4为弓形虫的新基因序列 (GenBank中的登录号为AY34916 2 ) ,命名为T .gP4。T .g -P4编码 96个氨基酸的跨膜蛋白。PROSCAN分析显示T .g -P4含有 4个蛋白激酶C磷酸化位点 ,3个N -肉豆酸酰化位点 ,1个核糖体蛋白L2 9信号。P1和P6为新基因片段。阳性克隆的分子鉴定正在进行中。结论 阳性克隆的筛选和鉴定为抗弓形虫病疫苗的研制奠定基础。

To screen and identify the potential candidates related to the natural resistance to Toxoplasma gondii for the development of Toxoplasma vaccine.The normal rat serum was used as a probe to screen Toxoplasma gondii tachyzoite cDNA expression libraries.The posifive clones were analyzed by PCR amplification and DNA sequencing.It was found 5 positive clones were obtained from about 2×10 5 phage plagues after three rounds of screening.The size of the inserts ranged from 0.55 kb to 1.8kb.A BLAST search of all available sequence databases using the partial sequences from 4 positive clones(P1,P4,P6,P8)showed that the sequence of P8 clone was identical with T.gondii rhoprty protein 2 (ROP2)antigen gene.However,there was no significant hit of any sequences to clone P4,suggested that P4 could be a novel gene (GenBank accession number Ay349162),named T.G P4,which encodes a transmembrane protein with 96 amino acid open reading frame.Proscan analysis of the T.g P4 amino acid sequence showed that this gene product contains 4 protein kinase C phosphorylation site,3 N myristoylation site,one ribosomal protein L29 signature.Clone P1 and P6 were small partial fragments.The further characterization of these positive clones are under investigation.It concludes that the identification of positive clones will lay a foundation for the development of toxoplasmosis vaccine.