弓形虫14-3-3信号转导蛋白基因序列测定

CLONING AND IDENTIFICATION OF 14-3-3 SIGNAL TRANSDUCTION PROTEIN GENE OF TOXOPLASMA GONDII

目的 克隆与鉴定弓形虫 14- 3- 3(Toxo14- 3 - 3)信号转导蛋白基因 ,用于寻找弓形虫新的诊断和候选疫苗分子 ,并在寄生虫入侵宿主细胞中寻找分子干预环节。方法 设计合成引物 ,以弓形虫RH株速殖子RNA为模板逆转录合成cDNA链 ,用PCR扩增出弓形虫 14- 3 - 3蛋白编码基因序列 ,克隆入 pGEM -T载体 ,并用双酶切法、以质粒为模板PCR法和DNA测序进行鉴定。结果 Toxo14- 3 - 3具有一个长度为 798bp的完整开放阅读框 ,与GenBank收录 (编号为ABO12 775 )Toxo14- 3 - 3蛋白编码基因一致 ,2个碱基出现密码简并。结论 本实验获得了完全正确的Toxo14- 3 - 3蛋白基因克隆 。

Aim To clone and identify 14-3-3 signal transduction protein gene of Toxoplasma gondii (Toxo14-3-3) for characterization of new candidate vaccine and diagnostic molecules and for search for a metabolic interference with parasite invasion to host cells.Methods A pair of primers were designed and synthesized based on the DNA sequence cloned from the enteroepithelial stage of T.gondii (strain:Beverley).The DNA encoding Toxo14-3-3 protein was amplified by RT-PCR.The PCR products were cloned into pGEM-T vector and the inserted DNA fragments were confirmed by both restriction endonuclease digestion and PCR followed by DNA sequencing.Results A complete open reading frame(ORF)of 789bp was verified,which was identical to Toxo14-3-3 submitted to GenBank(Accession No.ABO 12775),with 2 mutants of degenerate codons.Conclusions Gene encoding Toxo 14-3-3 signal transduction protein has been constructed to clone vector,which lay the foundation of further research..