CN116440269A - Slc35a1基因作为靶点在防治猪流行性腹泻病中的应用 - Google Patents
Slc35a1基因作为靶点在防治猪流行性腹泻病中的应用 Download PDFInfo
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Abstract
本发明公开了SLC35A1基因作为靶点在抗猪流行性腹泻病中的应用，通过实验证实，相比于对照组，敲除SLC35A1基因后，猪流行性腹泻病毒（Porcine epidemic diarrheavirus，PEDV）在宿主Vero细胞的复制能力被显著抑制，因此特异靶向编辑该基因或干扰其蛋白表达可抑制PEDV侵染宿主细胞，从而防治PEDV感染所引发的猪流行性腹泻病。
Description
技术领域
本发明属于生物技术领域，具体涉及SLC35A1作为靶点在抗猪流行性腹泻病中的应用。
背景技术
猪流行性腹泻(Porcine epidemic diarrhea,PED)是由冠状病毒科的α冠状病毒亚科猪流行性腹泻病毒(PEDV)引起的一种高度接触性肠道传染病，以呕吐、腹泻和食欲下降为基本特征，各种年龄的猪均易感。猪流行性腹泻病会导致仔猪生长缓慢、成活率低、饲料报酬低。猪流行性腹泻病目前没有特效药，尽管有疫苗，但是其预防效果并不理想。此外，目前PEDV致病机制还不是十分清楚，特别是缺乏有效的宿主抗病毒靶点，从而阻碍了猪流行性腹泻的预防与治疗。因此，鉴定新的参与病毒感染关键宿主因子对开展宿主靶向抗病毒研究尤为重要。
SLC35A1是胞苷5'-单磷酸-唾液酸转运蛋白(CMP-sialic acid transporter)，可以将CMP-唾液酸从胞质溶胶转运到高尔基囊泡中。研究人员发现SLC35A1 基因编码蛋白负责将唾液酸转运至高尔基体，并在高尔基体内完成该多糖链的组装，而流感病毒正是利用唾液酸作为受体入侵宿主细胞，通过CRISPR敲除文库筛选以及单独敲除SLC35A1基因，发现SLC35A1基因敲除细胞可阻止病毒吸附到宿主细胞，从而有效抑制流感病毒的复制。然而，也有研究发现，敲除 SLC35A1基因后，反而能显著增强水疱性口炎病毒感染所诱发的细胞死亡和凋亡作用。由此可见，SLC35A1基因在不同病毒侵染其宿主细胞的过程中发挥的作用可能不同。
尽管PEDV严重危害养猪业健康发展，但参与PEDV复制相关的宿主因子还不清楚，本发明旨在发掘防控PEDV的新靶点。
发明内容
为了克服现有技术存在的不足，本发明的目的在于提供一种新的靶点，能够有效地应用于猪流行性腹泻病的预防和治疗。
为了实现上述目的，本发明采用以下技术方案：
本发明公开了SLC35A1基因作为靶点用于制备防治猪流行性腹泻病毒感染的药物，所述药物为靶向编辑SLC35A1基因或干扰其蛋白表达的物质。具体地，所述的药物包括靶向敲除SLC35A1基因的sgRNA或其表达载体，用于RNA干扰的dsRNA，反义寡核苷酸、小分子抑制剂。
进一步优选地，靶向敲除SLC35A1基因的sgRNA序列为 5’-CCATAGCTTTAAGATACACA-3’。
本发明还公开了一种药盒，包含靶向敲除SLC35A1基因的sgRNA序列、 CRISPR/Cas9质粒，该药盒可用于制备防治猪流行性腹泻病的药物。
本发明还公开了SLC35A1基因作为靶点在制备抗流行性腹泻病毒感染的基因编辑细胞或动物模型中的应用。具体地，包括：(1)利用慢病毒载体，通过 HEK293T细胞进行包装，获得CRISPR/Cas9系统慢病毒颗粒；(2)将慢病毒颗粒感染目的细胞株，流式分选法传代筛选，获得SLC35A1基因缺失的细胞株。
进一步地，本发明发现SLC35A1与猪流行性腹泻病毒复制具有相关性。在本发明的具体实施例中，当敲除SLC35A1基因时，可以降低猪流行性腹泻病毒感染Vero细胞的能力。
综上所述，本发明的有益效果在于：本发明提供SLC35A1基因在防治猪流行性腹泻病中的应用。本发明发现猪流行性腹泻病毒感染宿主细胞后SLC35A1 基因表达水平上调。并且，通过特异靶向编辑SLC35A1基因或干扰其蛋白表达可有效抑制猪流行性腹泻病毒侵染宿主细胞，从而防止PEDV感染所引发的猪流行性腹泻病。因此，SLC35A1可以作为猪流行性腹泻病防治的潜在靶点，具有重要的临床应用价值。
附图说明
图1为实施例1中sanger测序检测SLC35A1敲除细胞株的基因型。WT为野生型细胞，SLC35A1-/-为采用CRISPR/Cas9慢病毒策略构建的SLC35A1基因敲除细胞，△52bp代表缺失52个碱基。PAM为protospacer adjacent motif的缩写， sgRNA为small guide RNA的缩写。
图2为实施例1中检测SLC35A1 mRNA表达水平的荧光定量PCR结果。 GAPDH为内参基因。
图3为实施例2中利用EdU细胞增殖实验评估SLC35A1基因敲除对细胞增殖的影响。WT为野生型细胞，SLC35A1-/-代表SLC35A1基因敲除细胞。蓝色荧光为DAPI染色的细胞核，红色荧光为EdU染色阳性细胞。
图4为实施例3中利用荧光定量PCR检测PEDV感染不同时间点，Vero细胞中SLC35A1的表达水平。GAPDH为内参基因，对照组为未感染PEDV的Vero 细胞，hpi为感染时间。
图5为实施例4中利用免疫荧光检测PEDV感染不同时间点，SLC35A1基因敲除细胞与野生型细胞中PEDV编码N蛋白的表达情况。病毒感染复数(MOI) 为0.01，绿色荧光为PEDV编码的N蛋白，蓝色荧光为DAPI染色的细胞核， hpi为感染时间。
图6为实施例4中利用荧光定量PCR检测PEDV感染不同时间点，SLC35A1 基因敲除细胞与野生型细胞中PEDV的拷贝数。hpi为感染时间，*表示p<0.05， **表示p<0.01，***表示p<0.001。
图7为实施例4中利用免疫荧光检测不同MOI感染条件下，SLC35A1基因敲除细胞与野生型细胞中PEDV编码N蛋白的表达情况。感染时间为24hpi，绿色荧光为PEDV编码的N蛋白，蓝色荧光为DAPI染色的细胞核，MOI为感染复数。
图8为实施例4中利用荧光定量PCR检测不同MOI感染条件下，SLC35A1 基因敲除细胞与野生型细胞中PEDV的拷贝数。感染时间为24hpi，MOI为感染复数，*表示p<0.05，**表示p<0.01，***表示p<0.001。
具体实施方式
为了本发明方案更好地被本技术领域的人员理解，下面将结合附图对本发明实施例中的技术方案进行详细的描述，显然，所描述的实施例并不仅限于此。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都应当属于本发明保护的范围。
实施例1：利用CRISPR/Cas9技术构建SLC35A1基因敲除细胞株
1.1 sgRNA设计及表达载体构建
针对SLC35A1基因(Ensembl:ENSCSAG00000017346)外显子序列设计 sgRNA序列为5’-CCATAGCTTTAAGATACACA-3’，PAM序列为“AGG”。送公司合成sgRNA引物对为SLC35A1-sgR-F:5’-caccgCCATAGCTTTAAGATACAC A-3’，SLC35A1-sgR-R:5’-aaacTGTGTATCTTAAAGCTATGGc-3’。随后，取合成的sgRNA引物F和R(10pmol)各5μL，于PCR仪中进行退火反应：95℃， 10min；65℃，60min。接着，将退火产物与经BsmBI(NEB)线性化的lentiCRISPR v2(addgene:#52961)载体连接。进一步，转化、涂板、过夜培养，第二天挑取单菌落进行sanger测序鉴定。选取阳性克隆菌落扩大培养，并使用去内毒素试剂盒提取质粒，获得sgRNA正确表达的载体命名为“lentiCRISPR v2-SLC35A1- KO”。
1.2 SLC35A1基因敲除细胞株的制备与鉴定
按照pMD2.G：psPAX2：lentiCRISPR v2-SLC35A1-KO质量比为1：2：3 的比例，转染HEK293T细胞进行慢病毒包装。随后，接种慢病毒感染Vero细胞，利用流式分选术挑取单个细胞至96孔细胞培养板进行扩大培养，接着，参考天根DNA提取试剂盒的方法(KG203)提取单克隆细胞的基因组DNA。靶向sgRNA 靶标基因组区域，利用NCBI-BLAST(https://blast.ncbi.nlm.nih.gov/Blast.cgi)设计扩增引物对为SLC35A1-PCR-F：5’-CGCTCATGTAATTGGCAAGCAT-3’， SLC35A1-PCR-R：5’-ACAGTTTGCCAACTCCTACTCT-3’。然后，以基因组DNA 为模板，利用扩增引物对进行PCR反应。进一步，将PCR纯化产物克隆至 pMD19-T载体中，转化、涂板、过夜培养，挑取单菌落进行sanger测序鉴定单克隆细胞的基因型。结果显示，与野生型细胞相比，SLC35A1-/-细胞的基因组中 sgRNA靶标区域存在52个碱基缺失(图1)，表明SLC35A1基因编码序列发生移码突变。进一步，提取细胞总RNA，利用荧光定量PCR的方法检测SLC35A1-/-细胞中SLC35A1的mRNA表达情况。如图2所示，与野生型细胞相比，基因敲除细胞中SLC35A1的表达水平显著下调，表明成功构建了SLC35A1敲除细胞株。
实施例2：敲除SLC35A1不影响细胞正常增殖
利用EdU细胞增殖实验评估SLC35A1基因敲除对Vero细胞正常增殖情况的影响。首先，取等体积37℃预热的2×EdU工作液(20μM)于细胞培养板中与原培养液混匀，使EdU终浓度为1×，孵育2h；接着用预冷的4％多聚甲醛室温固定15min，随后漂洗细胞，预冷0.3％TritonX-100室温通透10min，再次漂洗细胞后加入Click反应液室温避光孵育30min；最后PBS清洗3遍，加入DAPI 染液，避光孵育10min。荧光显微镜下观察发现，SLC35A1基因敲除细胞增殖情况与野生型细胞无显著差异(图3)。
实施例3：PEDV感染Vero细胞后SLC35A1基因表达水平上调
按照MOI为0.01，接种PEDV(JS-A毒株，NCBI ID：MH748550，以下相同)感染Vero细胞，按照TRIZOL法提取12hpi、24hpi和36hpi的细胞总RNA，利用荧光定量PCR检测SLC35A1的mRNA表达水平的变化。其中SLC35A1基因的定量检测引物为：SLC35A1-qPCR-F：GAAAATGTCTTGGGGAGC， SLC35A1-qPCR-R：GCATAAAGCAGTACACGGA。结果显示，受PEDV感染后，Vero细胞中SLC35A1的mRNA表达水平显著上调(图4)。
实施例4：敲除SLC35A1能显著抑制PEDV在宿主细胞内的复制
接种PEDV感染SLC35A1基因敲除细胞与野生型细胞，收集不同时间点或不同MOI条件下的细胞样品，通过间接免疫荧光检测PEDV编码N蛋白的表达。取PEDV感染的细胞样品，用预冷的4％多聚甲醛室温固定10min，然后加入预冷0.3％TritonX-100室温通透10min，接着用含有BSA的封闭液室温封闭1.5h。 PBS漂洗3遍后，加入稀释的PEDV-N抗体，4℃过夜孵育。第二天，加入二抗室温摇床孵育2h。最后，用DAPI染液对细胞核进行室温避光染色10min，漂洗干净后，在荧光显微镜下观察荧光表达情况。
参考2019年发表于《吉林畜牧兽医》的“猪流行性腹泻病毒(PEDV)荧光定量RT-PCR检测方法的建立”一文构建PEDV绝对定量PCR标准质粒。靶向PEDV基因组，设计扩增引物对PEDV-186-F：TACTAAGCGTAACATCCTGCC， PEDV-186-R：GTAGTACCAATAACAACCGAAGC。以编码PEDV基因组的cDNA 为模板，PCR扩增、纯化后克隆至pMD19-T载体，挑取单个菌落进行sanger测序获得标准质粒。以标准质粒为模板，倍比稀释后荧光定量PCR扩增，获得到 Ct值与病毒拷贝数间的相互关系，绘制标准曲线y＝-3.2511x+39.778(R2＝0.9632)，其中y为Ct值，x为Log10(拷贝数)。进一步，收集PEDV感染不同时间点或不同MOI条件下的样品，提取病毒RNA(Takara，9766)，按照PrimeScript RT reagent Kit with gDNA Eraser试剂盒(Takara，RR047A)操作反转录获得cDNA，以cDNA为模板，利用PEDV-186-F和PEDV-186-R引物对进行荧光定量PCR 扩增反应。
结果显示，与野生型细胞相比，PEDV感染12hpi、24hpi和36hpi时，SLC35A1 基因敲除细胞中PEDV病毒粒子拷贝数及PEDV编码N蛋白的表达水平均显著降低(图5-6)。在MOI分别为0.01、0.05、0.1和0.2的条件下，PEDV感染24hpi 的SLC35A1基因敲除细胞中PEDV病毒粒子拷贝数及PEDV编码N蛋白的表达水平均显著低于野生型细胞组(图7-8)。表明敲除SLC35A1基因能显著抑制 PEDV在宿主细胞内的复制。
上述步骤中，未详尽描述、未特别指明的技术，均为现有技术中已有的常规技术手段，均可按照常规分子生物学和细胞生物学实验条件或按照制造厂商说明书建议的条件进行。
Claims (8)
1.SLC35A1基因作为靶点在制备防治猪流行性腹泻病毒感染的药物中的应用。
2.用于防治猪流行性腹泻病的药物，其特征在于，所述药物为靶向编辑SLC35A1基因或干扰其蛋白表达的物质。
3.根据权利要求2所述的药物，其特征在于，所述的药物包括靶向敲除SLC35A1基因的sgRNA或其表达载体，用于RNA干扰的dsRNA，反义寡核苷酸、小分子抑制剂。
4.根据权利要求3所述的药物，其特征在于，靶向敲除SLC35A1基因的sgRNA序列为5’-CCATAGCTTTAAGATACACA -3’。
5.一种药盒，其特征在于，包括靶向敲除SLC35A1基因的sgRNA序列、CRISPR/Cas9质粒。
6.根据权利要求5所述的药盒，其特征在于，sgRNA序列为5’-CCATAGCTTTAAGATACACA -3’。
7.权利要求5或6所述的药盒在制备防治猪流行性腹泻病的药物中的应用。
8.SLC35A1基因作为靶点在制备抗流行性腹泻病毒感染的基因编辑细胞或动物模型中的应用。
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