弓形虫p35蛋白的基因克隆及其分子特征

CLONING AND MOLECULAR CHARACTERIZATION OF TOXOPLASMA GONDII P35 PROTEIN

目的 探讨 p35蛋白作为一个鉴别急性感染的诊断抗原和疫苗候选的应用价值。 方法 从弓形虫RH株分离总的RNA ,应用弓形虫EST基因库合成扩增p35基因片段引物 ,结合 5‘和 3‘末端cDNA快速扩增法进行 p35基因 5‘和 3‘末端cDNA的扩增 ,TA克隆RT -PCR产物及DNA顺序分析 ;用蛋白质分析软件解析 p35基因编码的氨基酸的亲水性和可能存在的T、B细胞的表位。结果 p35基因含有 15 37个碱基 ,编码 378个氨基酸 ,其氨基酸的亲水性区域分布在氨基酸的 2 0 1到2 2 5和 30 1到 340号 ,且含有多个T、B细胞的表位。结论 p35蛋白的C端区有高的亲水性 ,并含有T、B细胞的表位 ,显示了p35蛋白应用于诊断和疫苗开发的可行性。

S:Aim p35 protein of Toxoplasma gondii(T.gondii)was cloned and expressed,so as to develop it into a vaccine candidate and diagonose antigen which differentiate between recently acquired infection and that acquired in the more distant past.Methods Total RNA was isolated from T.gondi,accoding to T.gondi expressd sequence Tags(EST)database,primers was designed for amplification p35 gene fragment by RT-PCR,and complete cDNA of p35 gene was amplified by 5` rapid amplification of cDNA Ends(RACE)and 3`RACE.The PCR products were cloned and sequenced,hydrophilicity regions of p35 amino acid,and epitopes of T and B cell were analylized by computer.Results cDNA of p35 from T.gondii RH strain was 1537bp in length,including 1134bp of open reading frame,and code 378 amino acid.The hydrophilicity regions were located from 201-225 and 301-340 amino acid,and p35 protein showed several epitopes of T and B cell.Conclusion Hydrophilicity regions of p35 protein were located at C termination of peptide,and included epitopes of T and B cell,and p35 protein seems to be not only a candidate vaccine,but also a target antigen for immunodiagonosis of T.gondii.