CN110172505A - Trim33基因作为prrsv感染相关因子的应用 - Google Patents
Trim33基因作为prrsv感染相关因子的应用 Download PDFInfo
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Abstract
本发明提供TRIM33基因作为PRRSV感染相关因子的应用。本发明首次从细胞水平上探明TRIM33基因影响PRRSV感染的机制。实验表明,TRIM33基因促进PRRSV的感染,同时,PRRSV感染可上调TRIM33基因的表达,进一步研究表明,TRIM33通过影响NF‑κB信号通路来影响IFNβ1的产生。本发明为抗蓝耳病转基因猪的生产打下坚实基础。
Description
技术领域
本发明涉及基因工程技术领域,具体地说,涉及TRIM33基因作为PRRSV感染相关因子的应用。
背景技术
猪繁殖与呼吸综合征(Porcine Reproductive And Respiratory Syndrome,PRRS),又称蓝耳病,是由PRRSV引起的,临床上以母猪的繁殖障碍和仔猪的呼吸道症状为该病的主要特征,母猪感染后表现为流产、弱胎、死胎和木乃伊胎等症状,仔猪感染后表现为精神不振、厌食以及呼吸困难等症状(Rossow,1998)。1987年美国首次报道了PRRS的发生,1990年欧洲发现了类似的疾病。1996年在我国首次出现,自发现之后,这种疾病就一直伴随着中国的养猪业。2006年夏我国大面积爆发了一种高致病型的蓝耳病,波及全国多达25个省份,表现为传播速度快、出现持续高热以及高致病率和高死亡率等特点,给养猪业造成了严重的打击(Li et al.,2007;Tian et al.,2007)。加之我国目前PRRSV毒株的多样性和临床的复杂程度日益加剧,因此,猪繁殖与呼吸综合征预防与控制势在必行。
PRRSV属于套式病毒目、动脉炎病毒科、动脉炎病毒属,是具有囊膜包被的单股、正链、不分节段的RNA病毒。PRRSV除了具有广泛变异和毒株多样性的特性外,还对宿主的免疫系统具有抑制作用,最终导致针对PRRSV中和抗体的产生不但存在延迟和不足,而且诱导产生的非特异性的中和抗体对病毒复制还具有增强效应,也就是抗体依赖性增强(antibody-dependent enhancement,ADE)(Yoon et al.,1996)。因此,不仅不能有效清除病毒,反而引起PRRSV的持续性感染,而且常常出现并发或继发其他病毒和细菌的感染,譬如圆环病毒、猪流行性腹泻病毒等(Pol et al.,1997)。PRRSV具有的这些生物学特性及其复杂致病机理,导致目前仍然没有有效的疫苗和防治策略。归根结底是对于PRRSV与宿主互作信息以及PRRSV感染相关因子的信息了解太少。
前期基于全基因组水平,利用piggyBac转座子系统和二代测序技术鉴定PRRSV感染相关的宿主基因,发现TRIM33基因属于候选基因之一。TRIM33(Tripartite motif-containing protein 33)是一个E3泛素化连接酶,属于进化上比较保守的TRIM家族(Letizia Venturini1,1999)。TRIM家族是由N端保守的结构包括RING,两个B-box以及一个coiled-coil结构域(Hatakeyama,2011)组成的。TRIM33与TRIM24,TRIM28和TRIM66同属于一个TRIM亚家族,包括C端的PHD和BRD结构域(Herquel et al.,2011)。这些TRIM蛋白不直接结合DNA而是被DNA结合蛋白招募,诸如核受体SMAD,PU.1,TAL-1等来作为转录调控因子(Herquel et al.,2013;Kusy et al.,2011)。目前报道TRIM33涉及多个生物过程,包括胚胎发育,造血系统发育,DNA修复,细胞循环调控,炎症激活和免疫反应等过程均发挥重要功能(Bai et al.,2010;Doisne et al.,2009;Kusy et al.,2011;Weng et al.,2014)。然而对于TRIM33是否会影响PRRSV感染尚未见报道。
发明内容
本发明的目的是提供TRIM33基因作为PRRSV感染相关因子的应用。
为了实现本发明目的,第一方面,本发明提供TRIM33基因的以下任一应用:
1)作为PRRSV感染相关因子的应用;
2)作为标志物在制备PRRSV感染检测和/或疗效评价试剂中的应用。
本发明中,所述TRIM33基因来自于猪或灵长类动物(如猴子)。
第二方面,本发明提供TRIM33基因抑制剂的以下任一应用:
1)用于制备抗PRRSV药物或组合物;
2)作为JAK-STAT信号通路的激活剂;
3)作为IFNβ1基因以及受IFNβ1基因调控的下游基因的激活剂;
4)作为NF-κB信号通路的激活剂。
本发明中,所述抑制剂是能够从转录或翻译水平上抑制TRIM33基因表达的物质,所述抑制剂选自shRNA、siRNA、dsRNA、miRNA、cDNA、反义RNA/DNA、低分子化合物、肽、抗体等中的至少一种。
优选地,所述抑制剂为siRNA,其核苷酸序列为5’-UUGUCUUUGGAGGUGUGGCTT-3’(SEQ ID NO:5)。
本发明中,所述受IFNβ1基因调控的下游基因包括但不限于IFIT1,IFIT2,MX2,OAS1和OAS3。
第三方面,本发明提供靶向编辑TRIM33基因的CRISPR-Cas9系统的以下任一应用:
1)用于制备抗PRRSV药物或组合物;
2)作为JAK-STAT信号通路的激活剂;
3)作为IFNβ1基因以及受IFNβ1基因调控的下游基因的激活剂;
4)作为NF-κB信号通路的激活剂;
5)用于制备TRIM33基因敲除的猪肺泡巨噬细胞系;
6)用于制备抗蓝耳病猪。
第四方面,本发明提供一种抗PRRSV药物或组合物,其有效成分为TRIM33基因抑制剂和/或靶向编辑TRIM33基因的CRISPR-Cas9系统。
第五方面,本发明提供一种JAK-STAT信号通路的激活剂,其有效成分为TRIM33基因抑制剂和/或靶向编辑TRIM33基因的CRISPR-Cas9系统。
第六方面,本发明提供IFNβ1基因以及受IFNβ1基因调控的下游基因的激活剂,其有效成分为TRIM33基因抑制剂和/或靶向编辑TRIM33基因的CRISPR-Cas9系统。
第七方面,本发明提供一种NF-κB信号通路的激活剂,其有效成分为TRIM33基因抑制剂和/或靶向编辑TRIM33基因的CRISPR-Cas9系统。
第八方面,本发明提供TRIM33基因敲除的猪肺泡巨噬细胞系,所述细胞系按如下方法制备得到:根据猪TRIM33基因序列,构建基于CRISPR-Cas9系统的sgRNA表达载体,以此作为TRIM33基因打靶载体,转入猪肺泡巨噬细胞中,获得的中靶阳性细胞克隆,即为TRIM33基因敲除的猪肺泡巨噬细胞系。
优选地,gRNA作用位点的DNA序列为:5’-CAGCGCGCCGGTAACTGCCG-3’和5’-GGCAGCGCGCCGGTAACTGC-3’(SEQ ID NO:6和7)。
第九方面,本发明提供所述细胞系在制备抗蓝耳病转基因猪中的应用。
借由上述技术方案,本发明至少具有下列优点及有益效果:
本发明首次从细胞水平上探明TRIM33基因影响PRRSV感染的机制。实验表明,TRIM33基因促进PRRSV的感染,同时,PRRSV感染可上调TRIM33基因的表达,进一步研究表明,TRIM33通过影响NF-κB信号通路来影响IFNβ1的产生。本发明在细胞水平验证了TRIM33敲除能够抑制PRRSV感染,可进一步制备TRIM33基因敲除猪来达到抗蓝耳病的效果,因此,本发明为抗蓝耳病转基因猪的生产打下坚实基础。此外,TRIM33敲除可以上调IFNβ1表达,可能有助于抵抗其它病原的感染。
附图说明
图1为本发明实施例1中PCR扩增TRIM33cDNA片段结果。用高保真酶Q5进行PCR扩增,扩增PCR产物进行凝胶电泳检测。1-4:扩增得到的TRIM33cDNA全长片段,5:1Kb Marker。
图2为本发明实施例1中Western blot检测Marc145细胞中TRIM33外源表达水平。分别用pcDNA3.1-TRIM33和pcDNA3.1空载体转染Marc145细胞,转染48h后,Western blot检测胞内的TRIM33的外源表达水平。利用anti-Myc鼠源单克隆抗体检测外源Myc-TRIM33的表达,anti-GAPDH兔源单克隆抗体检测内参GAPDH的表达。NC代表转染空载体组。
图3为本发明实施例1中qPCR检测TRIM33过表达胞内病毒mRNA水平。用CH-1aPRRSV以MOI=1.0感染TRIM33过表达以及野生型Marc145细胞。感染36h后收取细胞,用qPCR方法检测胞内的PRRSV mRNA含量。GAPDH作为内参。数据为3次独立实验的结果,结果用t-test分析(mean±SEM),*p<0.05,**p<0.01和***p<0.001。
图4为本发明实施例1中两种TRIM33敲除单克隆细胞系和野生型Marc145细胞靶点序列比对结果。利用CRISPR/Cas9技术构建TRIM33敲除细胞系,分别命名为40-8-1和40-11-11,靶点位置的突变情况分别是插入碱基A和缺失碱基A。翻译均为提前终止。
图5为本发明实施例1中qPCR检测TRIM33敲除细胞中的病毒mRNA水平。用CH-1aPRRSV以MOI=1.0感染两个TRIM33敲除单克隆细胞系和野生型的Marc145细胞。感染12h,24h,36h,48h收取细胞,用qPCR实验方法检测胞内的PRRSV N mRNA含量。GAPDH作为内参。40-8-1和40-11-11是TRIM33敲除细胞系。数据为3次独立实验的结果,结果用t-test分析(mean±SEM),*p<0.05,**p<0.01和***p<0.001。
图6为本发明实施例1中Western blot检测TRIM33敲除细胞中的病毒蛋白水平。用CH-1a PRRSV以MOI=1.0感染TRIM33敲除细胞系和野生型的Marc145细胞。感染36h或48h后收取细胞,用Western blot实验方法检测胞内的PRRSV蛋白含量。GAPDH作为内参。T1和T11是TRIM33敲除细胞系。使用本实验室自己制备的兔抗PRRSV N蛋白的血清(1:500)来检测PRRSV N蛋白,以兔抗GAPDH的单克隆抗体检测内参GAPDH的含量。
图7为本发明实施例1中免疫荧光染色实验检测TRIM33敲除细胞中的病毒蛋白含量。用CH-1a PRRSV和XH-GD PRRSV两种毒株感染野生型Marc145细胞和TRIM33敲除细胞(40-8-1和40-11-11),接毒36h后,进行免疫荧光检测实验,其中PRRSV N蛋白杂交使用鼠源SDOW17anti-PRRSV N蛋白的单克隆抗体,Alexa Fluor 488标记的二抗,信号在荧光显微镜下呈现绿色。DAPI(蓝色)染细胞核。图中GFP列显示PRRSV N蛋白的染色结果,overlay列显示为GFP与DAPI图像的重叠图像。图中标尺为1000μm。
图8为本发明实施例1中检测不同PRRSV毒株在TRIM33敲除细胞中的生长动力学。分别用CH-1a、XH-GD和WUH3 PRRSV三种毒株分别感染野生型Marc145细胞和TRIM33敲除细胞(40-8-1和40-11-11),每种细胞的每个时间点作三个重复孔,接毒剂量MOI为0.1,分别于接毒后12h,24h,36h,48h,将细胞反复冻融裂解后,测定病毒滴度,绘制病毒生长曲线。横坐标代表感染的时间点,纵坐标代表Ig(TCID50/mL)。
图9为本发明实施例1中qPCR检测PRRSV感染Marc145细胞后病毒和TRIM33的mRNA含量变化。将CH-1a PRRSV感染野生型Marc145细胞,MOI为1.0,分别于接毒后12h,24h,36h,48h,60h收集细胞,用qPCR方法检测胞内的PRRSV N和TRIM33 mRNA含量。GAPDH作为内参。左图为随着时间点的推移胞内PRRSV N mRNA的含量变化,右图为随着时间点的推移胞内TRIM33 mRNA的含量变化。数据为3次独立实验的结果,结果用t-test分析(mean±SEM),*p<0.05,**p<0.01和***p<0.001。
图10为本发明实施例1中双荧光素酶检测不同剂量PRRSV感染对TRIM33启动子活性的影响。将pGL3-TRIM33双荧光素酶报告基因载体与海参荧光素酶报告基因载体(pRL-TK)共转入Marc145细胞中,pRL-TK作为转染效率的内参,pGL3-Basic作为空白对照。转染12h后,不同剂量的CH-1a PRRSV(MOI为0.01,0.1,1.0)感染24h后,收集细胞并检测荧光素酶的活性。数据为3次独立实验的结果,结果用t-test分析(mean±SEM),*p<0.05,**p<0.01和***p<0.001。
图11为本发明实施例1中qPCR检测TRIM33对IFNβ1及下游干扰素刺激基因的表达水平。将CH-1a PRRSV感染野生型的Marc145细胞(NC)、Marc14TRIM33-/-细胞(40-8-1和40-11-11)和TRIM33过表达细胞,24h后,用poly(I:C)(25μg/mL)刺激细胞24h后收集细胞,用qPCR方法检测胞内的IFNβ1mRNA含量(a)。同时qPCR方法检测野生型的Marc145细胞(NC)、Marc14TRIM33-/-细胞(40-8-1和40-11-11)中IFIT1,IFIT2,MX2,OAS1和OAS3的mRNA含量(b)。GAPDH作为内参。数据为3次独立实验的结果,结果用t-test分析(mean±SEM),*p<0.05,**p<0.01和***p<0.001。
图12为本发明实施例1中双荧光素酶实验检测IFNβ1启动子活性。将pGL3-IFNβ1双荧光素酶报告基因载体与海参荧光素酶报告基因载体(pRL-TK)共转入野生型Marc145细胞(NC)、Marc14TRIM33-/-细胞(40-8-1和40-11-11)和TRIM33过表达细胞,pRL-TK作为转染效率的内参,pGL3-Basic作为空白对照。转染12h后,不同剂量的CH-1a PRRSV(MOI为0.01,0.1,1.0)感染24h后,收集细胞并检测荧光素酶的活性。数据为3次独立实验的结果,结果用t-test分析(mean±SEM),*p<0.05,**p<0.01和***p<0.001。
图13为本发明实施例1中双荧光素酶实验检测TRIM33抑制IFNβ1的产生的通路。将全长IFNβ1启动子进行突变,即分别删除IRF1/2,IRF3,NF-κB和ATF-2/c-Jun转录因子的结合位点(a)和单独的NF-κB转录因子结合位点(b)克隆到pGL3-Basic荧光素酶报告基因载体上,与pRL-TK共转入TRIM33敲除细胞(40-8-1和40-11-11)和野生型Marc145细胞,转染24h用PRRSV进行感染,感染24h后,用25μg/mL的poly(I:C)进行刺激处理,24h后收集细胞并检测荧光素酶的活性。ns代表统计学上不显著。
图14为本发明实施例2中在改造过的3D4/21细胞中验证TRIM33与PRRSV之间的关系。a:Western blot检测pTRIM33外源蛋白表达水平。分别用Myc-pTRIM33和空载体转染YHC-PAM细胞,转染48h后,Western blot检测胞内蛋白的表达水平。利用anti-Myc鼠源单克隆抗体检测外源蛋白的表达,anti-GAPDH兔源单克隆抗体检测内参GAPDH的表达。NC代表3D4/21细胞中转染空载体组。b:qPCR检测TRIM33过表达细胞中的病毒含量。利用WUH3PRRSV毒株进行感染上述细胞,24h后收集细胞并提取细胞总RNA。用qPCR方法检测胞内的PRRSV N mRNA含量。GAPDH作为内参。c:qPCR检测siRNAs对TRIM33基因的敲降效率。将设计好的针对TRIM33的siRNA以及阴性对照siNC转染YHC-PAM细胞,48h收集细胞,并提取细胞总RNA。以设计好的针对TRIM33定量引物对TRIM33mRNA水平进行qPCR检测。d:qPCR检测siRNAs敲降细胞中的病毒mRNA含量。使用WUH3PRRSV毒株对TRIM33敲降细胞进行感染,24h后收集细胞并提取细胞总RNA。用qPCR方法检测胞内的PRRSV N mRNA含量。GAPDH作为内参。数据为3次独立实验的结果,结果用t-test分析(mean±SEM),*p<0.05,**p<0.01和***p<0.001。
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明的范围。若未特别指明,实施例均按照常规实验条件,如Sambrook等分子克隆实验手册(Sambrook J&Russell DW,Molecular Cloning:a Laboratory Manual,2001),或按照制造厂商说明书建议的条件。
实验细胞和病毒:
Marc-145细胞(ATCC NO.CRL-12231),非洲绿猴肾上皮细胞系,由华中农业大学张淑君教授实验室惠赠;
改造过的3D4/21细胞由中国农业大学动物医学院杨汉春教授实验室惠赠;
PRRSV传统毒株CH-1a(GenBank accession NO.AY032626)由中国农业大学生物学院封文海教授实验室惠赠;
PRRSV高致病性毒株WUH3(GenBank accession No.HM853673.2)由华中农业大学张淑君教授实验室惠赠;
PRRSV高致病性毒株XH-GD(GenBank accession No.EU624117.1)由华南农业大学张桂红教授实验室惠赠。
菌株和质粒:
克隆载体pMD19-T购自Takara公司;
克隆载体pcDNA3.1(+)由中国农业大学农业生物技术国家重点实验室提供;
打靶载体CRISPR/Cas9质粒pX330由中国农业大学农业生物技术国家重点实验室提供;
双荧光素酶报告载体系统由中国农业大学农业生物技术国家重点实验室提供;
大肠杆菌(Escherichia coli)DH5α由中国农业大学农业生物技术国家重点实验室提供,-80℃保存。
常用实验试剂:
PCR引物合成由北京六合华大基因科技有限公司合成;DNA测序由北京六合华大基因生物公司和上海美吉生物医药科技有限公司完成;
2×M5Taq HiFi PCR mix DNA聚合酶购自聚合美公司,-20℃保存;Q5DNA高保真聚合酶购自NEB公司,-20℃保存;dNTPs购自Sigma公司,-20℃保存;100bp和1Kb DNA marker购自中科瑞泰(北京)公司;琼脂糖购自HydraGene公司;核酸染料购自北京雷根生物技术有限公司;T4连接酶和各种限制性内切酶购自NEB公司;氨苄青霉素购自Solarbio公司;TRIzol购自Magen公司;
蛋白marker购自Thermo Fisher Scientific公司;RIPA裂解液(强)购自碧云天公司;蛋白酶抑制剂PMSF购自碧云天公司;蛋白酶K购自Roche公司;PVDF膜购自Millipore公司;ECL Western blot发光底物购自Thermo Fisher Scientific公司;
DMEM细胞培养基、PBS、HEPES均购自Gibco公司;1640培养基、优化培养基(Opti-MEM)、双抗(青链霉素)、非必需氨基酸均购自中科迈晨(北京)公司;胰酶购自Neuronbc公司;胎牛血清购自Foundation公司;DMSO购自MP公司;克隆环、硅胶、poly(I:C)均购自Sigma公司;jetPRIME脂质体转染试剂购自Polyplus公司;HiPerFect购自QIAGEN公司;
鼠源PRRSV N蛋白单克隆抗体(SDOW17)购自RTI公司;兔抗PRRSV N蛋白多克隆抗体为本实验制备;鼠源c-Myc标签抗体购自Cell Signal Techonology公司;兔源GAPDH抗体购自Cell Signal Techonology公司;HRP标记的山羊抗小鼠IgG(H+L)抗体购自中杉金桥公司;HRP标记的山羊抗兔IgG(H+L)抗体购自中杉金桥公司;Alexa Fluor 488的标记山羊抗小鼠IgG(H+L)抗体、DAPI购自碧云天公司;BSA购自Sigma公司。
实验所用试剂盒:
DNA纯化试剂盒购自Magen公司;
去内毒素质粒小量提取试剂盒(M5Endofree Plasmid Mini Kit)购自北京聚合美公司;
细胞及血液DNA提取试剂盒(DNeasy Blood and Tissue Kit)购自Magen公司;
DNA小量提取试剂盒购自Qigen公司;
总RNA提取试剂盒购自Magen公司;
反转录试剂盒(M-MLV Reverse Transcriptase)购自Promega公司;
定量PCR试剂盒(SYBR Green PCR Master Kit)购自Yeasen公司;
BCA蛋白浓度测定试剂盒购自碧云天公司;
双荧光素酶检测试剂盒购自Promaga公司。
实验方法:
酶切、连接、回收、转化、PCR扩增等常规分子生物学实验操作步骤详见《分子克隆(第三版)》。
一、间接免疫荧光:
1、样品准备:接种PRRSV 24-48h后,吸弃上清,PBS洗两次;
2、固定:固定液(甲醇)室温固定10min(甲醇覆盖细胞即可),PBS洗两次;
3、封闭:加入1%BSA,并于室温下封闭30min-1h;
4、一抗孵育:弃封闭液,并加入抗PRRSV N蛋白的SDOW17抗体(1:100稀释),于室温下孵育1h。PBS清洗3次,每次10min;
5、二抗孵育:加入Alexa Fluor 488标记的羊抗兔IgG(1:1000稀释),室温避光孵育1h。PBS清洗3次,每次10min;
6、DAPI染色:加入DAPI(1:1000稀释),避光孵育3-5min,PBS清洗3次;
7、观察拍照:在荧光倒置显微镜下观察,并进行拍照记录。
二、双荧光酶报告基因检测:
双荧光素酶报告基因检测系统(Dual-Luciferase Report gene assay system,DLR)是以完全不同的两种荧光素为底物来区分萤火虫荧光素酶和海肾荧光素酶化学发光活性的报告系统。萤火虫荧光素酶(61KDa)能够催化荧光素氧化发光。同时,在辅酶A(CoA)的作用下,可检测到持续的发光信号。而海肾荧光素酶(36KDa)可以利用O2和海肾荧光素产生发光信号。最后可以通过荧光检测仪定量荧光素氧化过程中释放的生物荧光。DLR检测实验中,将连接了两种荧光素酶的载体分别转入细胞中,依次检测其活性,其中海肾荧光素酶的活性作为实验的内参。DLR系统可以极灵敏,极高效地检测基因的表达。具体实验步骤如下:
1、转染:将连接了目的基因启动子的萤火虫荧光素酶报告基因载体与pRL-TK海肾荧光素酶基因载体共同转染细胞;
2、收集细胞:转染24-48h后,对于贴壁不紧的细胞来说直接吹打下来,对于贴壁较紧的细胞来说,使用胰酶进行消化,收集细胞。PBS清洗细胞,用150μL的DMEM重悬细胞;
3、裂解细胞并检测萤火虫荧光素酶活性:将150μL的细胞悬液平均分到两个孔中,分别加入75μL的荧光虫荧光素酶底物,混匀后于室温避光孵育10min,立即检测萤火虫荧光素酶活性;
4、检测海参荧光素酶活性:加入75μL的海肾荧光素酶底物,混匀后于室温避光孵育10min,立即检测海肾荧光素酶活性;
5、计算:海肾荧光素酶活性作为内参,计算两者之间的比值,即为相对荧光素酶活性。
三、PRRSV的繁殖:
本发明所用的PRRSV毒株是CH-1a、WUH3、和XH-GD。下面是在Marc145细胞(猴肾细胞)上进行繁殖具体步骤:
1、准备细胞:在25cm2细胞培养瓶中培养细胞,待细胞密度达到80-90%汇合度时,可以用于接种病毒;
2、接毒:吸弃旧培养基,PBS清洗细胞,吸弃培养瓶中的PBS,随后将融化的病毒液接种细胞。PRRSV的接毒量刚好覆盖细胞即可,一般加1mL,轻轻摇晃以利于病毒粒子充分接触培养细胞;
3、补液:1h后补加维持培养基至5mL,尽量保证每隔15min轻轻摇晃培养瓶;
4、观察细胞病变:当观察到80%左右的细胞出现细胞病变,将细胞转移至-80℃冰箱,反复冻融3次之后,使细胞充分裂解以释放病毒粒子;
5、将细胞裂解液转移入15mL离心管中,4℃,5000g离心10min;
6、将上清液于生物安全柜中进行分装,并于-80℃保存。
四、TCID50法测定病毒滴度:
1、准备细胞:将生长状态良好的细胞铺至96孔细胞培养板中,培养12-24h,待细胞汇合度达到80-90%时准备接毒;
2、稀释病毒:将病毒液作10倍梯度稀释(从10-1稀释到10-8);
3、接种病毒:吸弃旧培养液,PBS洗一次,然后将稀释好的病毒液接种到细胞中。每个稀释度作8个重复孔。同时设置阳性对照孔和不感染病毒的阴性对照孔;
4、观察细胞病变:48h后观察细胞病变状态并做记录;
5、计算病毒滴度:统计每个稀释度下的细胞病变的孔数(也可通过间接免疫荧光方法检测阳性感染孔数),用Reed-Muench的方法来计算病毒半数致死感染量(Mediantissue culture infective dose,TCID50)。
实施例1在Marc145细胞中验证TRIM33与PRRSV感染之间的关系
(1)TRIM33基因真核表达载体构建
根据Ensembl非洲绿猴(Chlorocebus sabaeus)TRIM33的cDNA序列(ENSCSAT00000016882.1),设计扩增全长TRIM33cDNA的全长引物,并分别在上、下游分别添加Sal I和BamH I酶切位点(用于连接到真核表达载体pcDNA3.1(+)上),为了能够增强真核基因的翻译效率,在5’酶切位点下游加入KOZAK序列,同时考虑到后期蛋白的表达情况,在KOZAK序列后面添加Myc标签序列(上游引物:5’-gtcgacgccaccATGGAACAAAAACTCATCTCAGAAGAGGATCTGGCGGAAAACAAAGGCGGC-3’,下游引物:5’-ggatccTTACTTTATATGTACTGGTC-3’),扩增出完整的Myc-TRIM33序列(图1),测得TRIM33cDNA实际序列如SEQ ID NO:1所示,PCR产物胶回收纯化后,经过Sal I和BamH I酶切后,最后连接到真核表达载体pcDNA3.1(+)(5428bp),测序正确后,表明成功构建真核表达载体pcDNA3.1(+)-Myc-TRIM33。
(2)检测TRIM33过表达细胞中的病毒含量
将上述构建好的融合有Myc标签的真核表达载体pcDNA3.1-Myc-TRIM33转染至Marc145细胞。转染48h后,收集并提取过表达细胞的总蛋白。采用anti-Myc标签的鼠源抗体进行蛋白水平的检测,GAPDH作为内参指示上样量的多少。Western blot结果如图2所示,表明外源TRIM33蛋白在Marc145细胞中高表达。同时将CH-1a PRRSV(MOI=1.0)感染TRIM33过表达细胞,感染36h后通过定量PCR实验检测胞内的病毒含量。结果如图3所示,TRIM33过表达细胞中的病毒mRNA含量显著高于对照组细胞。
(3)检测TRIM33基因敲除细胞中的病毒含量
CRISPR/Cas9系统是利用靶点特异性的sgRNA将Cas9核酸酶带到基因组上的具体靶点,从而对特定基因位点进行切割导致突变。为了敲除TRIM33基因,本发明分别在基因的5’端设计靶点,即针对TRIM33基因的第一个外显子序列设计靶点,在网站(http://www.addgene.org/crispr/zhang/)提交序列,系统自动设计相应的靶位点,同时包括打分排序。根据打分结果选取几个靶点进行效率的验证。首先将基因的sgRNA连接到pX330载体中,测序正确后,将载体转染至Marc145细胞中,48h后收集细胞,提取细胞的总的基因组。以基于靶点设计的测序引物进行PCR扩增,并对PCR产物进行测序,发现其中一个sgRNA打靶后靶点位置存在明显的套峰,说明靶点处确实发生了打靶(5’-GGCGGCAGGGCCGGCGCTGAGGG-3’)。
将上述验证打靶的细胞消化离心,重悬成为单细胞悬液。计算后以1:100-1:400的稀释比例接种在10cm细胞培养皿中。一到两周后可以观察到单细胞克隆,PBS清洗细胞,随后用粘有硅胶的克隆环小心按压覆盖住细胞克隆边缘,随后用胰酶消化,反复吹吸后将其转移至96孔板中,随后依次将细胞转移到48孔板、24孔板、12孔板、6孔板中进行传代扩繁,并在收取细胞之前,将部分单克隆细胞进行冻存。利用此方法我们第一次筛选得到55个克隆,但是均为混合克隆,重新稀释挑取单克隆后,得到42个克隆,最终选择2个单克隆进行后续实验,细胞标号是40-8-1和40-11-11,突变类型是插入碱基A和删除碱基A(图4)。两种突变均会造成蛋白翻译提前终止。所以说,本实验利用CRISPR/Cas9技术成功得到了TRIM33基因敲除细胞系,可以用于下一步的验证实验。
接着,我们用传统型毒株CH-1a PRRSV分别同时感染野生型的Marc145细胞、Marc14TRIM33-/-细胞,首先将细胞接种于12孔细胞培养板中,每种细胞的每个时间点作三个重复孔,接毒剂量MOI为1.0,分别于接毒后12h,24h,36h,48h收取细胞总RNA,qPCR定量检测胞内的病毒mRNA含量。结果如图5所示,相比于野生型细胞,在PRRSV感染的不同的时间点,Marc14TRIM33-/-细胞内的病毒mRNA含量显著低于野生型细胞。这个结果与上述TRIM33过表达细胞中的病毒定量结果一致。
为了进一步检测TRIM33敲除细胞中的病毒蛋白含量是否发生同样的变化,进一步采用传统型毒株CH-1a PRRSV同时感染野生型Marc145细胞和Marc14TRIM33-/-细胞后进行蛋白水平检测。首先将上述四种细胞接种于12孔细胞培养板中,接毒剂量MOI为1.0,分别于接毒后36h或48h收取细胞总蛋白,使用本实验室自己制备的兔抗PRRSV N蛋白的血清进行病毒蛋白含量的检测,设置GAPDH蛋白为内参。如图6所示,相比于野生型细胞,在PRRSV感染的36h后,在PRRSV感染的36h和48h后,Marc14TRIM33-/-细胞内的病毒蛋白含量均显著低于野生型细胞。这个结果与qPCR的结果也是一致的。
上述实验结果表明TRIM33确实与PRRSV抗性相关,本发明希望通过免疫荧光染色,进一步验证TRIM33与PRRSV抗性之间的相关关系。分别用传统型毒株CH-1a PRRSV和高致病毒株XH-GD PRRSV感染野生型Marc145细胞和TRIM33敲除细胞(40-8-1和40-11-11),接毒36h后,将细胞进行免疫荧光显色实验,其中PRRSV N蛋白杂交使用鼠源SDOW17anti-PRRSVN蛋白的单克隆抗体,Alexa Fluor 488标记的二抗,信号在荧光显微镜下呈现绿色。结果如图7所示。结果显示,相较野生型Marc145细胞,TRIM33敲除细胞中的绿色荧光信号显著弱于野生型细胞,是说明TRIM33敲除确实抑制了PRRSV的感染,这与上述Western blot结果一致。同时也进一步说明TRIM33与PRRSV抗性的相关性不依赖于特定的毒株。
(4)TRIM33影响PRRSV生长曲线检测
为了研究TRIM33对PRRSV生长曲线的影响,本发明分别用传统型毒株CH-1a和高致病毒株XH-GD和WUH3 PRRSV感染野生型Marc145细胞和TRIM33敲除细胞,首先将细胞接种于12孔细胞培养板中,每种细胞的每个时间点作三个重复孔,接毒剂量MOI为1.0,分别于接毒后12h,24h,36h,48h,将细胞反复冻融裂解后,测定病毒TCID50,并进一步绘制病毒生长曲线。结果如图8所示。结果显示,CH-1a PRRSV和XH-GD PRRSV感染48h,也就是在感染后期,PRRSV在TRIM33敲除细胞的生长受到显著的抑制。而对于WUH3 PRRSV来说,各个时间点(12h,24h,36h,48h),PRRSV在TRIM33敲除细胞中的生长均受到显著的抑制。总之,TRIM33的敲除确实能够抑制PRRSV的感染。
(5)PRRSV感染影响TRIM33的表达水平检测
本发明进一步验证了PRRSV感染与TRIM33基因表达水平之间的关系。首先将野生型Marc145细胞接种于12孔细胞培养板中,每个时间点作三个重复孔,将CH-1a PRRSV感染野生型Marc145细胞,接毒剂量MOI为1.0,分别于接毒12h,24h,36h,48h,60h后收取细胞总RNA,qPCR检测胞内的病毒mRNA含量以及TRIM33 mRNA含量,结果如图9所示,随着感染时间的延长,细胞内的病毒含量呈现显著上调的趋势,直到48h达到最高值,48h后胞内的病毒含量呈现下调的趋势。有意思的是,随着PRRSV的感染,TRIM33的表达趋势与胞内病毒含量的变化趋势保持一致,即在感染后48h胞内的TRIM33含量达到最高值。由此推测,PRRSV感染上调TRIM33的表达。
为了进一步验证TRIM33的上调受到PRRSV的调控,本发明克隆了TRIM33转录起始位点上游1930bp启动子序列(SEQ ID NO:2),并连接到pGL3-Basic荧光素酶报告基因载体上。同时与海参荧光素酶报告基因载体(pRL-TK)共转入Marc145细胞中,作为转染效率的内参。转染12h后,接种不同剂量的CH-1a PRRSV,分别是MOI为0.01,0.1,1.0,病毒感染24h后,收集细胞检测荧光素酶的活性。结果图10所示,表明随着PRRSV感染剂量的增加,TRIM33启动子的活性呈现显著上调的趋势,因此说明PRRSV上调TRIM33的表达呈现剂量依赖的方式。
(6)TRIM33影响IFNβ1的产生和信号水平检测
首先,本发明检测TRIM33是否能够影响IFNβ1 mRNA水平。用PRRSV感染野生型的Marc145细胞、Marc14TRIM33-/-细胞(40-8-1和40-11-11)和TRIM33过表达细胞,24h后,用poly(I:C)(25μg/mL)刺激细胞。处理24h后收集细胞,提取细胞的总RNA,用qPCR的方法检测IFNβ1mRNA的水平,结果如图11a所示。表明TRIM33敲除细胞中的IFNβ1 mRNA的水平显著高于野生型细胞,TRIM33过表达细胞中的IFNβ1 mRNA的水平显著低于野生型细胞。
由于I型干扰素能够激活JAK-STAT信号通路,进而刺激下游大量干扰素刺激基因的表达,最终抑制病毒的感染。本发明通过qPCR定量检测了几个重要的干扰素刺激基因(Interferon stimulating genes,ISGs)IFIT1,IFIT2,MX2,OAS1和OAS3的表达,进一步探究TRIM33是否能够抑制IFNβ1下游的信号。结果如图11b所示,表明TRIM33敲除细胞中的干扰素刺激基因IFIT1,IFIT2,MX2,OAS1和OAS3的mRNA的水平显著高于野生型细胞。这也就说明TRIM33同样抑制IFNβ1下游的信号通路。
上述实验结果表明TRIM33抑制了IFNβ1mRNA的水平。进一步地,分析了TRIM33基因对IFNβ1启动子活性的影响。本发明克隆了IFNβ1转录起始位点上游357bp启动子序列(SEQID NO:3),并连接到pGL3-Basic荧光素酶报告基因载体上。同时与海参荧光素酶报告基因载体(pRL-TK)共转入野生型Marc145细胞、Marc14TRIM33-/-细胞和TRIM33过表达细胞中。然后用PRRSV进行感染,24h后,用25μg/mL的poly(I:C)进行刺激处理,24h后收集细胞并检测荧光素酶活性。结果如图12所示,与野生型Marc145细胞相比,TRIM33敲除细胞中IFNβ1启动子活性显著高于野生型细胞,TRIM33过表达细胞中IFNβ1启动子活性显著低于野生型细胞,这个结果与上述IFNβ1定量结果是一致的。
(7)TRIM33通过影响NF-κB信号通路来影响IFNβ1的产生
为了进一步研究TRIM33通过哪些信号通路抑制IFNβ1的表达,本实验分析了前面提到的IFNβ1启动子序列,发现这段启动子区域上存在多种常见的转录调控因子的结合位点,包括IRF1/2,IRF3,NF-κB和ATF-2/c-Jun。本发明将全长的IFNβ1启动子进行突变,即分别删除IRF1/2,IRF3,NF-κB和ATF-2/c-Jun转录因子的结合位点,将测序正确的IFNβ1突变启动子分别克隆到pGL3-Basic荧光素酶报告基因载体上,与海肾荧光素酶报告基因载体pRL-TK共同转入TRIM33敲除细胞和野生型Marc145细胞,转染24h后,用PRRSV进行感染,感染24h后,用25μg/mL的poly(I:C)进行刺激处理,24h后收集细胞并进行双荧光素酶活性的检测。结果图13a所示,TRIM33敲除细胞中IFNβ1启动子活性的显著升高与NF-κB相关。为了进一步说明TRIM33通过NF-κB来抑制IFNβ1的表达,本发明只将NF-κB在IFNβ1上反应元件序列克隆到pGL3-Basic荧光素酶报告基因载体上,成功构建pGL3-NF-κB载体,并重复上述实验步骤,结果如图13b所示,表明转染pGL3-NF-κB载体后,TRIM33敲除细胞中IFNβ1启动子活性的显著升高的确与NF-κB转录调控因子相关。
实施例2在改造过的3D4/21细胞中验证TRIM33与PRRSV感染之间的关系
本发明中使用的Marc145细胞,来源于猴,并非PRRSV感染自然宿主,因此受限于自然宿主相关研究,而且PRRSV感染进入细胞途径不同,很容易在Marc145细胞上产生适应性。进一步地,在猪源细胞中进一步验证候选基因与PRRSV抗性之间的相关性。猪TRIM33基因的cDNA序列如SEQ ID NO:4所示。
文献报道PRRSV具有显著的单核/巨噬细胞嗜性,原代猪肺泡巨噬细胞(porcinealveolar macrophage,PAMs)是其体内主要的靶细胞,然而PAMs只局限于原代培养,因此获得成本高,制备过程繁琐,不同批次细胞存在差异,以及分离的细胞容易被其他微生物污染等缺点也限制了原代PAMs无法得到广泛的应用。本实施例中利用构建好的PRRSV易感的猪的肺泡巨噬细胞(修饰过的3D4/21细胞),即在永生化的猪的肺泡巨噬细胞中稳定转染CD163受体和CD169受体,使其对PRRSV易感。
为了在猪源细胞系中(修饰过的3D4/21细胞)初步验证候选基因TRIM33与PRRSV抗性的相关性,本发明还成功构建Myc-pig-TRIM33真核表达载体。首先设计扩增猪源全长TRIM33cDNA序列的引物,并分别在上、下游分别添加Sal I和BamH I酶切位点进行扩增,并连接到真核表达载体pcDNA3.1(+)上,测序验证正确后转染修饰过的3D4/21细胞,培养48h后,Western blot检测胞内TRIM33的表达情况。将利用anti-Myc标签的单克隆抗体通过检测Myc的表达情况来反映候选蛋白的表达情况。结果如图14a所示,TRIM33蛋白在细胞中过表达。接着,选择WUH3 PRRSV毒株感染上述过表达细胞,感染24h后,利用qPCR检测胞内的PRRSV mRNA含量。结果如图14b显示,TRIM33过表达之后,胞内病毒含量显著上升。
随后,本发明在吉玛公司设计针对TRIM33的siRNAs(5’-UUGUCUUUGGAGGUGUGGCTT-3’),将siRNAs转染细胞48h之后,通过qPCR方法检测不同siRNAs对各个候选基因的敲降效率,结果如图14c所示,表明检测的siRNA对内源的TRIM33具有显著的敲降效率。接着,选择WUH3PRRSV毒株感染上述siRNAs处理后的细胞,感染24h后,利用qPCR检测胞内的PRRSVmRNA含量。结果如图14d所示,TRIM33敲降之后,胞内病毒含量显著下降。这个结果与之前Marc145细胞中验证的结果是一致的,说明本发明中筛选到的候选基因在猪源细胞中仍然是有效的。
虽然,上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述,但在本发明基础上,可以对之做一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
参考文献
[1]Bai,X.,Kim,J.,Yang,Z.,Jurynec,M.J.,Akie,T.E.,Lee,J.,LeBlanc,J.,Sessa,A.,Jiang,H.,DiBiase,A.,et al.(2010).TIF1gamma controls erythroid cellfate by regulating transcription elongation.Cell 142,133-143.
[2]Doisne,J.M.,Bartholin,L.,Yan,K.P.,Garcia,C.N.,Duarte,N.,Le Luduec,J.B.,Vincent,D.,Cyprian,F.,Horvat,B.,Martel,S.,et al.(2009).iNKT celldevelopment is orchestrated by different branches of TGF-beta signaling.J ExpMed 206,1365-1378.
[3]Hatakeyama,S.(2011).TRIM proteins and cancer.Nat Rev Cancer 11,792-804.
[4]Herquel,B.,Ouararhni,K.,and Davidson,I.(2011).The TIF1alpha-related TRIM cofactors couple chromatin modifications to transcriptionalregulation,signaling and tumor suppression.Transcription 2,231-236.
[5]Herquel,B.,Ouararhni,K.,Martianov,I.,Le Gras,S.,Ye,T.,Keime,C.,Lerouge,T.,Jost,B.,Cammas,F.,Losson,R.,et al.(2013).Trim24-repressedVL30retrotransposons regulate gene expression by producing noncodingRNA.Nature structural&molecular biology 20,339-346.
[6]Kusy,S.,Gault,N.,Ferri,F.,Lewandowski,D.,Barroca,V.,Jaracz-Ros,A.,Losson,R.,and Romeo,P.H.(2011).Adult hematopoiesis is regulated by TIF1gamma,a repressor of TAL1and PU.1transcriptional activity.Cell stem cell 8,412-425.
[7]Letizia Venturini1,Jun You3,5,Michael Stadler1,2,Rene′Galien1,6,Vale′rie Lallemand1,Marcel HM Koken1,Marie G Mattei4,Arnold Ganser2,PierreChambon3,Re′gine Losson3and Hugues de The′*,1(1999).TIF1g,a novel member ofthe transcriptional intermediary factor 1family.ONCOGENE.
[8]Li,Y.,Wang,X.,Bo,K.,Wang,X.,Tang,B.,Yang,B.,Jiang,W.,and Jiang,P.(2007).Emergence of a highly pathogenic porcine reproductive and respiratorysyndrome virus in the Mid-Eastern region of China.Vet J 174,577-584.
[9]Pol,J.M.,van Leengoed,L.A.,Stockhofe,N.,Kok,G.,and Wensvoort,G.(1997).Dual infections of PRRSV/influenza or PRRSV/Actinobacilluspleuropneumoniae in the respiratory tract.Veterinary microbiology 55,259-264.
[10]Rossow,K.D.(1998).Porcine reproductive and respiratorysyndrome.Veterinary pathology 35,1-20.
[11]Tian,K.,Yu,X.,Zhao,T.,Feng,Y.,Cao,Z.,Wang,C.,Hu,Y.,Chen,X.,Hu,D.,Tian,X.,et al.(2007).Emergence of Fatal PRRSV Variants:Unparalleled Outbreaksof Atypical PRRS in China and Molecular Dissection of the UniqueHallmark.PLoS One 2,e526.
[12]Weng,L.,Mitoma,H.,Trichot,C.,Bao,M.,Liu,Y.,Zhang,Z.,and Liu,Y.J.(2014).The E3ubiquitin ligase tripartite motif 33is essential for cytosolicRNA-induced NLRP3inflammasome activation.Journal of immunology(Baltimore,Md:1950)193,3676-3682.
[13]Yoon,K.J.,Wu,L.L.,Zimmerman,J.J.,Hill,H.T.,and Platt,K.B.(1996).Antibody-dependent enhancement(ADE)of porcine reproductive and respiratorysyndrome virus(PRRSV)infection in pigs.Viral immunology 9,51-63.
序列表
<110> 中国农业大学
<120> TRIM33基因作为PRRSV感染相关因子的应用
<130> KHP191111769.6
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3384
<212> DNA
<213> 猴(Chlorocebus sabaeus)
<400> 1
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gtgctggtgg aggaggagga ggaggaaggc ggcagggccg gcgctgaggg cggcgcggcc 180
gggcccgacg acgggggggt ggccgcggcc tcctcgggct ctgcccaggc tgcttcatct 240
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tcagcctcgg ctcccgctcc gggtccctcg gcggggccgc ctcctggacc gccagcctcg 360
ctcttggaca cctgcgccgt gtgtcagcag agcttgcaga gccggcgtga ggcggagccc 420
aagctgctgc cctgtcttca ctccttctgc ctgcgctgcc tgccggagcc ggagcgccag 480
ctcagcgtgc ccatcccggg gggcagcaac ggcgacatcc agcaagttgg tgtaatacgg 540
tgcccagtat gccgccaaga atgcagacag atagaccttg tggataatta ttttgtgaaa 600
gacacatctg aagctcctag cagttctgat gaaaaatcag aacaggtatg tactagttgt 660
gaagacaatg caagtgcagt tggcttttgt gtagaatgtg gagagtggct atgtaagaca 720
tgtatcgaag cacatcaaag agtaaaattt actaaagatc acttgatcag gaagaaagaa 780
gatgtctcag agtctgtcgg agcatctggt caacgccctg ttttctgccc tgtacacaaa 840
caagaacagt tgaaactttt ctgtgaaaca tgtgatagac tgacatgtag agactgtcag 900
ctattggaac acaaagaaca taggtatcag tttttggaag aagcttttca aaatcagaaa 960
ggtgcaattg agaatctact ggctaaactt cttgagaaga agaattatgt tcattttgca 1020
gccactcagg tgcagaatag gataaaagaa gtaaatgaga ctaacaaacg agtagaacag 1080
gaaattaaag tggccatttt cacccttatc aatgaaatta ataagaaagg aaaatctctc 1140
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gcaagtggca gcagcacagc actactgtac agcaagcgac tgattacttt ccagttgcgt 1320
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tgtgatccca ccttctgggc aaagaatgta gtcaatttag gtaatctagt aatagagagt 1440
aaaccaactc ctggttatac tcctaatgtt gtagttgggc aagttcctcc agggacaaac 1500
cacattagta aaacccctgg acagattaac ttagcacagc ttcgacttca gcacatgcaa 1560
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gcacctgtac caactacaac aacaacaaca caacagcatc ctagacaagc agcccctcag 1680
atgttacagc aacagcctcc tcgattgatc agtgtgcaaa caatgcaaag aggcaacatg 1740
aactgtggag cttttcaagc ccatcagatg agactcgctc agaatgctgc cagaataccg 1800
gggataccca ggcacagcgg ccctcagtat tccatgatgc agccacacct ccaaagacaa 1860
cactcaaacc cagggcatgc tggacccttt cccgtagtat cggtacacaa caccacaatc 1920
aacccaacga gccctactac agcaactatg gcaaatgcaa accgaggtcc caccagccca 1980
tctgttacag caatagagct aatcccctca gttaccaatc cagaaaacct tccatcgctg 2040
ccagatattc cacccataca gttggaagat gctggctcaa gtagtttaga taatctacta 2100
agtagataca tctcaggcag tcacctaccc ccacagccta caagcaccat gaatccctcc 2160
ccaggaccct ctgccctttc tccgggatca tcaggtttat ccaattctca cacacctgtg 2220
agacccccaa gtacttctag tactggcagt cgaggcagct gtggctcatc aggaagaact 2280
gctgagaaga caagtcttag tttcaaatct gatcaggtga aggtcaagca agaacctggg 2340
actgaagatg aaatatgtag cttttcagga ggtgtaaaac aagaaaaaac agaggatggc 2400
aggaggagtg cttgcatgtt gagcagtcct gagagtagct tgacaccacc tctctcaacc 2460
aacctgcatc tagaaagtga gttggatgca ttggcaagcc tggaaaacca tgtgaaaact 2520
gaacctgcag atatgaatga aagctgcaaa cagtcagggc tcagcagcct tgttaatgga 2580
aagtccccaa ttcgaagcct catgcacagg tcggcaagga ttggaggaga tggcaacaat 2640
aaagatgatg acccaaatga agactggtgt gctgtctgcc aaaatggagg agatctcttg 2700
tgctgcgaaa aatgtccaaa ggtctttcat ctaacttgtc atgttccaac actacttagc 2760
tttccaagtg gggactggat atgcacattt tgtagagata ttggaaagcc agaagttgaa 2820
tacgattgtg ataatttgca acatagtaag aaggggaaaa ctgcacaggg gttaagcccc 2880
gtggaccaaa ggaaatgtga acgtcttctg ctttacctct attgccatga attaagtatt 2940
gaattccagg agcctgttcc tgcttcgata ccaaactact ataaaattat aaagaaacca 3000
atggatttat ccaccgtgaa aaagaagctt cagaaaaaac attcccaaca ctaccaaatc 3060
ccagatgact ttgtggctga tgtccgtttg atcttcaaga actgtgaaag gtttaatgaa 3120
atgatgaaag ttgttcaagt ttatgcagac acacaagaga ttaatttgaa ggctgattca 3180
gaagtagctc aggcagggaa agcagttgca ttgtactttg aagataaact cacagagatc 3240
tactcagaca ggaccttcgc acctttgcca gagtttgagc aggaagagga tgatggtgag 3300
gtaactgagg actctgatga agactttata cagccccgca gaaaacgcct aaagtcagat 3360
gagagaccag tacatataaa gtaa 3384
<210> 2
<211> 1930
<212> DNA
<213> 猴(Chlorocebus sabaeus)
<400> 2
cctaaggcta gggtatgagt actcctgggg tacttcagtg aaggaaaatt ttccaagggg 60
tacataaaag tgtagtttta agggaattaa tctatcatat atgtgagtgt ataactacac 120
acatgtatac atagttgtaa atgcaagagt taagtctgtg acaatgaaaa atggagttga 180
tacaggacct catctcattc tagcaatgtt atatgcattc atgcctactc aaactaatta 240
ggggaagggg gaagactccc agccatctca ttaaaacatt cccaacactt tttcatgttt 300
aaaaaaatac attttagtat actttaaaaa aaatggatag tattaagcca ctttagtgtt 360
tactttccta tttaaaataa ttttaaaatg tcagtattac aaaatgtcac aaattacact 420
ctgcaactat ttaaacttct gaaaaaattt agtcaactta caatgttttg aggcaataaa 480
acagctttac aaaattcttt taaagggtac aaaagttaaa aaaaaaaatt taagaccatt 540
tctgtacatg acctggctcc cttaataact ctcttatctc atgtcctact attctctctc 600
ttgttcaagc cactcaagcc acactgactc ctgtaacaag ccaagaatca gtttccctca 660
ggccctttgc acttgctatg tgttccgcta cctgctcttc tgcaagacag gttccttcat 720
ccgatcaata ggggccttct tcagtcacct ctaatccttt ctaccccact tactcttttc 780
ttcacagcac ttaccactcc ctggcatata tctgttctca atctttctct attagattgt 840
aaatcccttg aatgcaggga gcgcctctgt tttgtaatcc ctgtgtatat ctaaaacact 900
atgacaataa agaggtcagg aaatattttt gaataaattt taaaaggttt tcaggaggcc 960
aggcgcagtg gctcgtgcct gtaattccag cactttcagc ggctgaggcg ggccaattgc 1020
ttgggcccag gagcttcagc ctgggcaaca tagtgagacc caagcctctt caaggcaagg 1080
gtggggggga agatcaccta aggggtggtg gcgtgcatct acagtcccag gtaattggga 1140
ggagcacggc ttgagcctgg gaggtcgagg ctgcagtgaa atgtgatcac gccactgcac 1200
tccagccagg gcgataaacg aggtctcaaa aaaaaaaaaa aaagttttca cgtggtttaa 1260
gcgcccttct tgggaacact aacaggagag catttcatga tcttaacact gagacagttg 1320
ttgcattaca taaaatcctc caaatacttt ttttccccaa ataaaacggc aaatttctga 1380
aagcttttat ttattttttt tgagacggag tttcgctctg tcgcccggct gggtgcagcg 1440
gcgcaatctc ggttcactgc agcctccgcc tccagccggg ttcaagcaat tctcctgcct 1500
cagcctcctg agtatctcgg attacaggcg cccgcaacca cacccggctg atttttatgt 1560
ttttagtgga gacggggttt caccgtattg gccaggctgg ccacgaactc ctgacctcaa 1620
gtgatctgcc ctcctcggct tcccgaagtg ctgggattac aggcttgagc caccatgccc 1680
ggcctgaaag cttctactac cattgttgta agggcatttg tcacccagct caaaccagga 1740
cacttaaaat gggacctcag aaggacgcag tattggttac aagccgtggc tctccaaact 1800
gaaatacatt agaatcagcc gaagggactt ttccccactc cgtcggctct ctccctcact 1860
tcctgattca ggtctgggga ctggccctgg catctctgct ttgaaaaact cctccgttga 1920
ttctgatacg 1930
<210> 3
<211> 357
<212> DNA
<213> 猴(Chlorocebus sabaeus)
<400> 3
tggctgtttt gaggttcttg aattctcagg tcatttgctt tcctttgctt tctcccaagt 60
cttttttcac aatttgcttt agtcattcac tgaaacttta aaaaacatta gaaaatctta 120
gaatttgtat atcttttttt ctaatatata taacataaga taggagttta aataaagagt 180
tttagaaact actacatatg caaatgacat aggaaaactg aaagggagaa ctgaaagtgg 240
gaaattcctc tgaatagaga gaggaccagg tcatataaat aggccatacc catggagaaa 300
ggacattcta actgcaaact ttcgaagcct tcgctctggc acaacaggta gtaggcg 357
<210> 4
<211> 3384
<212> DNA
<213> 猪(Sus scrofa)
<400> 4
atggcggaaa acaaaggcgg cggcgaggct gagagcggcg gcgggggcag cggcagcgcg 60
ccggtaactg ccggggccgc cgggcccgcg gcgcaggagg cggagccgcc tctcgccgct 120
gtgctggtgg aggaggagga ggaggaaggc agcagggccg gcgcggagag cggcgcggcc 180
gggcccgacg acggtggggt agccgcggcc tcctcgggct cggccccggc tgcctctgcc 240
cctgcagcct ccgtggcccc tggagttcca gggggtgcgg tatcgacccc ggccccagct 300
gcagcctcgg ctccggctcc ggctccctcg gcggggccgc ctcctggacc gccagcctcg 360
ctcctggaca cctgcgccgt gtgtcagcag agcctgcaga gccgacgtga ggcggagccc 420
aagctgctgc cctgtcttca ctccttctgc ctgcgctgcc ttcccgaacc cgagcgccag 480
ctcaacgtgc ccatcccggg gggcagtaac ggcgacatcc agcaagttgg tgtgatacgg 540
tgcccagtat gccgccaaga atgcagacag atagaccttg tggataatta ttttgtgaaa 600
gatacatctg aagctcctag cagttctgat gagaaatcag aacaggtatg tactagttgt 660
gaagacaatg caagtgcagt tggcttttgt gtagaatgtg gagaatggct gtgtaagaca 720
tgtattgaag cacatcaaag agtaaaattc actaaagatc acttgatcag gaagaaggaa 780
gatgtctcag agtctgttgg agcatctggt cagcgccctg tcttctgccc tgtacacaaa 840
caagagcagt tgaaactttt ctgtgaaaca tgtgatagac tgacatgtag agactgtcag 900
ctattggaac acaaagaaca taggtatcag tttttggaag aagcttttca aaaccagaaa 960
ggtgcaattg agaatctact ggctaagctt cttgagaaga agaattatgt tcattttgca 1020
gctactcagg tgcaaaatag gataaaagaa gtaaatgaaa ctaacaaacg agtagaacag 1080
gaaattaaag tggccatttt cacccttatc aatgaaatta ataagaaagg aaaatctctc 1140
ttacagcagc tagagaatgt tacaaaagag agacagatga agttactgca gcaacagaat 1200
gacatcacag gcctttcccg gcaggtgaag catgttatga actttacaaa ctgggcaatt 1260
gcaagtggca gcagcacggc actactgtac agcaagcgat tgattacttt tcaattgcgc 1320
catattttga aagcacggtg tgatcctgtc cctgctgcta atggagcaat acgtttccat 1380
tgtgatccca ccttctgggc aaagaatgta gtcaatttag gtaatctagt aatagagagt 1440
aaaccagctc ctggttatac tcctaatgtt gtagttgggc aagttcctcc aggaacaaac 1500
cacattagta aaacccctgg gcagattaac ttagcacagc ttcgacttca gcacatgcaa 1560
caacaagtgt atgcacagaa gcatcagcag ttgcaacaga tgaggatgca gcaaccacct 1620
gcacctgtac caacaacaac agcaacaaca cagcagcacc ctagacaagc agcccctcag 1680
atgttacaac aacagcctcc aagattgatc agtgtgcaag caatgcaaag aggcaacatg 1740
aactgtggag ctttccaggc ccatcagatg agattggctc agaatgctgc ccgaatacca 1800
gggttaccca ggcacagtgg ccctcagtat tccatgatgc agccacacct ccaaagacaa 1860
cactcaaacc cggggcatgc tggacccttt cctgttgtgt cggtgcacaa caacccagtc 1920
aatccaacca gccctactac agctaccatg gcaaatgcaa atcgaggtcc caccagcccg 1980
tctgttacag caatagaact aattccctca gtgaccaatc cagaaaacct tccatcgctt 2040
ccagatattc cacccataca gttggaagat gctggttcaa gtagtttaga taatctacta 2100
agtagatata tctcaggcag tcacctaccc ccacagccca caagcaccat gaatccctcc 2160
ccaggaccct ctgccctgtc tccaggatca tcaggtttat ccaattctca tacacctgtg 2220
aggcccccta gtacttctag tactggtagt cgaggcagct gtgggtcatc aggaagaact 2280
gctgagaaga caagtcttag tttcaagtct gatcaagtga aggtcaagca agaacatggc 2340
actgaagatg aaatatgtag cttttcagga gctgtgaaac aagaaaagac agaggatggc 2400
aggaggagtg cctgcatgtt gagcagtcct gagagtagct tgacaccacc tctctcaacc 2460
aacctgcatc tagagagtga gctggatgcg ttagcaagtc tcgaaaacca tgtgaaaact 2520
gaacccacgg atatgaatga aagctgcaaa caatcagggc tcagtagcct tgttaacgga 2580
aagtccccag ttcgaagcct catgcacagg tcggcaagga tcggaggaga tggcagtaac 2640
aaagatgacg acccaaatga agactggtgt gctgtctgcc aaaatggagg ggatctcttg 2700
tgctgtgaaa aatgtccaaa ggtctttcat ctaacttgtc atgttccaac acttcttagc 2760
tttccaagtg gggactggat atgcacattt tgcagagata ttgggaaacc agaagttgaa 2820
tatgattgtg ataatttaca gcatagcaaa aaggggaaaa ctgtacaggg gttaagcccc 2880
gtggaccaaa ggaaatgtga acgtcttctg ctttacctct attgccatga attaagtatt 2940
gaatttcagg agccagttcc tgcttcgata ccaaactact ataaaattat aaagaaacca 3000
atggatttat ccaccgtgaa aaagaagctt cagaaaaaac attcccaaca ctaccaaatc 3060
ccggatgact ttgtggctga tgttcgtttg atcttcaaga actgtgaaag gtttaatgaa 3120
atgatgaaag ttgttcaagt ttatgcagaa acacaagaga ttaatttgaa ggctgattca 3180
gaagtagctc aggcagggaa agcagttgca ttgtactttg aagataaact cacagagatc 3240
tactcagaca ggaccttcgc acctttgcca gagtttgagc aggaagagga tgatggtgag 3300
gtaactgagg actctgatga agactttata cagccccgca gaaaacgcct aaagtcagat 3360
gagagaccag tacatataaa gtaa 3384
<210> 5
<211> 21
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 5
uugucuuugg agguguggct t 21
<210> 6
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
cagcgcgccg gtaactgccg 20
<210> 7
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ggcagcgcgc cggtaactgc 20
Claims (10)
1.TRIM33基因的以下任一应用:
1)作为PRRSV感染相关因子的应用;
2)作为标志物在制备PRRSV感染检测和/或疗效评价试剂中的应用;
优选地,所述TRIM33基因来自于猪。
2.TRIM33基因抑制剂的以下任一应用:
1)用于制备抗PRRSV药物或组合物;
2)作为JAK-STAT信号通路的激活剂;
3)作为IFNβ1基因以及受IFNβ1基因调控的下游基因的激活剂;
4)作为NF-κB信号通路的激活剂;
优选地,所述TRIM33基因来自于猪;
其中,所述抑制剂是能够从转录或翻译水平上抑制TRIM33基因表达的物质,所述抑制剂选自shRNA、siRNA、dsRNA、miRNA、cDNA、反义RNA/DNA、低分子化合物、肽、抗体中的至少一种;
优选地,所述抑制剂为siRNA,其核苷酸序列为5’-UUGUCUUUGGAGGUGUGGCTT-3’。
3.根据权利要求2所述的应用,其特征在于,其中,所述受IFNβ1基因调控的下游基因包括IFIT1,IFIT2,MX2,OAS1和OAS3。
4.靶向编辑TRIM33基因的CRISPR-Cas9系统的以下任一应用:
1)用于制备抗PRRSV药物或组合物;
2)作为JAK-STAT信号通路的激活剂;
3)作为IFNβ1基因以及受IFNβ1基因调控的下游基因的激活剂;
4)作为NF-κB信号通路的激活剂;
5)用于制备TRIM33基因敲除的猪肺泡巨噬细胞系;
6)用于制备抗蓝耳病猪;
优选地,所述TRIM33基因来自于猪。
5.抗PRRSV药物或组合物,其特征在于,有效成分为TRIM33基因抑制剂和/或靶向编辑TRIM33基因的CRISPR-Cas9系统,其中,所述抑制剂的定义同权利要求2中所述。
6.JAK-STAT信号通路的激活剂,其特征在于,有效成分为TRIM33基因抑制剂和/或靶向编辑TRIM33基因的CRISPR-Cas9系统,其中,所述抑制剂的定义同权利要求2中所述。
7.IFNβ1基因以及受IFNβ1基因调控的下游基因的激活剂,有效成分为TRIM33基因抑制剂和/或靶向编辑TRIM33基因的CRISPR-Cas9系统,其中,所述抑制剂的定义同权利要求2中所述,所述受IFNβ1基因调控的下游基因包括IFIT1,IFIT2,MX2,OAS1和OAS3。
8.NF-κB信号通路的激活剂,其特征在于,有效成分为TRIM33基因抑制剂和/或靶向编辑TRIM33基因的CRISPR-Cas9系统,其中,所述抑制剂的定义同权利要求3中所述。
9.TRIM33基因敲除的猪肺泡巨噬细胞系,其特征在于,所述细胞系按如下方法制备得到:根据猪TRIM33基因序列,构建基于CRISPR-Cas9系统的sgRNA表达载体,以此作为TRIM33基因打靶载体,转入猪肺泡巨噬细胞中,获得的中靶阳性细胞克隆,即为TRIM33基因敲除的猪肺泡巨噬细胞系;
优选地,gRNA作用位点的DNA序列为:5’-CAGCGCGCCGGTAACTGCCG-3’和5’-GGCAGCGCGCCGGTAACTGC-3’。
10.权利要求9所述细胞系在制备抗蓝耳病转基因猪中的应用。
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Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105518137A (zh) * | 2015-06-11 | 2016-04-20 | 深圳市第二人民医院 | CRISPR-Cas9特异性敲除猪SALL1基因的方法及用于特异性靶向SALL1基因的sgRNA |
CN106414740A (zh) * | 2015-06-11 | 2017-02-15 | 深圳市第二人民医院 | CRISPR‑Cas9特异性敲除猪SLA‑3基因的方法及用于特异性靶向SLA‑3基因的sgRNA |
-
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105518137A (zh) * | 2015-06-11 | 2016-04-20 | 深圳市第二人民医院 | CRISPR-Cas9特异性敲除猪SALL1基因的方法及用于特异性靶向SALL1基因的sgRNA |
CN106414740A (zh) * | 2015-06-11 | 2017-02-15 | 深圳市第二人民医院 | CRISPR‑Cas9特异性敲除猪SLA‑3基因的方法及用于特异性靶向SLA‑3基因的sgRNA |
Non-Patent Citations (1)
Title |
---|
JIANHUI BAI,ET AL: "A high-throughput screen for genes essential for PRRSV infection using a piggyBac-based system", 《VIROLOGY》 * |
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