本文關(guān)鍵詞： 惡性瘧原蟲 CRISPR/Cas系統(tǒng) 基因編輯　出處：《中國寄生蟲學(xué)與寄生蟲病雜志》2017年03期 　論文類型：期刊論文

【摘要】：目的利用單鏈引導(dǎo)RNA(sg RNA)串聯(lián)表達框架編輯惡性瘧原蟲(Plasmodium falciparum)K13和NUP116基因。方法根據(jù)惡性瘧原蟲K13和NUP116基因序列設(shè)計引物,PCR擴增K13和NUP116基因的同源臂,并引入突變位點。PCR串聯(lián)基因K13和NUP116的sg RNA,將同源臂和串聯(lián)的sg RNA與載體p L6cs連接,構(gòu)建用于電擊轉(zhuǎn)染實驗的載體p L6-K13-NUP116,將其與表達Cas9蛋白的質(zhì)粒一同通過電擊轉(zhuǎn)染法,轉(zhuǎn)入惡性瘧原蟲體內(nèi),利用成簇的規(guī)律間隔的短回文重復(fù)序列及其相關(guān)蛋白9(CRISPR/Cas9)系統(tǒng)對基因進行編輯修飾,通過篩選藥物二氫葉酸還原酶抑制劑(WR)和殺稻瘟菌素(BSD)篩選轉(zhuǎn)基因瘧原蟲,提取轉(zhuǎn)基因瘧原蟲的基因組,對其進行PCR檢測,最終通過測序鑒定K13和NUP116基因是否成功被突變修飾。結(jié)果 PCR擴增出K13和NUP116串聯(lián)的同源臂(K13全長557 bp,NUP116全長569 bp)以及串聯(lián)的sg RNA,與載體p L6cs連接后成功獲得用于電擊轉(zhuǎn)染實驗的表達載體p L6-K13-NUP116。電擊轉(zhuǎn)染后通過藥物篩選轉(zhuǎn)基因瘧原蟲,培養(yǎng)至第28天涂片鏡檢發(fā)現(xiàn)瘧原蟲。PCR檢測結(jié)果顯示,轉(zhuǎn)基因瘧原蟲K13和NUP116基因突變修飾成功。測序表明,K13和NUP116基因的目標位點被成功突變。結(jié)論基于CRISPR/Cas9編輯技術(shù)的串聯(lián)sg RNA表達載體可同時對惡性瘧原蟲K13和NUP116基因進行編輯。
[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.
【作者單位】： 蚌埠醫(yī)學(xué)院病原生物學(xué)教研室安徽省感染與免疫重點實驗室;同濟大學(xué)醫(yī)學(xué)院;
【基金】：國家自然科學(xué)基金(No.81630063) 安徽高�？蒲袆�(chuàng)新平臺團隊項目(No.2016-40)~~
【分類號】：R382.31
【正文快照】： *Corresponding author,E-mail:qfzhangsh@aliyun.com瘧疾主要流行于非洲和東南亞等熱帶地區(qū)。隨著經(jīng)濟發(fā)展和公共衛(wèi)生條件的改善,以及消除瘧疾行動計劃的實施,目前我國僅云南等少數(shù)地區(qū)有本地感染病例[1-2]。但是,近年來隨著我國與瘧疾流行地區(qū)經(jīng)濟交往和人員往來逐漸頻繁,輸

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