CN117343932B - 靶向抑制MS4A7基因表达的siRNA及其应用 - Google Patents
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Abstract
本发明公开了靶向抑制MS4A7基因表达的siRNA及其应用。其中,优选的,所述的靶向抑制MS4A7基因表达的siRNA的序列如下所示:正义链:5’‑CCGCUGGAGUAGGCCUCUUTT‑3’;反义链:5’‑AAGAGGCCUACUCCAGCGGTT‑3’。实验证明,当MS4A7表达受到抑制,病毒复制水平也明显下降,与对照组相比CDV拷贝数下降高达3.09倍,说明干扰下调MS4A7表达能够显著抑制CDV在星形胶质细胞中的复制,同时si‑cMS4A7转染对细胞毒性低,不会影响细胞正常存活与增殖。因此,本发明的提出为CDV的预防和治疗提供了有效的技术手段。
Description
技术领域
本发明涉及一种靶向抑制MS4A7基因表达的siRNA,还涉及该siRNA在抑制犬瘟热病毒复制中的应用。本发明属于生物技术领域。
背景技术
犬瘟热病毒(canine distemper virus,CDV)属于副粘病毒科(Paramyxoviridae)麻疹病毒属(Morbillivirus,MV)成员,可感染犬以及多种食肉动物,导致高度接触性传染病。CDV侵害神经系统,在脑白质形成的脱髓鞘病变,是研究神经退行性疾病的模式病毒。与麻疹病毒相似,CDV感染后在脑部形成持续性感染,主要感染的靶细胞为星形胶质细胞。即使康复也会导致终生的神经症状,给动物健康造成巨大的损害。
跨膜4A基因家族(membrane-spanning 4A gene family,MS4A)是近年来发现的在细胞增殖和分化过程中起重要作用的基因家族。MS4A基因家族成员参与细胞信号转导。目前已经发现在人类基因组中,此家族成员有超过14个。MS4A7是Gingras MC等人在2001 年发现的MS4A家族新的成员。此家族成员在氨基酸序列同源性、蛋白质空间结构及染色体定位上都有共同特性。MS4A7蛋白除了有4个保守的跨膜疏水区之外,还有许多独特的特点Marie-ClaudeGingras 等通过斑点分子杂交法证实了MS4A7在许多正常组织和癌组织中的表达有差异。其在FAB1、FAB5、U-937等淋巴系统疾病的细胞或细胞系中,随着细胞分化程度的提高,MS4A7的表达也增加。这表明了MS4A7在细胞成熟过程或在成熟细胞中有重要的作用。
近期有研究利用生物信息学分析推测MS4A7基因簇可能参与了AD(阿尔茨海默病)的神经炎症致病机制,这提示MS4A7可能参与了神经炎症调控。MS4A7 与单核细胞系的成熟细胞功能有关,Northern斑点杂交法检测其在多种正常组织和癌组织中均有不同程度的表达。目前其成为单核细胞表面标记物在疾病中得到了应用,推测与其同族成员MS4A1(CD20)和MS4A2(FcεRIβ)一样,可能是参与信号转导的受体复合体的一个组成部分。
本发明在前期对CDV感染星形胶质细胞转录组信息分析基础上,发现MS4A7在CDV感染DA细胞中表达升高,因此,本发明以MS4A7为研究对象,观察RNA干扰(RNAinterfering,RNAi) 抑制MS4A7表达对CDV感染星形胶质细胞的影响,并进一步研究其作用机制,以期为CDV的预防和治疗提供技术手段。
发明内容
本发明的目的在于提供一种靶向抑制MS4A7基因表达的siRNA及其在抑制犬瘟热病毒复制中的应用。
为了达到上述目的,本发明采用了以下技术手段:
本发明首先提出了靶向抑制MS4A7基因表达的siRNA在制备抑制犬瘟热病毒复制的药物中的应用。
其中,优选的,所述的靶向抑制MS4A7基因表达的siRNA的序列如下所示:
正义链:5’-CCGCUGGAGUAGGCCUCUUTT-3’;
反义链:5’-AAGAGGCCUACUCCAGCGGTT-3’。
一种靶向抑制MS4A7基因表达的siRNA也在本发明的保护范围之内,所述的靶向抑制MS4A7基因表达的siRNA的序列如下所示:
正义链:5’-CCGCUGGAGUAGGCCUCUUTT-3’;
反义链:5’-AAGAGGCCUACUCCAGCGGTT-3’。
相较于现有技术,本发明的有益效果是:
首先,本发明提出了 MS4A7作为靶点在抑制犬瘟热病毒复制的药物中的应用。其次,本发明还提出了一种靶向抑制MS4A7基因表达的siRNA。实验证明,当MS4A7表达受到抑制,病毒复制水平也明显下降,与对照组相比CDV拷贝数下降高达3.09倍,说明干扰下调MS4A7表达能够显著抑制 CDV在星形胶质细胞中的复制,同时si-cMS4A7 转染对细胞毒性低,不会影响细胞正常存活与增殖。因此,本发明的提出为CDV的预防和治疗提供了有效的技术手段。
附图说明
图1为荧光定量PCR检测siRNA敲减MS4A7的转录水平;
图2为CCK-8法检测si-cMS4A7敲减对细胞活力的影响;
图3为siMS4A7对CDV复制的影响;
其中,A 干扰60h后RT-qPCR检测CDV结果图;B Western blot检测CDV-N蛋白表达图;C 干扰后 CDV-N蛋白表达的灰度值扫描结果图。
具体实施方式
下面结合具体实施例来进一步描述本发明,本发明的优点和特点将会随具体实施例的描述而更为清楚。但实施例仅是范例性的,并不对本发明的范围构成任何限制。本领域技术人员应该理解的是,在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换,但这些修改和替换均落入本发明的保护范围内。
实施例1
1材料和方法
1.1 病毒、细胞及主要试剂和仪器
CDV病毒Snyder Hill(ATCC:VR-1587TM)由本实验室保存,犬星形胶质细胞由本实验室自行制备。DMEM培养基和LipofectamineTM 2000 均购自Invitrogen; CCK-8 细胞活力检测试剂盒购自美伦生物公司;阴性对照NC、MS4A7 siRNA购自吉玛基因公司。
1.2 方法
1.2.1细胞培养及转染
犬星形胶质细胞常规使用含10% FBS 的DMEM培养液,在5% CO2、37 ℃培养箱培养。转染前1 d,胰酶消化对数生长期的细胞,接种细胞于含10% FBS 的DMEM培养基的24孔板,每孔0.5 mL( 约7 × 104 细胞) ,使细胞在转染日约为70% 融合度。参照LipofectamineTM2000 制备Lipo 2000-siRNA复合物,将复合物加入24 孔板,轻柔来回摇晃培养板使之混匀,培养箱内常规孵育4-5 h,更换为完全培养基培养,继续培养24 h,进行转染后的其他检测步骤。实验分为阴性对照组(negative control,NC),si-cNC组(转染阴性对照si-cNC)和si-cMS4A7组(转染MS4A7的特异性siRNA)。
si-cMS4A7序列为5’-3’:CCGCUGGAGUAGGCCUCUUTT(SEQ ID NO. 1);AAGAGGCCUACUCCAGCGGTT(SEQ ID NO. 2)。si-cNC序列为5’-3’:UUCUCCGAACGUGUCACGUTT(SEQ ID NO. 3);ACGUGACACGUUCGGAGAATT(SEQ ID NO. 4)。
1.2.2 siRNA干扰MS4A7表达效率的检测
采用qPCR法检测转染si-MS4A7 后犬星形胶质细胞中MS4A7蛋白表达情况,简要步骤如下:
将24孔板中培养的犬星形胶质细胞弃去上清,PBS洗一次,每孔中加入试剂盒中的R-I试剂300μL,反复轻轻吹打细胞孔中样品至细胞完全裂解,接下来按照试剂盒说明书进行RNA的提取。使用PrimeScript Ⅱ Reverse Transcript反转录试剂盒和Primer 6反转录成cDNA。使用荧光定量预混液SYBR Green Master Mix进行荧光定量PCR,犬MS4A7引物序列(5’-3’) :上游引物CAGGAATGACCAGTAGGAAGTT(SEQ ID NO. 5);下游引物:AGAAGAGTCACAGATTCCCTTG(SEQ ID NO. 6)。犬GAPDH引物序列(5’-3’):上游引物AACGAGAAGAGCTATGATCCTG(SEQ ID NO. 7),下游引物GGTGGCAGTATAGGAATGAACT(SEQ IDNO. 8)。检测及溶解曲线程序为预混液说明书中推荐,检测体系为20μL:SYBR GreenMaster Mix 10 μL,ddH2O 5 µL,上游引物 1 µL,下游引物 1 µL,cDNA 3μl。实验重复3次。
1.2.3 CCK-8 法检测各组细胞活力
将转染siRNA后的犬星形胶质细胞以每孔1.75 × 104 接种于96 孔板,每组6个复孔,放置在5%体积分数CO2、37 ℃培养箱培养,分别在培养1 h、12h、24 h、36 h、48h以及60 h时,每孔中加入CCK-8 试剂10μL,培养箱内继续孵育3 h后,酶标仪测定450 nm 波长处的吸光度(A) 值。实验重复3 次。
1.2.4 siRNA干扰下调MS4A7表达对CDV复制的检测
使用反转录试剂盒,对样品提取总RNA进行反转录,反转录合成cDNA ,针对CDV-N和犬MS4A7进行RT-qPCR(荧光定量PCR引物由吉林省库美生物科技公司合成)。CDV-N引物序列(5’-3’) :上游引物CAACGGCCCTAAATTAACTG(SEQ ID NO. 9);下游引物:CCTCTACTAACTTGATGCTT(SEQ ID NO. 10)。犬MS4A7引物序列(5’-3’) :上游引物ATCCATTATGGTGACCACTAGC(SEQ ID NO. 11);下游引物:TCCATCTGTGTGTTCCGTATAG(SEQ IDNO. 12)。
采用Western blot 法检测siRNA干扰下调MS4A7表达对CDV复制的影响,简要步骤如下: 适量RIPA(PMSF)裂解液提取细胞总蛋白,BCA 法测定蛋白含量,蛋白样品加上样缓冲液,100 ℃煮沸变性10 min,按照每孔50 μg上样,经10% SDS-PAGE,电泳后电转移蛋白至PVDF 膜,室温条件将膜用5%脱脂奶粉封闭1 h,洗膜3次,每次5min。将CDV N抗体(1∶1000,SANTA CRUZ) 和内参GAPDH抗体( 1∶ 2 000,中杉金桥),抗体孵育液加入至孵育盒,4 ℃孵育过夜,洗膜, IRDye 800CW羊抗兔/鼠荧光二抗(1:8000,LI-COR)室温孵育1 h,洗膜3次,每次5min。Image-ProPlus 6. 0 软件进行灰度值扫描分析,实验重复3次。
1.2.5 统计学方法
所有实验数据采用SPSS 21. 0 软件进行分析,计量资料用均数±标准差( mean± SD) 表示,多组差异比较采用单因素方差分析,两两比较采SNK-q 检验,以P < 0. 05为差异有统计学意义。
2 结果
2.1 CCK-8 法检测各组犬星形胶质细胞的活力
对96孔板中的犬星形胶质细胞分别转染 si-cMS4A7和si-cNC,转染后检测MS4A7抑制效果,收集1hpi-60hpi样品使用CCK-8 试剂检测转染后细胞存活情况。由于缺少针对犬种属MS4A7的抗体,所以用qPCR检测MS4A7转录本敲减情况,由结果可知MS4A7转录本降低了72.64%,表明MS4A7基因被成功敲减(图1)。同时对转染后0~60h的细胞每隔12h时间点的细胞活力进行检测,结果表明si-cMS4A7敲减对星胶质细胞活力没有影响(图2)。说明siRNA敲减能在不影响细胞活力的情况下降低细胞内MS4A7转录水平。说明si-cMS4A7和si-cNC转染对细胞毒性低,不会影响细胞正常存活与增殖。
2.2 干扰下调 MS4A7表达抑制 CDV在犬星形胶质细胞中的复制
为了检测下调MS4A7分子对CDV复制的影响,将si-cMS4A7和si-cNC分别转染犬星形胶质细胞,转染后24h,再以5MOI(病毒在Vero细胞上复制测定结果)的CDV感染,在感染后1hpi-60hpi收取细胞RNA和细胞蛋白, 进行RT-qPCR和Western Blot方法检测。RT-qPCR检测结果显示,与对照组相比,在60hpi时,MS4A7表达受到抑制时,CDV拷贝数下降高达3.09倍(见图3A)。Western Blot结果显示,与对照组相比,MS4A7表达受到抑制时,病毒复制水平也明显下降,见图3B。灰度值扫描进一步分析结果表明,当MS4A7分子表达受到抑制时,CDV N蛋白表达明显降低(见图3C)。
Claims (2)
1.靶向抑制MS4A7基因表达的siRNA在制备抑制犬瘟热病毒(caninedistempervirus,CDV)复制的药物中的应用;所述的靶向抑制MS4A7基因表达的siRNA的序列如下所示:
正义链:5’-CCGCUGGAGUAGGCCUCUUTT-3’;
反义链:5’-AAGAGGCCUACUCCAGCGGTT-3’。
2.一种靶向抑制MS4A7基因表达的siRNA,其特征在于,所述的靶向抑制MS4A7基因表达的siRNA的序列如下所示:
正义链:5’-CCGCUGGAGUAGGCCUCUUTT-3’;
反义链:5’-AAGAGGCCUACUCCAGCGGTT-3’。
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---|
Differential transcriptional profiles identify microglial- and macrophage-specific gene markers expressed during virus-induced neuroinflammation;Ana Beatriz DePaula-Silva 等;Journal of Neuroinflammation;第16卷(第152期);第1-20页 * |
犬瘟热病毒细胞受体研究进展;徐淑娟 等;特产研究(03);第63-66页 * |
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