弓形虫重组质粒pBudCE4.1-AMA1-ROP18的构建及鉴定

目的 构建pBudCE4.1-AMA1-ROP18 DNA重组质粒，并对其进行克隆及鉴定。方法 采用PCR法对目的基因进行扩增，构建pBudCE4.1-AMA1-ROP18重组质粒，转染至人包皮成纤维细胞（HFF）进行表达并用SDS-PAGE和β-actin进行鉴定。 结果 经测序、双酶切和PCR鉴定，重组质粒pBudCE4.1-AMA1-ROP18构建正确；经RT-PCR和SDS-PAGE鉴定， AMA1、ROP18基因均在哺乳动物细胞中正确转录与表达。结论 成功构建pBudCE4.1-AMA1-ROP18重组质粒，并可在HFF细胞中稳定表达。

Construction and identification of the recombinant plasmid pBudCE4.1-AMA1-ROP18 from Toxoplasma gondii

Objective To construct, clone and identify the DNA recombinant plasmid pBudCE4.1-AMA1-ROP18. Methods Amplify the target genes by PCR, construct the eukaryotic recombinant plasmid pBudCE4.1-AMA1-ROP18 and express it in human foreskin fibroblast (HFF) cells by transfection. Then identify the plasmid with SDS-PAGE and beta actin. Results We proved that the recombinant plasmid pBudCE4.1-AMA1-ROP18 was constructed correctly by identification of sequencing, double enzyme digestion and PCR.We proved that AMA1 gene and ROP18 gene both transcribed and expressed correctly in mammalian cells by identification of RT-PCR and SDS-PAGE. Conclusion The recombinant plasmid pBudCE4.1-AMA1-ROP18 is constructed successfully and expressed in HFF cells stably.