弓形虫重组质粒pBudCE4.1-AMA1-ROP18的构建及鉴定  

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

作　　者：石娜[1] 何深一[1] 陈琳[1] 周怀瑜[1] 丛华[1] 白杨[1] 赵广会[1] 赵群力[1] 

机构地区：[1]山东大学医学院寄生虫学教研室,济南250012

出　　处：《山东大学学报（医学版）》2012年第10期56-60,67,共6页Journal of Shandong University:Health Sciences

基　　金：国家自然科学基金(81071373);山东省自然科学基金(ZR2009CM079);家畜疫病病原生物学国家重点实验室开放基金(SKLVEB2011KFKT005);山东省优秀中青年科学家科研奖励基金(BS2009SW008)

摘　　要：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。Objective To construct, clone and identify the DNA recombinant plasmid pBudCE4. 1-AMA1-ROPI8. Methods Amplify the target genes by PCR, construct the eukaryotic recombinant plasmid pBudCE4. I-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 tran- scribed and expressed correctly in mammalian cells by identification of RT-PCR and SDS-PAGE. Conclusion The re- combinant plasmid pBudCFA. 1-AMA1-ROP18 is constructed successfully and expressed in HFF cells stably.

关 键 词：弓形虫属 质粒 细胞转染 弓形虫顶端膜抗原1 棒状体蛋白18 

分 类 号：R382.5[医药卫生—医学寄生虫学]