CN116445530B - 磷酸核糖焦磷酸激酶prps基因在制备抗弓形虫病药物中的应用 - Google Patents
磷酸核糖焦磷酸激酶prps基因在制备抗弓形虫病药物中的应用 Download PDFInfo
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Abstract
本发明提供磷酸核糖焦磷酸激酶PRPS基因在制备抗弓形虫病药物中的应用。本发明通过分析磷酸核糖焦磷酸激酶(PRPS)在弓形虫生长中的作用,显示通过CRISPR/Cas9系统敲除野生型弓形虫的TgPRPS基因后,缺失TgPRPS基因减缓虫体体外的复制和虫体体外空斑的形成,可以显著减缓虫体体外生长,并在体外传代培养15d后虫体完全死亡。表明TgPRPS基因对弓形虫生长非常重要,能够作为药物作用靶点,在治疗药物的开发和弓形虫病的防治中具有重要的意义。
Description
技术领域
本发明属于基因工程技术领域。更具体地,涉及磷酸核糖焦磷酸激酶PRPS基因在制备抗弓形虫病药物中的应用。
背景技术
弓形虫是一种专性细胞内寄生原虫,可以感染包括人在内的几乎所有温血动物,猫为其终末宿主。弓形虫感染引起弓形虫病。孕妇感染弓形虫可导致流产、产死胎或胎儿畸形等。儿童和老人感染弓形虫易患脑膜炎和肺炎等疾病;免疫缺陷患者感染弓形虫会导致死亡。弓形虫感染猪、牛、羊等家畜也会引起繁殖障碍或流产,严重危害畜牧业的发展,造成巨大的经济损失。
目前临床上采用磺胺类药物来抗弓形虫感染,如乙胺嘧啶、磺胺嘧啶等药物,但这些只对急性感染期的速殖子有效,对慢性感染期的缓殖子或组织包囊无效。因此迫切需要发掘更多的药物靶点,研制出新型抗弓形虫病的药物。代谢途径与虫体的生长和繁殖紧密关联,其代谢关键酶可以成为潜在的药物设计靶点。现有技术公开在药物靶标发掘方面做了诸多尝试,如敲除弓形虫支链酮酸脱氢酶(BCKDH),尽管影响TCA循环,造成严重的生长缺陷,但是虫体依然可以存活。此外,抑制丙酮酸激酶1(PYK1)导致碳代谢紊乱,降低ATP水平,积累大量支链淀粉,造成严重的生长缺陷,但是虫体也能够传代培养。尽管这些基因显示了一定的药靶潜力,但是因其缺失不会导致虫体死亡,在作为药物靶点时有一定局限性。因此,发掘更多的潜在药物靶点,对筛选抗弓形虫感染药物具有重要的意义。
发明内容
本发明提供磷酸核糖焦磷酸激酶PRPS基因在制备抗弓形虫病药物中的应用。
本发明的目的是提供弓形虫磷酸核糖焦磷酸激酶TgPRPS基因的应用。
本发明另一目的是提供抑制或下调弓形虫磷酸核糖焦磷酸激酶TgPRPS基因的物质的应用。
本发明又一目的是提供一种弓形虫病药物。
本发明又一目的是提供一种弓形虫TgPRPS基因敲除虫株的构建方法。
本发明再一目的是提供弓形虫TgPRPS基因敲除虫株的应用。
本发明上述目的通过以下技术方案实现:
本发明通过分析磷酸核糖焦磷酸激酶(PRPS)在弓形虫生长中的作用,发现在Ⅰ型野生型虫株DiCre中缺失TgPRPS(TgPRPS基因编号:TGGT1_220100)后,可以显著减缓虫体体外生长,并在体外传代培养15d后虫体完全死亡,显示TgPRPS对弓形虫生长非常重要,能够作为弓形虫的药物作用靶点。
因此,以下应用均在本发明的保护范围内:
弓形虫磷酸核糖焦磷酸激酶TgPRPS基因在调控弓形虫生长中的应用。
弓形虫磷酸核糖焦磷酸激酶TgPRPS基因作为靶点在制备或筛选抗弓形虫病药物中的应用。
进一步地,通过敲除弓形虫TgPRPS基因来减缓弓形虫的生长速率。
抑制或下调弓形虫磷酸核糖焦磷酸激酶TgPRPS基因的物质在制备抗弓形虫病的药物中的应用。
抑制或下调弓形虫磷酸核糖焦磷酸激酶TgPRPS基因的物质在降低弓形虫的生长速率或在制备降低弓形虫生长速率的制剂中的应用。
本发明提供一种抗弓形虫病药物,含抑制或下调弓形虫磷酸核糖焦磷酸激酶TgPRPS基因的物质。
本发明提供一种弓形虫TgPRPS基因敲除虫株的构建方法,利用CRISPR/Cas9系统敲除弓形虫的TgPRPS基因。
上述构建方法包含以下步骤:
S1.设计敲除TgPRPS基因的靶点sgRNA序列;
S2.将步骤S1中得到的sgRNA序列构建到CRISPR/Cas9敲除质粒中;
S3.构建TgPRPS同源片段,并与步骤S2的敲除质粒共转染到野生型弓形虫虫株中;
S4.采用反向筛选技术,通过乙胺嘧啶药物筛选、PCR鉴定和IFA鉴定获得敲除PRPS基因的弓形虫遗传改造虫株。
优选地,步骤S1中采用的靶点sgRNA序列依次如SEQ ID NO.1~4所示;步骤S3中TgPRPS同源片段的序列如SEQ ID NO.13所示。
另外,作为一种可优选的实施方案,本发明提供具体的弓形虫TgPRPS基因敲除虫株的构建方法:
以pSAG1-Cas9-TgU6-ccdb-sgMIC3质粒为模板,构建pSAG1-Cas9-TgU6-dg TgPRPS质粒;制备TgPRPS-5UTR::DHFR*::TgPRPS-3UTR同源模板;将二者共电转至野生型DiCre虫株中,通过乙胺嘧啶药物筛选、PCR鉴定获得弓形虫遗传改造虫株TgPRPS-cKD。之后加入50nM雷帕霉素(Rapamycin,RAPA)处理24h后,在倒置荧光显微镜下观察YFP荧光基因的表达情况,YFP阳性(YFP+)的速殖子即为TgPRPS基因敲除虫株。
本发明提供一种弓形虫TgPRPS基因敲除虫株,由上述方法构建得到。
本发明具有以下有益效果:
本发明提供磷酸核糖焦磷酸激酶基因在制备抗弓形虫病药物中的应用。本发明通过分析磷酸核糖焦磷酸激酶(PRPS)在弓形虫生长中的作用,显示通过CRISPR/Cas9系统敲除野生型弓形虫的TgPRPS基因后,缺失TgPRPS基因减缓虫体体外的复制和虫体体外空斑的形成,可以显著减缓虫体体外生长,并在体外传代培养15d后虫体完全死亡。表明TgPRPS基因对弓形虫生长非常重要,能够作为药物作用靶点,在治疗药物的开发和弓形虫病的防治中具有重要的意义。
附图说明
图1为敲除弓形虫TgPRPS的策略示意图;
图2为TgPRPS-cKD单克隆虫株的PCRs鉴定图;
图3为TgPRPS基因敲除虫株的IFA鉴定图;
图4为TgPRPS-cKD虫株体外复制实验结果图;
图5为TgPRPS-cKD虫株体外空斑实验结果图。
具体实施方式
以下结合说明书附图和具体实施例来进一步说明本发明,但实施例并不对本发明做任何形式的限定。除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明,以下实施例所用试剂和材料均为市购。
实施例1质粒的构建
在弓形虫体内经磷酸戊糖代谢途径催化生成的核糖-5-磷酸(R5P)在磷酸核糖焦磷酸激酶(PRPS)催化下生成5-磷酸核糖-1-焦磷酸(PRPP),为嘌呤核苷酸和嘧啶核苷酸代谢提供中间产物和前体物质。本发明研究发现磷酸核糖焦磷酸激酶(PRPS)与弓形虫生长相关,具体研究如下:
1、pSAG1-Cas9-TgU6-sg-in5’-TgPRPS和pSAG1-Cas9-TgU6-sg-in3’-TgPRPS质粒构建
(1)用gRNA在线设计网站(http://www.e-crisp.org/E-CRISP/designcrispr.html)对TgPRPS(网址为https://toxodb.org/toxo/app的ToxoDB数据库中TgPRP S基因编号:TGGT1_220100)打靶位点进行设计,设计得到的靶点引物序列如下表1所示。
表1构建TgPRPS特异性CRISPR/Cas9质粒所使用的引物
以商业化的pSAG1-Cas9-TgU6-ccdb-sgMIC3质粒为模板,按照碧云天Anne alingBuffer for DNA Oligos(5×)试剂盒说明进行操作,分别进行TgPRPS特异性CRISPR/Cas9质粒的构建,进行PCR扩增,按照下表2中的反应体系,将各试剂液体混匀后,置于PCR仪中反应,反应程序如下表3所示。
表2PCR扩增反应体系
表3PCR扩增反应程序
(2)双片段连接:按照诺唯赞双片段克隆试剂盒说明书进行操作,反应体系配置如下表4所示。
表4双片段克隆反应体系
注:表中(1)为gRNA1-PRPS-5UTR引物对扩增体系,(2)为gRNA2-PR PS-3UTR引物对扩增体系。
每片段最适使用量=[0.02×片段碱基对数]ng,将上述液体混匀后,PCR仪中37℃反应30min,冰浴5min后做转化。取10μL反应产物转化入100μL的DH5α大肠杆菌感受态细胞中,涂LB/Amp+平板,37℃倒置培养8-12h;挑取单菌落置于1mL LB/Amp液体培养基中,37℃/180rpm振荡培养至菌液浑浊;取500μL菌液进行测序分析,若测序结果显示靶点序列已完全被替换,即pSA G1-Cas9-TgU6-sg-in5’-TgPRPS和pSAG1-Cas9-TgU6-sg-in3’-TgPRPS质粒均构建成功。
2、pSAG1-Cas9-TgU6-dgTgPRPS质粒构建
(1)PCR扩增目的片段:以pSAG1-Cas9-TgU6-sg-in3’-TgPRPS质粒为模板,利用如下表5设计合成的引物PCR扩增相应目的片段,PCR扩增所采用的反应体系和程序如下表6和表7所示。
表5构建特异性CRISPR/Cas9质粒所使用的引物
表6 PCR扩增反应体系
表7 PCR扩增程序
(2)DpnⅠ消化:为了除去模板DNA,利用如下表8和表9所示体系和程序进行消化。
表8 Dpn Ⅰ消化体系
表9 Dpn Ⅰ消化程序
(3)目的片段回收:DNA电泳,将目的条带片段进行切胶,放入2mL EP管中,使用DNA回收试剂盒对目的片段回收。
(4)PCR扩增载体骨架:以pSAG1-Cas9-TgU6-sg-in5’-TgPRPS质粒为模板,利用如下表10设计合成的引物PCR扩增相应载体骨架,PCR扩增所采用的反应体系和程序同表6和7所示。
表10构建特异性CRISPR/Cas9质粒所使用的引物
(5)DpnⅠ消化:为了除去模板DNA,采用的反应体系和程序同表8和表9所示进行消化。
(6)载体骨架回收:DNA电泳,将目的条带进行切胶,放入2mL EP管中,使用DNA回收试剂盒对载体骨架回收。
(7)双片段连接:按照诺唯赞双片段克隆试剂盒说明书进行操作,反应体系配置如下表11所示。
表11双片段克隆反应体系
每片段最适使用量=[0.02×片段碱基对数]ng,将上述液体混匀后,PCR仪中37℃反应30min,冰浴5min后做转化。取10μL反应产物转化入100μL的DH5α大肠杆菌感受态细胞中,经氨苄青霉素(Amp)筛选、PCR鉴定和DN A测序,获得正确的pSAG1-Cas9-TgU6-dgTgPRPS质粒。
实施例2弓形虫基因敲除虫株TgPRPS-cKD的构建
1、TgPRPS-5UTR::DHFR*::TgPRPS-3UTR同源模板的制备
(1)弓形虫RNA的提取与cDNA的制备
将纯化收集的弓形虫虫体5000rpm离心6min,弃上清,加入1mL Trizol重悬沉淀,吹打混匀10次后将样品转入2mL无RNAse的EP管中,涡旋3min,使虫体充分裂解,放置10min;加入200μL氯仿,振荡15s,冰上静置5min,12000rpm,4℃,离心10min;用无RNAse的枪头吸取上层水相置于新的2mL无RNAse的EP管中,加入等体积的异丙醇,充分混匀后,置于-20℃沉淀1h;12000rpm,4℃,离心10min,弃去上清,留管底白色RNA沉淀,加入1mL用DEPC水配成的75%乙醇,洗涤沉淀;8000rpm,4℃,离心5min,弃上清,在超净台中风干,加入50μL无RNAseDEPC水,溶解RNA沉淀。
使用Nanodrop 2000紫外分光光度计测定总RNA浓度和纯度,并对所提取的总RNA质量用1%琼脂糖凝胶电泳鉴定;再取1μg总RNA进行反转录,先去除gDNA,采用表12所示体系去除gDNA,在PCR仪中42℃反应2min,室温30min,冰上冷却后;接着按照表13所示体系进行反转录为cDNA,在PCR仪中37℃ 15min,85℃ 5s,产物-80℃储存待用。
表12去除gDNA反应体系
表13反转录cDNA反应体系
(2)PCR扩增目的片段
分别以cDNA和pTub::Loxp::GO::Loxp::YFP::Ter::DHFR*(Tub:启动子;Loxp:34bp序列;YFP:报告基因;Ter:终止子;DHFR:药筛标签)质粒为模板,利用如下表14设计合成的引物PCR扩增相应目的片段,PCR扩增所采用的反应体系和程序同实施例1中表6和下表15所示。
表14构建同源重组质粒所使用的引物
表15PCR扩增程序
(3)目的片段回收:DNA电泳,将目的条带片段进行切胶,放入2mL EP管中,使用DNA回收试剂盒对目的片段回收。
(4)双片段连接:按照诺唯赞双片段克隆试剂盒说明书进行操作,反应体系配置如下表16所示。
表16双片段克隆反应体系
每片段最适使用量=[0.02×片段碱基对数]ng,将上述液体混匀后,PCR仪中37℃反应30min,冰浴5min后做转化。取10μL反应产物转化入100μL的DH5α大肠杆菌感受态细胞中,经氨苄青霉素(Amp)筛选、PCR鉴定和DNA测序,获得正确的同源重组质粒pTub::Loxp::PRPS::Loxp::YFP::Ter::DHFR*。
(5)同源重组片段的扩增:用下表17设计合成的引物,使用高保真酶Phanta对鉴定正确的pTub::Loxp::PRPS::Loxp::YFP::Ter::DHFR*进行目的片段的PCR扩增,电泳、切胶回收,测定浓度,得到TgPRPS-5UTR::DHFR*::TgPRPS-3UTR同源片段。
表17同源重组片段扩增所使用的引物
TgPRPS-5UTR::DHFR*::TgPRPS-3UTR的具体序列如SEQ ID NO.13所示:GCTTGTCGAGGTTATGCGTCGTGTCCTTTTTTCCCTGCCGCGCTCTTGTGCGACGGCCAGTCTTAAGCTCGGGCCCCAAATAATGATTTTATTTTGACTGATAG
TGACCTGTTCGTTGCAACACATTGATGAGCAATGCTTTTTTATAATGCCAAC
TTTGTACAAAAAAGCAGGCTAGGCGATTAAGTTGGGTAACGCCAGGGTTTT
CCCAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGCGCGTAATACGAC
TCACTATAGGGCGAATTGGGTACCGGGCCCCCCCTCGACGGTATCGATAAG
CTTAACCACAAACCTTGAGACGCGTGTTCCAACCACGCACCCTGACACGC
GTGTTCCAACCACGCACCCTGAGACGCGTGTTCTAACCACGCACCCTGAG
ACGCGTGTTCTAACCACGCACCCTGAGACGCGTGTTCAAGCTTGCCTGCAT
TGGGTGCGGTTGGTGATCCTGGTTGGACCGGTGGAGATGCGCGCGCACGA
AGGGGATGTGTCAGAAACATTTTGTTTGTTCTCTGTGAACTTTTAGATGTGT
TAAAGGCGGCGAATATTAGCAGAGAGTCCTCCTTGTTCCATTCTCTCTTGAA
TTTCGCCCTTTCCTTCTCTTTGCGAGTGTGGTAGAGAACAAGCACTCGTTC
GCCGTCCCTGACGACGCAACCCGCGCAGAAGACATCCACCAAACGGTGTT
ACACAATCACCTTGTGTGAAGTTCTTGCGGAAAACTACTCGTTGGCATTTT
TTCTATAACTTCGTATAGCATACATTATACGAAGTTATAGAATTCCGACAAAA
TGTGGATCGGTTCCAGGAGCATTCGTTCGCATGCGGGCGAGCTTGCGCTCG
CTCGCCTGAGAGCGAGGGCAGACCATCTTTTCCTCGACTTCACTGCCGCGA
ACACGTCGGTGAAAAAGTACCATTTCATGACATCTCGTGTATCACCGCTTCT
TCGCCGTCCTTCGAGGGAACAGGCGGCCGCGGCAAAGTACTTATACGCCTC
GGTCCCTCCCCTCTGTCAAATTTCCGCGGCGACTCCTAGACGGCGTGGCGC
AGACGATAAGCGCGCGGGCGGCAACTCACGAGCAGGCAACAAGTCCTCG
GGAAACGAGAAGCGACACTTCTCTTTCGCTGCAGCAGGAGGCCTCGGATC
TGTCCTCCTGTGTGGCGCGCCTCTCGCTGGAGGCGAGCGACGCGACGCTG
CTCCGTCGTCAGCGGAAACGGAGAAACGAAACTCGTCAGCGTCAACTACC
ACCCGATGTTCTTCGTTCTTCCGCGCTCCTTCGTCGTCCGATGCATCTTTTAT
TGAATCGAAGTTTTACTTTTTCTCTAGGGCGTTTAGCCAGCAACCAGCCTTG
TGTCAAACCGCCGCGCCCGTGCCGAAAACCGACAAAGGACGAAACCAGC
AAGGCGCCCGACCAACGGACGACCATGAGGAGCCCGCTCACCACGACAGT
CCGTTCTGGCGTTCTCAGGAGAACCGTCCCTTCGACCGCAAGCTGGGAGA
CGCTCTGTTGTTCTGCGGCAACAGCAACGAGCCACTGGCGCGTGCGGTCG
CAGATCGCCTCTCCACAAAACTGGGGAAGGCAGTCGTCAAACGGTTTGCA
GACGGAGAGGTTAACATTCAGTTTGCGGACTCGCTCAGAGGCAAGGACGT
GTACATTATCCAACCTACCAGCACGCCCGTGAACGAACACCTCGTGGAGCT
TTTGCTGATGATCTCGACATGCCGACGCGCGAGCGCAAAAAAAATCACTGC
GGTCATTCCTTACTATGGCTATGCTCGACAGGACCGGAAACTGTCTAGCCGC
GTACCCATTTCTGCGGCTGACGTGGCCAGGATGATTGAGGCGATGGGTGTT
GATCGCGTTGTTGCGGTCGATCTGCACTGCGGACAGATCCAGGGTTTCTTT
GGCCCGCGGGTGCCTGTAGACAACCTCGAGGCGCAGATCATCGGCTTGGA
GTATTTCCACCACAAAGACCTCCACAAACCGGTGGTCGTGTCTCCTGATGC
AGGCGGTGTTTACAGAGCTCGGAAATTCCAAGAAGGTCTCATTGCTCGTGG
GTACAATGACTGCGGCATCGCCATGTTGATCAAGCAGCGCCTCCGAGCAAA
CGAGATCGAGCGGATGGATCTTGTTGGATCTGTCGCTGGCTCCGATGTCATT
ATAGTCGACGACATGATTGATACCGCCGGCACACTTTGCGAAGCTGCTCGT
GAGCTAAGGAAGAAAGGCGCTCGCCGAGTGTTTGCCTTCGCGACCCACGG
TCTCTTCAGCGGACCTGCCATTGAGAGAATCGAGGCCAGCCCTTTGGAGGA
AGTCGTCGTGACTGACAGCATCAAGGCTCGCCCCGAAGTGGCAGAGTGCC
CGCGCATCACTTCGCTGTCGATCAGTGTCCTTCTGGCAGATGCGATCCGCC
GCATTCACCAGAAGGAGAGTCTTAACGACCTTTTCAATGTTAAGTATGAGG
TCCACACGAACCAGGACCCGCTCGATTAACGAGGATATGCATAGATCTTAAT
TAATAACTTCGTATAGCATACATTATACGAAGTTATAAAATGGTGAGCAAGG
GCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGC
GACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGC
CACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGC
CCGTGCCCTGGCCCACCCTCGTGACCACCTTCGGCTACGGCCTGCAGTGCT
TCGCCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCA
TGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGC
AACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAA
CCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGG
GGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCG
ACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATC
GAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCAT
CGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCTACCAGT
CCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTG
GAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAA
GTAAATGCAGCCCACAGAAGCTGCCCGTCTCTCGTTTTCCTCTCTTTTCGG
AGGGATCAGGGAGAGTGCCTCGGGTCGGAGAGAGCTGACGAGGGGGTGC
CAGAGACCCCTGTGTCCTTTATCGAAGAAAAGGGATGACTCTTCATGTGGC
ATTTCACACAGTCTCACCTCGCCTTGTTTTCTTTTTGTCAATCAGAACGAAA
GCGAGTTGCGGGTGACGCAGATGTGCGTGTATCCACTCGGAATGCGTTATC
GTTCTGTATGCCGCTAGAGTGCTGGACTGTTGCTGTCTGCCCACGACAGCA
GACAACTTTCCTTCTATGCACTTGCAGGATGGTGCAGCGCAAACGACGGAG
AGAAAGGAGCACCCTCTCAGTTTCCCTACGATGTGCTGTCAGTTTCGACTC
TTCACCGCGAACGATTGGCGATACGTCTCTGTTGACTTGTTAGGCTCCGAC
CACGAAGCTCCCTTAACTAGATAAGCCGCGACACCTAAGTGTACACCATTT
GCAGATCGATAATCTGCGACCGCTGAATCCGTCCAGATCAGTAAAACCGCA
CCACCTAAGTGTAAACCTTGTTTAGGTCGATAAAATGCTACCAACCCCCAC
CCACAATCGAGCCTTGAGCGTTTCTGCGCACGCGTTGGCCTACGTGACTTG
CTGATGCCTGCCTCTGGCCATTCATGCCAGTCAGTGCGCATAAAAATGTGG
ACACAGTCGGTTGACAAGTGTTCTGGCAGGCTACAGTGACACCGCGGTGG
AGGGGGATCCACTAGTTCTAGACTCGAGCTGCAGCAGGCTGTAAATCCCGT
GAGTCGTCCTCACAAATCATCAAGCAGGTGTCCTCAGGGAGACTGCCTGA
CTGAGTTATGCTAATTCCTTTCTACTTTGGCGTGGTCACGGGGGCGCGCCGG
ATCCTTAATTAAGTCTAGCATGTCATTCGATTTTCACCCCCCGCGTAGTTCCT
GTGTGTCATTCGTTGTCGAGACAACTCTGTCCCGCCCCGGTGCTGTTCCATA
TGCGTGACTTTCCCGCAATTTTTTCAGACTTTCAGGAAAGACAGGCTCCGG
AACGATCTCGTCCATGACTGGTAAATCCACGACACCGCAATGGCCCCCAGC
ACCTCTATCTCTCGTGCCAGGGGACTAACGTTGTATGCGTCTGCGTCTTGTC
TTTTTGCATTCGCTTTCCAAAAAAGAGAGCCATCCGTTCCCCCGCACATTCA
ACGCCGCGAGTGCGGTTTTTGTCTTTTTTGAGTGGTAGGACGCTTTTCATGC
GCGAACTACGTGGACATTAAGTTCCATTCTCTTTTTCGACAGCACGAAACC
TTGCATTCAAACCCGCCCGCGGAAGATCCGATCTTGCTGCTGTTCGCAGTC
CCAGTAGCGTCCTGTCGGCCGCGCCGTCTCTGTTGGTGGGCAGCCGCTACA
CCTGTTATCTGACTGCCGTGCGCGAAAATGACGCCATTTTTGGGAAAATCG
GGGAACTTCATTCTTTAAAAGTATGCGGAGGTTTCCTTTTTCTTCTGTTCGT
TTCTTTTTCTCGGGTTTGATAACCGTGTTCGATGTAAGCACTTTCCGTCTCTC
CTCCGTGCTTTGTTCGACATCGAGACCAGGTGTGCAGATCCTTCGCTTGTC
GATCCGGAGACGCGTGTCTCGTAGAACCTTTTCATTTTACCACACGGCAGT
GCGGAGCACTGCTCTGAGTGCAGCAGGGACGGGTGAAGTTTCGCTTTAGT
AGTGCGTTTCTGCTCTACGGGGCGTTGTCGTGTCTGGGAAGATGCAGAAAC
CGGTGTGTCTGGTCGTCGCGATGACCCCCAAGAGGGGCATCGGCATCAAC
AACGGCCTCCCGTGGCCCCACTTGACCACAGATTTCAAACACTTTCGTCGT
GTGACAAAAACGACGCCCGAAGAAGCCAGTCGCCTGAACGGGTGGCTTCC
CAGGAAATTTGCAAAGACGGGCGACTCTGGACTTCCCTCTCCATCAGTCGG
CAAGAGATTCAACGCCGTTGTCATGGGACGGAAAAACTGGGAAAGCATGC
CTCGAAAGTTTAGACCCCTCGTGGACAGATTGAACATCGTCGTTTCCTCTT
CCCTCAAAGAAGAAGACATTGCGGCGGAGAAGCCTCAAGCTGAAGGCCA
GCAGCGCGTCCGAGTCTGTGCTTCACTCCCAGCAGCTCTCAGCCTTCTGGA
GGAAGAGTACAAGGATTCTGTCGACCAGATTTTTGTCGTGGGAGGAGCGG
GACTGTACGAGGCAGCGCTGTCTCTGGGCGTTGCCTCTCACCTGTACATCA
CGCGTGTAGCCCGCGAGTTTCCGTGCGACGTTTTCTTCCCTGCGTTCCCCG
GAGATGACATTCTTTCAAACAAATCAACTGCTGCGCAGGCTGCAGCTCCTG
CCGAGTCTGTGTTCGTTCCCTTTTGTCCGGAGCTCGGAAGAGAGAAGGAC
AATGAAGCGACGTATCGACCCATCTTCATTTCCAAGACCTTCTCAGACAAC
GGGGTACCCTACGACTCCGTGGTTCTCGAGAAGAGAAGGAAGACTGACGA
CGCAGCCACTGCGGAACCGAGCAACGCAATGAGCTCCTTGACGTCCACGA
GGGAGACAACTCCCGTGCACGGGTTGCAGGCTCCTTCTTCGGCCGCAGCC
ATTGCCCCGGTGTTGGCGTGGATGGACGAAGAAGACCGGAAAAAACGCGA
GCAAAAGGAACTGATTCGGGCCGTTCCGCATGTTCACTTTAGAGGCCATGA
AGAATTCCAGTACCTTGATCTCATTGCCGACATTATTAACAATGGAAGGACA
ATGGATGACCGAACGGGCGTTGGTGTCATCTCCAAATTCGGCTGCACTATG
CGCTACTCGCTGGATCAGGCCTTTCCACTTCTCACCACAAAGCGTGTGTTC
TGGAAAGGGGTCCTCGAAGAGTTGCTGTGGTTCATTCGCGGCGACACGAA
CGCAAACCATCTTTCTGAGAAGGGCGTGAAGATCTGGGACAAGAATGTGA
CACGCGAGTTCCTCGATTCGCGCAATCTCCCCCACCGAGAGGTCGGAGACA
TCGGCCCGGGCTACGGCTTCCAGTGGAGACACTTCGGCGCGGCATACAAA
GACATGCACACAGACTACACAGGGCAGGGCGTCGACCAGCTGAAGAATGT
GATCCAGATGCTGAGAACGAATCCAACAGATCGTCGCATGCTCATGACTGC
CTGGAATCCTGCAGCGCTGGACGAAATGGCGCTGCCGCCTTGTCACTTGTT
GTGCCAGTTCTACGTGAACGACCAGAAGGAGCTGTCGTGCATCATGTATCA
GCGGTCGTGCGATGTCGGCCTCGGCGTCCCCTTCAACATCGCTTCCTATTCG
CTTTTGACGCTCATGGTTGCACACGTCTGCAACCTAAAACCTAAGGAGTTC
ATTCACTTCATGGGGAACACGCATGTCTACACGAACCATGTCGAGGCTTTA
AAAGAGCAGCTGCGGAGAGAACCGAGACCGTTCCCCATTGTGAACATCCT
CAACAAGGAACGCATCAAGGAAATCGACGATTTCACCGCCGAGGATTTTG
AGGTCGTGGGCTACGTCCCGCACGGACGAATCCAGATGGAGATGGCTGTCT
AGCGGAAATACAGAAGCTGCCCGTCTCTCGTTTTCCTCTCTTTTCGGAGGG
ATCAGGGAGAGTGCCTCGGGTCGGAGAGAGCTGACGAGGGGGTGCCAGA
GACCCCTGTGTCCTTTATCGAAGAAAAGGGATGACTCTTCATGTGGCATTTC
ACACAGTCTCACCTCGCCTTGTTTTCTTTTTGTCAATCAGAACGAAAGCGA
GTTGCGGGTGACGCAGATGTGCGTGTATCCACTCGTGAATGCGTTATCGTTC
TGTATGCCGCTAGAGTGCTGGACTGTTGCTGTCTGCCCACGACAGCAGACA
ACTTTCCTTCTATGCACTTGCAGGATGAATTCCTACCCAGCTTTCTTGTACA
AAGTGGTCGTCTCTAGTTTTTTTGACAGACCGCTGACGGAATCGGCTTTCTCGTATACCGCCTTTCTGCATGTTGTTTCCATTTTGTGTGTCG(其中Tub:启动子(280-803bp),Loxp:34bp序列(770-803bp),PRPS:CDS区(818-2488bp),Loxp:34bp序列(2547-2580bp),YFP:荧光报告基因(2584-3303bp),DHFR:药筛标签(4097-7312bp))。
2、弓形虫基因敲除虫株TgPRPS-cKD的构建
敲除弓形虫TgPRPS的策略示意图如图1所示,pSAG1-Cas9-TgU6-dgTgPRPS CRISPR质粒可以特异性地打靶TgPRPS位点,与TgPRPS-5UTR::DHFR*::TgPRPS-3UTR同源片段共转染,通过乙胺嘧啶药物筛选,得到弓形虫基因敲除虫株TgPRPS-cKD,加入雷帕霉素后,缺失TgPRPS基因。具体实验方法如下:
体外培养弓形虫DiCre速殖子,待形成较大纳虫泡时,收集新鲜的弓形虫DiCre速殖子,并用3μm无菌的滤膜过滤纯化虫体,3000rpm离心10min;弃上清,用Cytomix(120mMKCl,0.15mM CaCl2,10mM K2HPO4/KH2PO4,25mM HEPES,2mM EGTA,5mM MgCl2,pH=7.6)重悬虫体沉淀至虫体量约为1×107个速殖子/mL。
将250μL虫体悬液加入4mm电转杯中,再加入1500ng TgPRPS-5UTR::DHFR*::TgPRPS-3UTR同源片段和7500ng实施例1制备得到的pSAG1-Cas9-TgU6-dgTgPRPS CRISPR质粒,混匀并除去气泡,静置2min。将电转杯置于Bio-Rad电转仪内,设置1600V、25μF、50Ω、4mm条件,电击一次,1500V、25μF、50Ω、4mm条件,电击两次,电转后的虫体在人源成纤维细胞(HFF)中培养和观察。
待电转后有40-60%虫体逸出细胞后,用5mL注射器吹打细胞,将细胞裂解并释放虫体;取500μL虫体悬液加入新鲜的HFF细胞中,并加入1μM乙胺嘧啶进行筛选。
在乙胺嘧啶作用3d后,可见宿主细胞内虫体增多,待虫体50%逸出后,裂解细胞,释放虫体,取500μL虫体悬液加入新鲜的HFF细胞中,再次药筛。
在药筛3代后,将虫体置于长满HFF细胞的96孔板中进行单克隆筛选,每孔接种1个弓形虫速殖子,在37℃、5% CO2条件下培养7d;将筛选获得的单克隆用无菌枪头刮下,接种到长满HFF细胞的24孔板中扩大培养,利用PCR对所有单克隆进行鉴定;将确定为TgPRPS-cKD的虫株继续扩大培养至T25培养瓶,并再次进行PCR1,PCR2和PCR3鉴定。
进行PCR1、PCR2、PCR3鉴定的引物如下表18所示,其采用的扩体系和反应程序如下表19和20所示。
表18PCR鉴定单克隆敲除株所使用的引物
表19PCR1、PCR2、PCR3反应体系
表20PCR1、PCR2、PCR3反应程序
PCR产物鉴定:扩增完成后,取PCR产物8μL,加2μL的6×核酸上样缓冲液混匀,点样到1.5%的琼脂糖凝胶中,120V,30min,用凝胶成像系统观察,PCR鉴定结果如图2所示,表明TgPRPS-cKD的单克隆虫株构建成功。
PCR鉴定正确的单克隆虫株,加入雷帕霉素后,得到TgPRPS基因敲除虫株,用IFA方法对该虫株进行鉴定。将TgPRPS-cKD和TgPRPS-cKD+RAPA 5d的虫株,分别用灭菌的3μM滤膜过滤、纯化,加入到长满HFF细胞的爬片中,并加入500μL 2% FBS DMEM培养基在标准生长条件(37℃,5% CO2)培养24h。之后用4%的多聚甲醛固定20min,洗涤后加入0.1% TritonX-100透化20min,用10% FBS封闭2h,后加入鼠源Ty抗体37℃30min,用Alexa Fluor 594结合山羊抗鼠IgG(Cell Signaling Technology,Danvers,MA,USA)和hoechst孵育30min,最后用倒置荧光显微镜观察和记录。
结果如图3所示,从图中可知TgPRPS-cKD+RAPA 5d的虫株显示出YFP绿色荧光标签,而TgPRPS-cKD虫株则不发绿光,表明雷帕霉素可以条件性敲除TgPRPS基因。
实施例3弓形虫TgPRPS-cKD虫株的体外实验
1、复制实验
(1)使用人源成纤维HFF细胞体外培养弓形虫TgPRPS-cKD速殖子,待有大量虫体逸出后,用RAPA处理5d;
(2)待TgPRPS-cKD±RAPA有30%虫体逸出时,用一次性细胞刮将细胞刮下来,5mL注射器吹打10-15次,再用灭菌的3μM滤膜过滤、纯化;
(3)收集虫体后,将TgPRPS-cKD+RAPA虫株用PBS重悬,在流式分选仪中将发YFP绿色荧光的敲除虫株收集起来;
(4)向长满HFF细胞的爬片中加入上述虫体,并加入相应的培养基(±RAPA,每组做3个平行),接种后的24孔板置于37℃、5% CO2培养箱中入侵HFF细胞1h;
(5)PBS洗2-3次,洗去未入侵的虫体,在37℃、5% CO2培养箱中继续培养虫体24h;
(6)4%的多聚甲醛固定20min,PBS洗3次;
(7)鼠抗弓形虫IgG孵育30min,PBS洗3次;
(8)0.1% Triton X-100透化20min,PBS洗3次;
(9)10% FBS封闭2h,PBS洗3次;
(10)猪抗弓形虫IgG抗体37℃孵育30min,PBS洗3次;
(11)采用Alexa Fluor 594结合山羊抗小鼠IgG(Cell Signaling Technology,Danvers,MA,USA)、山羊抗猪IgG(H+L)二抗,FITC(Thermo Fisher Scientific,USA)和hoechst避光孵育30min,最后用倒置荧光显微镜观察,并统计纳虫泡中虫体的个数,每组统计至少200个纳虫泡,实验单独重复3次。
结果如图4所示,由图可知加入雷帕霉素,缺失TgPRPS基因,纳虫泡中虫体个数变少,表明缺失TgPRPS基因减缓虫体体外的复制。
2、空斑实验
(1)使用人源成纤维HFF细胞体外培养弓形虫DiCre和TgPRPS-cKD速殖子,待有大量虫体逸出后,分别用RAPA处理5d;
(2)待DiCre±RAPA和TgPRPS-cKD±RAPA有30%虫体逸出时,用一次性细胞刮将细胞刮下来,5mL注射器吹打10-15次,再用灭菌的3μM滤膜过滤、纯化;
(3)收集虫体后,将TgPRPS-cKD+RAPA虫株用PBS重悬,在流式分选仪中将发YFP绿色荧光的敲除虫株收集起来;
(4)用细胞计数板对虫体进行计数,向长满HFF细胞的6孔板中接种虫体并加入2%FBS DMEM培养基(100Tg/每孔,每组做3个平行),接种后的6孔板置于37℃、5% CO2培养箱中培养7d;
(5)7d后,用PBS洗2-3次,4%多聚甲醛37℃固定20min,PBS洗2次;
(6)0.1%结晶紫染色20min,PBS洗1-2次后,将6孔板置于室温晾干。用扫描仪观察并记录空斑结果.
结果如图5所示,由图中可知加入雷帕霉素,缺失TgPRPS基因,不形成可视化的噬斑,表明TgPRPS基因对弓形虫存活非常关键。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (9)
1.弓形虫磷酸核糖焦磷酸激酶TgPRPS基因在制备调控弓形虫生长的药物中的应用,其特征在于,通过敲除弓形虫TgPRPS基因来减缓弓形虫的生长速率,所述TgPRPS基因编号为TGGT1_220100。
2.弓形虫磷酸核糖焦磷酸激酶TgPRPS基因作为靶点在制备或筛选抗弓形虫病药物中的应用,其特征在于,所述TgPRPS基因编号为TGGT1_220100;敲除TgPRPS基因的靶点sgRNA序列如SEQ ID NO.1~4所示。
3.敲除弓形虫磷酸核糖焦磷酸激酶TgPRPS基因的物质在制备抗弓形虫病的药物中的应用,其特征在于,所述TgPRPS基因编号为TGGT1_220100;所述敲除弓形虫磷酸核糖焦磷酸激酶TgPRPS基因的物质为包含序列如SEQ ID NO.1~4所示的sgRNA和/或采用CRISPR/Cas9系统对TgPRPS基因进行敲除的试剂。
4.敲除弓形虫磷酸核糖焦磷酸激酶TgPRPS基因的物质在制备降低弓形虫生长速率的制剂中的应用,其特征在于,所述TgPRPS基因编号为TGGT1_220100;所述敲除弓形虫磷酸核糖焦磷酸激酶TgPRPS基因的物质为包含序列如SEQ ID NO.1~4所示的sgRNA和/或采用CRISPR/Cas9系统对TgPRPS基因进行敲除的试剂。
5.一种抗弓形虫病药物,其特征在于,含敲除弓形虫磷酸核糖焦磷酸激酶TgPRPS基因的物质,所述TgPRPS基因编号为TGGT1_220100;所述敲除弓形虫磷酸核糖焦磷酸激酶TgPRPS基因的物质为包含序列如SEQ ID NO.1~4所示的sgRNA和/或采用CRISPR/Cas9系统对TgPRPS基因进行敲除的试剂。
6.一种弓形虫TgPRPS基因敲除虫株的构建方法,其特征在于,利用CRISPR/Cas9系统敲除弓形虫的TgPRPS基因,所述TgPRPS基因编号为TGGT1_220100。
7.根据权利要求6所述构建方法,其特征在于,包含以下步骤:
S1.设计敲除TgPRPS基因的靶点sgRNA序列;
S2.将步骤S1中得到的sgRNA序列构建到CRISPR/Cas9敲除质粒中;
S3.构建TgPRPS同源片段,并与步骤S2的敲除质粒共转染到野生型弓形虫虫株中;
S4.通过乙胺嘧啶药物筛选、PCR鉴定和IFA鉴定获得敲除TgPRPS基因的弓形虫遗传改造虫株。
8.根据权利要求7所述构建方法,其特征在于,步骤S1中靶点sgRNA序列依次如SEQ IDNO.1~4所示;步骤S2中构建CRISPR/Cas9敲除质粒所采用的引物序列依次如SEQ ID NO.5~8所示;步骤S3中构建的TgPRPS同源片段的序列如SEQ ID NO.13所示。
9.一种弓形虫TgPRPS基因敲除虫株,其特征在于,由权利要求6~8所述构建方法构建得到。
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