弓形虫529 bp重复序列基因的PCR阳性质控质粒的构建

目的：构建含有弓形虫529 bp高度重复序列基因的重组质粒,为基于该基因的弓形虫病分子生物学诊断建立标准阳性对照品。方法：以弓形虫RH株基因组DNA为模板,对529 bp的基因片段进行扩增,将纯化后的PCR产物与pMD18-T载体连接后转入大肠埃希菌DH-5α中,提取重组质粒PCR及测序鉴定。以弓形虫529 bp基因为靶基因设计的检测引物,扩增弓形虫基因组DNA及质粒DNA。结果：PCR产物序列与GENBANK中弓形虫529 bp基因序列完全一致。以弓形虫基因组DNA和质粒DNA为模板,成功扩增出预期的249bp基因片段。结论：成功构建可作为标准阳性对照品的含有弓形虫529 bp高度重复序列基因的重组质粒pMD-18-Tox,为经济实用的弓形虫诊断试剂盒的研制奠定了基础。

Construction of standard control plasmid with repetitive 529 bp DNA fragment of Toxoplasma gondii for PCR assay

Objective：To establish cloning vector and standard positive control of molecular biology diagnosis of toxoplasmosis based on the 529 bp repetitive fragment to detect the presence of Toxoplasma gondii（T.gondii）.Methods：The repetitive 529 bp DNA fragment from T.gondii was amplified by PCR and was inserted into pMD18-T vectors to construct recombinant plasmid and transformed to E.coli DH-5α.A pair of primers were designed and synthesized based on the 529 bp sequence to amplify the 249 bp fragment gene and plasmid DNA of T.gondii.Results：The 529 bp DNA fragment of T.gondii was successfully amplified and inserted into pMD18-T vectors after purification.The 249 bp fragment was amplified by PCR using T.gondii gemome DNA and recombinant plasmid DNA as template.Conclusions：Successfully cloned assay of 529 bp DNA fragment of T.gondii and amplified the 249 bp DNA fragment have laied the foundation of development of T.gondii diagnostic kit.