利用sgRNA串联表达框架编辑恶性疟原虫K13和NUP116基因的研究

发布时间：2018-01-19 11:14

本文关键词： 恶性疟原虫 CRISPR/Cas系统基因编辑　出处：《中国寄生虫学与寄生虫病杂志》2017年03期 　论文类型：期刊论文

【摘要】：目的利用单链引导RNA(sg RNA)串联表达框架编辑恶性疟原虫(Plasmodium falciparum)K13和NUP116基因。方法根据恶性疟原虫K13和NUP116基因序列设计引物,PCR扩增K13和NUP116基因的同源臂,并引入突变位点。PCR串联基因K13和NUP116的sg RNA,将同源臂和串联的sg RNA与载体p L6cs连接,构建用于电击转染实验的载体p L6-K13-NUP116,将其与表达Cas9蛋白的质粒一同通过电击转染法,转入恶性疟原虫体内,利用成簇的规律间隔的短回文重复序列及其相关蛋白9(CRISPR/Cas9)系统对基因进行编辑修饰,通过筛选药物二氢叶酸还原酶抑制剂(WR)和杀稻瘟菌素(BSD)筛选转基因疟原虫,提取转基因疟原虫的基因组,对其进行PCR检测,最终通过测序鉴定K13和NUP116基因是否成功被突变修饰。结果 PCR扩增出K13和NUP116串联的同源臂(K13全长557 bp,NUP116全长569 bp)以及串联的sg RNA,与载体p L6cs连接后成功获得用于电击转染实验的表达载体p L6-K13-NUP116。电击转染后通过药物筛选转基因疟原虫,培养至第28天涂片镜检发现疟原虫。PCR检测结果显示,转基因疟原虫K13和NUP116基因突变修饰成功。测序表明,K13和NUP116基因的目标位点被成功突变。结论基于CRISPR/Cas9编辑技术的串联sg RNA表达载体可同时对恶性疟原虫K13和NUP116基因进行编辑。
[Abstract]:Objective to edit Plasmodium falciparum from Plasmodium falciparum by using a single strand guided RNA(sg tandem expression frame. Methods primers were designed according to the sequence of K13 and NUP116 genes of Plasmodium falciparum. The homologous arm of K13 gene and NUP116 gene was amplified by PCR, and the mutation site. PCR tandem gene K13 and sg RNA of NUP116 were introduced. The recombinant plasmid pL6-K13-NUP116 was constructed by ligating the homologous arm and serial sg RNA with the vector pL6cs. The plasmids expressing Cas9 protein were transfected into Plasmodium falciparum by electroporation. The gene was edited and modified by a cluster of regular interval short palindrome repeats and its associated protein 9 / CRISPRP / Cas9 (CRISPRP / Cas9) system. The transgenic plasmodium was screened by screening dihydrofolate reductase inhibitor (WR) and rice blast fungicide (BSD). The genomes of transgenic plasmodium were extracted and detected by PCR. Finally, the K13 and NUP116 genes were sequenced to determine whether they were successfully modified by mutation. Results the homologous arm K13 of K13 and NUP116 was amplified by PCR, and the length of K13 was 557bp. The total length of NUP116 is 569 BP) and the series sg RNA. The expression vector pL6-K13-NUP116was successfully obtained by ligation with pL6cs. After electroporation, the transgenic plasmodium was screened by drugs. On the 28th day of culture, the results of microscopical examination of Plasmodium falciparum showed that the mutation of K13 and NUP116 gene of the transgenic plasmodium was successful, and the sequencing showed that. The target sites of K13 and NUP116 genes have been successfully mutated. Conclusion A series sg based on CRISPR/Cas9 editing technique is proposed. RNA expression vector can edit the gene of Plasmodium falciparum K13 and NUP116 simultaneously.
【作者单位】：蚌埠医学院病原生物学教研室安徽省感染与免疫重点实验室;同济大学医学院;
【基金】：国家自然科学基金(No.81630063) 安徽高校科研创新平台团队项目(No.2016-40)~~
【分类号】：R382.31
【正文快照】： *Corresponding author,E-mail:qfzhangsh@aliyun.com疟疾主要流行于非洲和东南亚等热带地区。随着经济发展和公共卫生条件的改善,以及消除疟疾行动计划的实施,目前我国仅云南等少数地区有本地感染病例[1-2]。但是,近年来随着我国与疟疾流行地区经济交往和人员往来逐渐频繁,输

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