基于CRISPR/Cas9的PALM基因敲除约氏疟原单克隆虫株的构建  

Construction of monoclonal PALM gene knockout strain of Plasmodium yoelii based on CRISPR/Cas9 technique

作　　者：曹玉洁 张玲红 苗昱辰 卢里 李春草 张璐 胡瑞 李江艳 夏惠[1] 陶志勇[1] 方强[1,2] CAO Yu-jie;ZHANG Ling-hong;MIAO Yu-chen;LU Li;LI Chun-cao;ZHANG Lu;HU Rui;LI Jiang-yan;XIA Hui;TAO Zhi-yong;FANG Qiang(Department of Microbiology and Parasitology,Bengbu Medical College,Bengbu Anhui 233030;School of Fundamental Sciences,Bengbu Medical College,Bengbu Anhui 233030;Department of Clinical Laboratory,The First Affiliated Hospital of Bengbu Medical College,Bengbu 233004,China)

机构地区：[1]蚌埠医学院病原生物学教研室,安徽蚌埠233030 [2]蚌埠医学院公共基础学院,安徽蚌埠233030 [3]蚌埠医学院第一附属医院检验科,安徽蚌埠233004

出　　处：《蚌埠医学院学报》2023年第1期60-65,共6页Journal of Bengbu Medical College

基　　金：安徽省高校自然科学研究重大项目(KJ2021ZD0077);安徽省“特支计划”创新人才项目;蚌埠医学院“512”人才项目(by51201101);安徽省高校自然科学研究重点项目(KJ2020A0567);蚌埠医学院研究生创新项目(Byycx21015);安徽省高校自然科学研究重大项目(2022AH040218)。

摘　　要：目的:基于CRISPR/Cas9技术构建并鉴定Plasmodium yoelii 17XNL PALM基因敲除单克隆虫株。方法:根据PlasmoDB数据库中的约氏疟原虫P.yoelii 17XNL PALM基因序列信息,设计引物与sgRNA。PCR扩增左、右同源臂,将同源臂和sgRNA与载体pYCm-golden-blue连接,构建用于基因敲除的CRISPR工具质粒pYCm-PALM KO。pYCm-PALM KO CRISPR质粒电转染至P.yoelii 17XNL裂殖体,尾静脉注射感染小鼠,24 h后采血查获疟原虫后,给予小鼠6 mg/mL乙胺嘧啶喂水进行药物压力筛选基因敲除疟原虫。此后每2天鼠尾采血涂片镜检疟原虫,喂药8 d时提取疟原虫基因组DNA进行PCR鉴定,最终经测序确定是否发生PALM基因敲除。将已证实存在PALM基因敲除疟原虫的鼠血采用有限稀释法接种小鼠,8 d后采血镜检疟原虫,PCR鉴定,筛选获取单克隆虫株。将获取的PALM基因敲除的单克隆虫株和P.yoelii 17XNL各接种至6只昆明小鼠体内,每只接种1×105感染疟原虫的红细胞(iRBC)。对感染后的小鼠每2天鼠尾取血制作血涂片,经吉姆萨染色后镜检计算原虫率,记录数据进行统计学分析。结果:PCR扩增出的PALM左右同源臂各600 bp以及sgRNA序列,与载体pYCm-golden-blue连接后成功获得用于电转染的PALM基因敲除的CRISPR质粒。该质粒电转染的疟原虫在小鼠体内经药物压力筛选8 d,用PALM基因内部引物经PCR检测显示PALM基因缺失,用基因组特异性引物经PCR检测出现阳性重组条带,片段长度1246 bp。随后将阳性重组条带切胶测序,测序结果进一步证实PALM基因敲除成功。在获得约氏疟原虫PALM基因敲除株的基础上,通过有限稀释感染12只小鼠,经PCR自2只小鼠血液中扩增出单一PALM基因敲除条带,检测不到野生型疟原虫的残留,获得了2个100%PALM基因敲除的单克隆虫株。PALM基因敲除单克隆株与P.yoelii 17XNL感染小鼠后均出现原虫血症,且2组小鼠原虫率逐日升高。感染后第10天,2组小鼠原虫血症水平�Objective:To construct and identify monoclonal Plasmodium yoelii 17XNL PALM gene knockout strain based on CRISPR/Cas9 technique.Methods:According to the sequence information of P.yoelii 17XNL PALM gene in PlasmoDB database,primers and sgRNA were designed,and the left and right homologous arms were amplified by PCR.The homologous arms and sgRNA were ligated with vector pYCm-golden-blue to construct CRISPR tool plasmid pYCm-PALM KO for gene knockout.pYCm-PALM KO plasmid was transfected into P.yoelii 17XNL by electroporation.Twenty-four hours after the transfected parasites were injected into the mice via tail vein,the blood of mice was collected for examination of Plasmodium.The Plasmodium-positive mice were fed 6 mg/mL pyrimethamine to screen PALM gene knockout malaria parasites.After that,the blood was collected from the mice every 2 days and smears were taken for microscopic examination of malaria parasites.On the 8th day of screening,PCR and sequencing were performed to identify whether PALM gene knockout was successful.The red blood cells infected with PALM gene knockout malaria parasites were injected into the mice by limited dilution method.Eight days later,the blood of mice were collected to detect Plasmodium with microscopic examination and identify by PCR to screen monoclonal malaria parasite strains.The obtained PALM gene knockout monoclonal strain and P.yoelii 17XNL were inoculated into 6 Kunming mice,each inoculated with 1×10~5 Plasmodium-infected red blood cells(iRBC).The infected mice were collected every two days to make blood smears,and the protozoa rate was calculated by Giemsa staining posterior microscopy,the data were recorded,and the SPSS 19.0 software was used for statistical analysis.Results:Plasmodium electrotransfected with this plasmid was screened by drug stress in mice for 8 days.The 600bp left and right homologous arms of PALM and sgRNA sequence amplified by PCR were ligated with the vector pYCm-golden-blue to obtain the CRISPR tool plasmid pYCm-PALM KO for PALM gene knockout,which wa

关 键 词：约氏疟原虫 PALM基因 基因敲除 CRISPR/Cas9 

分 类 号：R382.31[医药卫生—医学寄生虫学]