CN116617378A - 弓形虫PruΔgra76弱毒疫苗株及其应用 - Google Patents
弓形虫PruΔgra76弱毒疫苗株及其应用 Download PDFInfo
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Abstract
本发明公开了弓形虫PruΔgra76弱毒疫苗株及其应用,属于疫苗制备技术领域。该弓形虫PruΔgra76是一种潜在的弓形虫减毒活疫苗虫株,可为弓形虫急性和慢性感染提供部分、有效的免疫保护力。对免疫小鼠的血清抗体测定发现,PruΔgra76免疫可诱导小鼠产生高水平的总IgG、IgG1和IgG2a。因此,PruΔgra76免疫可刺激机体产生Th1和Th2免疫应答,且Th1应答占主导地位,本发明为抗弓形虫感染药物的开发奠定了基础。
Description
技术领域
本发明属于疫苗制备技术领域,尤其涉及弓形虫PruΔgra76弱毒疫苗株及其应用。
背景技术
刚地弓形虫(Toxoplasma gondii,T.gondii)简称弓形虫,隶属于顶复门(Apicomplexa)、孢子虫纲(Sporozoasida)、真球虫目(Eucoccidiida)、肉孢子虫科(Sarcocystidae)、弓形虫属(Toxoplasma),是一种严格的活细胞内寄生性原虫。弓形虫的感染在全世界范围内均有分布,可以感染几乎所有的温血动物(包括人类),给畜牧业生产和公共健康造成巨大隐患。因此,弓形虫是人兽共患病病原微生物中一种重要的机会致病性顶复门寄生虫。
针对人类弓形虫病的主要治疗方案包括联合使用抗叶酸药物乙胺嘧啶和磺胺嘧啶,但这种治疗方案的治愈率并未达到100%。其他治疗弓形虫病的方案有乙胺嘧啶联合克林霉素、克拉霉素、阿奇霉素或阿托伐醌,或使用甲氧苄啶-磺胺甲恶唑的单药治疗。但这些治疗方案均没有组合使用乙胺嘧啶-磺胺嘧啶的疗效好。然而,以上使用的所有临床治疗方案都仅对弓形虫快速分裂的速殖子阶段有效,对组织包囊内缓慢增殖的缓殖子没有任何显著的效果,也不能治愈持续感染。同时,目前的治疗方法可能出现多种副作用。针对弓形虫病的疫苗有助于有效控制长期的弓形虫病,同时减少了宿主对化学疗法的依赖和后果。
弱毒活弓形虫病疫苗能模拟弓形虫感染的过程,可为弓形虫感染提供有效的免疫保护力。研究表明,减毒活疫苗S48和T-263、TS-4,在防止猫卵囊脱落、降低绵羊流产率和减少家畜组织包囊形成方面显示出很高的效力。然而,在新西兰、英国和其他欧洲国家,只有一种基于速殖子S48减毒活株的商业疫苗Toxovax1获得使用许可,主要用于降低绵羊流产的发生率。然而,Toxovax1疫苗具有保质期相对较短的缺陷,不能完全消灭弓形虫,且并不适用于预防人弓形虫病。此外,由于这种活的减毒速殖子S48株的分子基础尚未完全了解,其可能会逆转为致病型虫株从而对易受伤害的个体造成威胁。因此,研制有效、安全、持久的抗弓形虫感染疫苗对于控制弓形虫病是非常必要的,而商业减毒活疫苗是弓形虫病疫苗研发的一个重点。
发明内容
本发明的目的之一在于提供弓形虫PruΔgra76弱毒疫苗株在制备抗弓形虫感染药物中的应用。
优选地,所述弓形虫PruΔgra76弱毒疫苗株是由PruΔku80虫株改造获得的。
优选地,所述弓形虫PruΔgra76弱毒疫苗株是利用CRISPR-Cas9技术敲除PruΔku80中的GRA76基因获得的。
优选地,所述的药物为弓形虫PruΔgra76弱毒疫苗。
本发明的目的之二在于提供一种弓形虫弱毒疫苗,其有效成分包括弓形虫PruΔgra76弱毒疫苗株。
优选地,所述弓形虫弱毒疫苗中还包括疫苗佐剂。
与现有技术相比,本发明具有如下有益效果:
本发明提供的弓形虫基因缺失株PruΔgra76是一种潜在的弓形虫减毒活疫苗虫株,可为弓形虫急性和慢性感染提供部分、有效的免疫保护力。对免疫小鼠的血清抗体测定发现,PruΔgra76免疫可诱导小鼠产生高水平的总IgG、IgG1和IgG2a。因此,PruΔgra76免疫可刺激机体产生Th1和Th2免疫应答,且Th1应答占主导地位,本发明为抗弓形虫感染药物的开发奠定了基础。
附图说明
图1A为实施例1中GRA76的结构预测图,GRA76全长含有545个氨基酸,包含1个信号肽(SP,浅灰色斜条纹)、5个蛋白无序区(IDR,黑色)和3个跨膜结构域(TM,黑色斜条纹)。
图1B为实施例1中PONDR对GRA76的蛋白无序区的预测结果。
图1C为实施例1中TMHMM 2.0对GRA76的跨膜结构域的预测结果。
图1D为实施例1中SignalIP 5.0对GRA76的信号肽的预测结果。
图2为实施例1中GRA76在弓形虫速殖子和缓殖子阶段的表达,其中(A)GRA76表位标签虫株的构建示意图;(B)GRA76表位标签虫株的PCR鉴定结果;(C)GRA76表位标签虫株的Western blotting鉴定结果;(D)GRA76在胞外速殖子中的定位;(E)GRA76在胞内速殖子中的定位;(F)GRA76在缓殖子阶段的亚细胞定位;(G)Western blotting证实GRA76在速殖子与缓殖子阶段表达量不同。
图3为实施例1中弓形虫基因缺失株PruΔgra76的鉴定及噬斑形成能力的评估图,其中(A)PruΔgra76虫株的构建示意图;(B)PruΔgra76的PCR鉴定结果;(C)Pru和PruΔgra76在HFF细胞中形成的噬斑;(D)PruΔgra76形成的噬斑面积及数量显著低于Pru形成的噬斑面积及数量(****P<0.0001)。
图4为实施例1中弓形虫基因缺失株PruΔgra76对缓殖子转化相关基因转录的影响结果图,其中(A)Pru和PruΔgra76的转录组数据火山图,在PruΔgra76中,GRA76缺失上调了与速殖子转化形成缓殖子相关的基因的转录;(B)在PruΔgra76中,转录水平上调的蛋白的亚细胞定位的预测;(C)Pru和PruΔgra76在未诱导条件(pH7.4)下的包囊转化率(***P<0.001);(D)Pru和PruΔgra76在诱导条件(pH8.2)下的包囊转化率(ns P>0.05)。
图5为实施例1中弓形虫基因缺失株PruΔgra76对小鼠毒力的影响和感染小鼠的脑包囊数统计结果,其中(A~F)感染2×102个(A)、5×102个(B)、5×103个(C)、5×104个(D)、5×105个(E)、5×106个(F)Pru或PruΔgra76速殖子的小鼠生存曲线;(G)存活小鼠的脑组织包囊数(****P<0.0001,**P<0.01)。
图6为实施例1中弓形虫基因缺失株PruΔgra76免疫可保护小鼠免受弓形虫急性和慢性感染的结果图,其中(A-D)免疫(5×104个PruΔgra76速殖子)第45天,免疫小鼠和空白小鼠感染1000个RH速殖子(A)、1000个PYS速殖子(B)、10个包囊(C)、40个包囊(D)的生存曲线。(E)感染包囊的小鼠的脑组织包囊数。
图7为实施例1中弓形虫基因缺失株PruΔgra76免疫诱导Th1和Th2免疫应答的结果图,其中(A)接种后45天的小鼠血清中抗弓形虫特异性的总IgG、IgG1和IgG2a,PruΔgra76疫苗可显著提升小鼠血清中的总IgG、IgG1和IgG2a(**P<0.01,****P<0.0001)。(B)PruΔgra76免疫小鼠的IgG2a水平显著高于IgG1水平(***P<0.001)。
具体实施方式
实施例1
1.GRA76的结构预测分析
从弓形虫全基因组TOXODB网站中获取GRA76的蛋白序列,使用PONDR(Predictorof Natural Disordered Regions)分析GRA76的蛋白无序区(protein intrinsicallydisordered regions,IDR),用SignalIP 5.0分析GRA76的信号肽(signal peptide,SP),用TMHMM分析GRA76的跨膜结构域(transmembrane domain,TMHMM)。结合预测的IDR、SP、TMHMM分析GRA76的结构。
2.GRA76在弓形虫速殖子和缓殖子阶段的表达特性
利用CRISPR-Cas9技术在Pru(PruΔku80虫株)中构建表位标签虫株Pru::GRA76-HA,通过间接免疫荧光实验证实GRA76是致密颗粒蛋白,及其在速殖子和缓殖子阶段的表达差异性。
3.PruΔgra76的构建及表型分析
虫株:弓形虫II型Pru(PruΔku80)虫株由中国农业科学院兰州兽医研究所保存。
利用CRISPR-Cas9技术构建PruΔgra76弓形虫株,具体过程如下:
细胞培养基:DMEM培养基分别加入100μg/mL的青霉素、100μg/mL的链霉素和10mM的HEPES溶液以及10%胎牛血清。
弓形虫培养基:除胎牛血清为2%,其余均与细胞培养基一致。
(1)GRA76的CRISPR-Cas9敲除质粒的构建
利用弓形虫全基因组TOXODB网站(http://www.toxodb.org/toxo/)获得GRA76的基因序列,并设计CRISPR-Cas9敲除质粒的SgRNA。
以pSAG1-Cas9-U6-SgUPRT为模板,以SEQ ID NO.1和SEQ ID NO.2为引物,利用PCR扩增方法将模板质粒pSAG1-Cas9-U6-SgUPRT中的SgUPRT分别替换为GRA76的CRISPR-Cas9敲除质粒的SgRNA。
其中用于构建GRA76的CRISPR-Cas9敲除质粒的引物见表1。
表1
用于构建GRA76的CRISPR-Cas9敲除质粒的PCR反应的反应体系见表2。
表2
用于构建GRA76的CRSPR-Cas9敲除质粒的PCR反应的反应程序见表3。
表3
根据Q5定点突变试剂盒,将以上PCR产物分别于37℃进行DpnI酶切,其中酶切反应体系见表4。
表4
将以上DpnI酶切产物于25℃进行KLD环化,其环化反应体系见表5。
表5
将以上KLD环化产物分别进行DH5α感受态转化,经涂平板后挑选单菌落,并将单菌落扩大培养后测序。将测序成功的CRISPR-Cas9敲除质粒再次转化、扩大培养后使用无内毒素质粒提取试剂盒提取质粒,备用。
(2)GRA76的同源DHFR片段的构建
在GRA76基因的起始序列前和终止序列后分别设计5'端和3'端同源臂的扩增引物(SEQ ID NO.3~SEQ ID NO.6)、扩增DHFR片段的引物(SEQ ID NO.7~SEQ ID NO.8)、扩增pUC19骨架的引物(SEQ ID NO.9~SEQ ID NO.10)、扩增GRA76的同源DHFR片段的引物(SEQID NO.11~SEQ ID NO.12)见表6。其中扩增5'端和3'端同源臂的模板的弓形虫基因组DNA,扩增DHFR片段的模板为pUPRT-DHFR-D质粒,扩增pUC19骨架的模板为pUC19质粒。其PCR反应体系同表2,反应程序同表3(扩增5'端和3'端同源臂的延伸时间为2min、扩增DHFR片段和pUC19骨架的延伸时间为3min)。
表6
将以上扩增的5'端同源臂、3'端同源臂、DHFR片段、pUC19骨架片段分别进行胶回收后,按比例混匀,并于37℃进行DpnI酶切,酶切体系见表4。根据ClonExpress MultiS一步克隆法,将以上DpnI酶切产物于37℃进行连接,连接体系见表7。
表7
将以上连接产物分别进行DH5α感受态转化,经涂平板后挑选单菌落,并将单菌落扩大培养后测序。以测序成功的同源DHFR质粒为模板,以SEQ ID NO.11和SEQ ID NO.12为引物,扩增同源DHFR片段并胶回收,备用。
(3)PruΔgra76的构建、筛选及鉴定
将HFF细胞接种于25T的细胞培养瓶中,加入6mL含有10%FBS的DMEM培养基,在37℃的CO2培养箱中培养,待细胞完全长满之后,换成6mL含2%FBS的DMEM培养基,加入1mL刚刚逸出的PruΔku80速殖子(下简称为Pru),培养65h后,收集及纯化速殖子用于弓形虫的电转染;将提取的CRISPR-Cas9敲除标签质粒与同源DHFR片段的胶回收产物混匀、灭菌后共同电转染入Pru速殖子;经乙胺嘧啶进行药物筛选、96孔板单克隆筛选后获得单克隆虫株;单克隆虫株放大培养后提取基因组DNA,进行PCR鉴定;使用引物SEQ ID NO.15和SEQ IDNO.16的PCR反应(PCR1)用于鉴定单克隆GRA基因缺失株中目的GRAs是否被敲除;使用引物SEQ ID NO.13和SEQ ID NO.14的PCR反应(PCR2)用于鉴定单克隆GRA基因缺失株中靶向敲除的GRA基因的5'端是否被替换为同源DHFR片段;使用引物SEQ ID NO.17和SEQ ID NO.18的PCR反应(PCR3)用于鉴定单克隆GRA基因缺失株中靶向敲除的GRA基因的3'端是否被替换为同源DHFR片段。其中反应体系如表7;反应程序如表8;引物序列见表9。
表7
表8
表9
(4)噬斑实验
从25T细胞瓶中接出少量已完全逸出的野生株Pru和PruΔgra76虫株,计数后使其终浓度达到2×103个/mL。在已铺满HFF的12孔板中每孔接入250μL,轻微摇匀使其速殖子均匀分布孔中,最后放置细胞培养箱;培养12d,弃上清,加入PBS溶液轻微吹打孔底部洗8次;加入1mL组织固定液固定20min,弃上清,PBS溶液洗8次;在每孔中加入1mL 0.25%结晶紫染色20min,弃上清,PBS溶液洗8遍,用吸水纸吸去在孔周边剩余的液体,风干后拍照观察,计算虫斑大小和数量。
4.PruΔgra76的转录组测定
Pru速殖子和PruΔgra76速殖子感染HFF细胞后,用预冷的PBS清洗,将含有胞内速殖子的HFF细胞刮下、离心收集样品。其中,Pru和PruΔgra76均制备3次独立的样品,每份样品对应一个75T细胞瓶收集的胞内速殖子。每个虫株的总RNA使用TRIzol方法(ThermoFisher Scientific,USA)提取,使用无RNA酶的DNA酶除去提取的RNA中的残余基因组DNA。使用Nano Drop和Agilent 2100生物分析仪(Thermo Fisher Scientific,USA)对每个样品中的总RNA进行鉴定和定量。经mRNA分离、mRNA片段化、cDNA合成、cDNA修饰、PCR和AMPureXP珠(Beckman Coulter)的清理后制备RNA文库。使用BGI-AEQ平台(中国深圳)对RNA文库进行测序。原始数据经SOAPnuke进行修整和过滤,以去除包含测序适配器、具有低质量碱基比(>20%)、未知碱基比大于5%的原始数据。使用HISAT(hierarchical indexing forspliced alignment oftranscripts)将修整的转录组数据与弓形虫ME49基因组数据进行比对分析。使用RSEM(v1.3.1)对转录组中基因的相对表达水平进行测定,基因的相对表达水平使用每百万个映射片段外显子模型的千碱基片段数(fragments per kilobaseofexon model per million mapped fragments,FPKM)表示。在Dr.Tom多组学数据挖掘系统上分析和挖掘转录组数据。使用DESeq2(v1.4.5)进行差异表达分析,转录水平变化log2≥1、Q值(经调整的P值)≤0.05的基因则为差异表达的基因,将其用火山图呈现。
5.包囊转化的评价
从25T细胞瓶中接出已完全逸出的野生株Pru和基因缺失株PruΔgra76速殖子,计数后稀释终浓度至1×105个/mL,分别接100μL到长满HFF的小皿。
诱导的包囊转化率试验为,其在正常培养基(pH 7.4)培养4h后,用预热的DMEM溶液洗去未入侵的速殖子;换成缓殖子诱导培养基(pH 8.2)继续培养48h;培养48h后弃上清;加入组织固定液固定20min,PBS溶液洗4次;加入0.2%Triton X-100,穿透20min;PBS溶液洗6次,加入3%BSA,封闭1h;PBS溶液洗6次,加入兔抗弓形虫IMC1单克隆抗体(1:500),4℃过夜;PBS溶液洗6次,加入Alexa Fluor 594山羊抗兔IgG(H+L)(1:1000)和FITC标记的Dolichos biflorus agglutinin(DBA)(1:500),37℃孵育1h;PBS溶液洗8次,在荧光显微镜下记录并统计DBA阳性虫斑的比例。
未诱导的包囊转化率试验为,接种到小皿后,其在正常培养基(pH 7.4)培养4h后,用预热的DMEM溶液洗去未入侵的速殖子,重新接入正常培养基(pH7.4)继续培养48h后,弃上清,进行间接免疫荧光试验,方法同上。
6.小鼠毒力的评价
(1)小鼠分组:试验开始前一周,将从兰州兽医研所实验动物中心购买的6-8周龄、SPF昆明雌鼠进行分组。以6只小鼠为一组,将其分为一笼,适应一周,以减少小鼠的应激反应。期间给小鼠提供充足的饲料和水,每2~3天更换垫料,每天早晚各观察一次小鼠。
(2)速殖子的准备:将速殖子终浓度稀释到1×103个/mL、2.5×103个/mL、2.5×104个/mL、2.5×105个/mL、2.5×106个/mL和2.5×107个/mL。
(3)攻鼠:将以上200μL稀释好的速殖子以腹腔注射的方式接种小鼠,Pru和PruΔgra76速殖子的攻鼠剂量为2×102个/只、5×102个/只、5×103个/只、5×104个/只、5×105个/只和5×106个/只,每种虫株接种6只小鼠。
(4)观察并记录:每天早晚分别观察并记录小鼠的发病症状及存活情况。
(5)小鼠脑包囊的计数方法:低剂量(2×102个/只)感染试验中,对感染30天仍然存活的小鼠安乐死后,无菌解剖后取脑组织。将脑组织无菌研磨后,加入1.5mL无菌PBS润洗后,将其转移至无菌2mL离心管中,显微镜下计数脑包囊数。
7.PruΔgra76的免疫效果的评价
(1)PruΔgra76对弓形虫急性和慢性感染的免疫保护力评价
①小鼠的免疫:经培养、收集、计数后,将新鲜逸出的PruΔgra76速殖子稀释至2.5×105个/mL,将200μL以上稀释好的速殖子以腹腔注射的方式免疫昆明小鼠。同时,以相同的饲养条件和饲养方式饲养相同数量的空白小鼠。
②小鼠的急性感染:小鼠免疫45天或空白小鼠饲养45天后,将1000个新鲜逸出的RH和PYS速殖子分别腹腔注射免疫鼠和空白鼠,每组6只小鼠,每天2次观察小鼠的发病及存活情况。
③弓形虫脑包囊的搜集:Pru虫株经HFF细胞培养、收集、计数后,将2×102个Pru速殖子腹腔注射昆明鼠,每天2次观察小鼠的发病及存活情况。待小鼠经PruΔgra76s免疫第45天,取Pru速殖子慢性感染的存活小鼠的脑组织,并将脑包囊经计数后稀释至10个/200μL和40个/200μL。
④小鼠的慢性感染:小鼠免疫45天或空白小鼠饲养45天后,将10个或40个包囊灌胃免疫鼠或空白鼠,每组6只小鼠,每天2次观察小鼠的发病及存活情况。感染第30天,取存活小鼠的脑组织并计数脑包囊数。
(2)PruΔgra76免疫小鼠的抗体检测
①采血:免疫第45天,采集免疫小鼠和空白小鼠的血清并置于1.5mL离心管中,4℃静置2h后,以4000g/min离心10min,吸取上清至1.5mL离心管中,-20℃冻存备用。
②抗原包被:将弓形虫特异性抗原(10μg/mL)加入ELISA板中,每孔添加100μL(即1μg/孔),37℃静置1.5~2h后,置于4℃过夜,0.5%PBST洗涤3次后,拍干水分。
③封闭:每孔加入100μL的5%BSA,37℃静置1~2h后,0.5%PBST洗涤3次后,拍干水分。
④孵育血清:用1%BSA将血清按照1:100比例稀释,每孔加入100μL的稀释的血清,37℃孵育1~2h,0.5%PBST洗涤3次后,拍干水分。
⑤孵育二抗:用1%BSA稀释二抗,每孔加入100μL。其中,羊抗鼠IgG1和羊抗鼠IgG2a的稀释比例为1:5000,IgG(HRP)的稀释比例为1:3000。37℃孵育1~2h,0.5%PBST洗涤3次后,拍干水分。
⑥显色剂中止:每孔加入TMB显色液100μL,待其全部显色稳定后,加入中止液(2%硫酸)100μL。
⑦测定OD450:于450nm下,测定OD值。
上述试验结果如下:
1.GRA76的结构预测分析
TGME49_299780(GRA76)在弓形虫的亚细胞定位预测为致密颗粒。对GRA76的蛋白序列分析发现,GRA76含有545个氨基酸,预测其N端含有一个信号肽、接近C端有3个跨膜结构域(图1A)。经PONDR分析预测,GRA76含有5个蛋白无序区(IDR)(图1B)。经TMHMM预测,GRA76的三个跨膜结构域分别预测在第372、438和475个氨基酸位点(图1C)。SignalIP 5.0预测GRA76的信号肽位于第28个氨基酸位点(图1D)。同时,经同源序列对比发现,GRA76与其他GRA蛋白一致与其他真核生物的蛋白无同源序列。
2.GRA76主要在弓形虫速殖子阶段表达
为探究GRA76在弓形虫Pru虫株速殖子和缓殖子阶段的亚细胞定位,构建了表位标签虫株Pru::GRA76-HA。图2A为表位标签虫株Pru::GRA76-HA的构建原理示意图;图2B~2C从DNA水平和蛋白质水平证实Pru::GRA76-HA的成功构建。间接免疫荧光实验表明,在胞外速殖子中,GRA76与GRA5、GRA12共定位(图2D);在HFF细胞内的速殖子阶段,GRA76与GRA5和GRA12定位于PV、部分定位于PVM(图2E);在HFF细胞内的缓殖子阶段,GRA76表达量低于其在速殖子阶段的表达量(图2F);图2G应用Western blotting证实GRA76在速殖子与缓殖子阶段表达的差异性。这说明,GRA76是一种主要在速殖子阶段表达的致密颗粒蛋白。
3.PruΔgra76的构建及表型分析
为探究弓形虫基因缺失株PruΔgra76相较于野生株Pru的胞内增殖速度变化,首先构建了基因缺失株PruΔgra76。图3A为PruΔgra76虫株的构建原理示意图;图3B从DNA水平证实PruΔgra76虫株的成功构建。噬斑形成试验表明,PruΔgra76虫株形成的噬斑面积和数量显著少于Pru虫株(图3C和3D)。这一结果说明,GRA76对弓形虫II型Pru虫株的胞内生长均至关重要。
4.PruΔgra76参与调控与缓殖子转化相关基因的转录
为研究弓形虫基因缺失株PruΔgra76的转录水平变化,搜集未经碱性诱导的Pru和PruΔgra76虫株的胞内速殖子,并采集RNA用于转录组的测序及分析。PruΔgra76中差异表达基因的选择原则为log2值≥1(或log2值≤-1)以及Q值(调整后的P值)≤0.05。与野生株Pru虫株相比,PruΔgra76中有280个基因的转录水平显著上调,49个基因的转录水平显著下调。有趣的是,在未经诱导的条件下,多种与缓殖子转化相关的基因的转录水平在PruΔgra76中均显著上调,包括BAG1、MAG2、ENO1、LDH2、AP2IX9和CST1(图4A)。但基于TOXODB数据库中hyperLOPIT预测的蛋白定位数据分析,多数在PruΔgra76中显著上调的蛋白的亚细胞定位仍然未知(140/280,图4B)。
为验证弓形虫基因缺失株PruΔgra76是否可提高弓形虫的缓殖子转化能力,对Pru和PruΔgra76速殖子的体外包囊转化能力进行了试验。结果表明,在未诱导的条件下,PruΔgra76的体外包囊转化水平显著高于野生株Pru(图4C)。在诱导条件下,野生株Pru、缺失株PruΔgra76的体外包囊转化力均接近100%,无显著性差异(图4D)。这说明,PruΔgra76有助于弓形虫速殖子向缓殖子的转化。鉴于GRA76缺失显著减缓了弓形虫的胞内复制能力,推测GRA76缺失可能给弓形虫的生长造成了一定的压力,促使Pru速殖子中参与缓殖子转化的基因的表达水平上调,进一步诱导了Pru速殖子向缓殖子的转化。
5.PruΔgra76的小鼠毒力评价
为探究弓形虫基因缺失株PruΔgra76对小鼠毒力的影响,分别将2×102个、5×102个、5×103个、5×104个、5×105个、5×106个Pru和PruΔgra76速殖子腹腔注射小鼠,并观察感染小鼠的生存情况及脑组织包囊数量(图5A~5G)。野生株Pru速殖子感染的小鼠中,除最低剂量(2×102个/只)感染的小鼠有37.5%存活外,其余小鼠均全部死亡(图5A~5F);缺失株PruΔgra76殖子感染的小鼠中,除最高剂量(5×106个/只)感染组的小鼠有50%死亡外,其余小鼠均全部存活(图5A~5F)。这表明,GRA76缺失可显著降低Pru虫株对小鼠的毒力(*P<0.05;**P<0.01)。同时,为评价弓形虫GRA76在小鼠脑包囊产生中的发挥的作用,取存活小鼠的脑组织并计数其组织包囊(图5G)。PruΔgra76速殖子感染小鼠的脑包囊数显著低于野生株Pru感染小鼠的脑包囊数(**P<0.01;****P<0.0001)。以上结果表明,GRA76缺失可影响弓形虫Pru虫株对小鼠的毒力,GRA76在小鼠脑包囊产生中发挥重要作用。
6.PruΔgra76可为弓形虫急性感染和慢性感染提供强有力的免疫保护力
GRA76缺失后,PruΔgra76的小鼠毒力显著降低。这一结果提示,PruΔgra76可能是一种潜在的弓形虫弱毒疫苗虫株。在PruΔgra76的小鼠毒力试验中,感染5×104个的小鼠全部存活,且感染小鼠的脑包囊数量较低。因此,在探究PruΔgra76是否可以保护小鼠免受弓形虫急性或慢性感染时,选择5×104个/只为小鼠的免疫剂量。昆明小鼠经PruΔgra76免疫后第45天(免疫剂量为5×104个/只),分别腹腔注射1000个RH速殖子或1000个PYS速殖子,以探究PruΔgra76免疫对弓形虫急性感染的免疫保护力。同时,免疫第45天,免疫小鼠和空白小鼠经口灌胃10个包囊或40个包囊,以评价PruΔgra76免疫对弓形虫慢性感染的免疫保护力。
试验结果表明,在急性感染阶段,空白小鼠均全部死亡;感染1000个RH速殖子的免疫小鼠仅50%死亡;感染1000个PYS的免疫小鼠全部存活(图6A和6B,**P<0.01)。在慢性感染阶段,感染10个或40个包囊的免疫小鼠均全部存活,而感染10个或40个包囊的空白小鼠的存活率分别为83.33%和33.33%(图6C和6D,*P<0.05,ns P>0.05)。同时,慢性感染中,PruΔgra76免疫小鼠的脑包囊数显著低于空白小鼠的脑包囊数(图6E,**P<0.01,****P<0.0001)。这表明,PruΔgra76的免疫可显著延长小鼠在弓形虫急性感染和慢性感染阶段的存活率,且可显著降低慢性感染小鼠的脑包囊数。
7.PruΔgra76可诱导产生Th1型为主的免疫反应
为探究PruΔgra76为免疫小鼠提供抗弓形虫感染的保护性免疫应答的潜在机制,在PruΔgra76免疫小鼠的第45天,分别取空白小鼠和免疫小鼠的血清,并通过ELISA测定血清中抗弓形虫的特异性IgG、IgG1和IgG2a的水平(图7)。相较于空白小鼠,免疫小鼠血清中的总IgG水平均显著增加(图7A,****P<0.0001)。为探究PruΔgra76的免疫引起的免疫反应的类型,进一步检测了血清中IgG1和IgG2a的水平。PruΔgra76的免疫可显著提高小鼠血清中IgG1和IgG2a的水平(图7A,**P<0.01,****P<0.0001);且IgG2a水平显著高于IgG1(图7B,***P<0.001)。这表明,PruΔgra76可刺激机体产生以Th1型为主的体液免疫反应,以限制弓形虫感染。
以上所述的实施例仅是对本发明的优选方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案做出的各种变形和改进,均应落入本发明权利要求书确定的保护范围内。
Claims (6)
1.弓形虫PruΔgra76弱毒疫苗株在制备抗弓形虫感染药物中的应用。
2.根据权利要求1所述的应用,其特征在于,所述弓形虫PruΔgra76弱毒疫苗株是由PruΔku80虫株改造获得的。
3.根据权利要求2所述的应用,其特征在于,所述弓形虫PruΔgra76弱毒疫苗株是利用CRISPR-Cas9技术敲除PruΔku80中的GRA76基因获得的。
4.根据权利要求1-3中任一项所述的应用,其特征在于,所述药物为弓形虫PruΔgra76弱毒疫苗。
5.一种弓形虫弱毒疫苗,其特征在于,其有效成分包括弓形虫PruΔgra76弱毒疫苗株。
6.根据权利要求5所述的弓形虫弱毒疫苗,其特征在于,还包括疫苗佐剂。
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