CN113509552B - 敲除或沉默猪的Kxd1基因在提高猪抗猪繁殖与呼吸综合征病毒中的应用 - Google Patents
敲除或沉默猪的Kxd1基因在提高猪抗猪繁殖与呼吸综合征病毒中的应用 Download PDFInfo
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Abstract
本发明属于基因工程及生物医药领域，公开了敲除或沉默猪的Kxd1基因在提高猪抗猪繁殖与呼吸综合征病毒中的应用，所述的Kxd1基因包括猪中的NCBI登录号为NC_010444.4的Kxd1基因或猪中与该基因同源的Kxd1基因。本发明发现该基因无论是在PK15‑CD163细胞中被敲除还是在Marc‑145细胞中被敲低之后都能够抑制PRRSV的增殖，故本基因可作为抑制PRRSV复制的疫苗药物的开发与抗病育种研究的新型靶点。
Description
技术领域
本发明属于基因工程及生物医药领域，具体涉及敲除或沉默猪的Kxd1基因在提高猪抗猪繁殖与呼吸综合征病毒中的应用。
背景技术
猪繁殖与呼吸综合征(porcine reproductive and respiratory syndrome，简称PRRS)是由猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndromevirus，简称PRRSV)引起的以母猪繁殖障碍、仔猪呼吸系统疾病和高死亡率为主要临床特征的重大病毒性传染病(Chand et al 2012；Van 1997)。自1987年首次在美国爆发以来，已成为影响全球养猪业发展的重要疾病(Keffaber et al 1989)。虽然目前已培育出PRRSV受体CD163基因缺失的抗PRRS的基因编辑猪(Burkardet al 2017；Whitworth et al 2015；Yanget al2018)，但仍存在基因编辑动物的生物安全、PRRSV变异较快、病毒与宿主(猪)互作机理不完全清楚以及可用于抗病育种的靶基因缺乏等问题，严重制约了靶向药物的开发及抗病育种的进程。
发明内容
本发明的目的在于提供了敲除或沉默猪的Kxd1基因在提高猪抗猪繁殖与呼吸综合征病毒中的应用，所述的Kxd1基因包括猪中的NCBI登录号为NC_010444.4的Kxd1基因或猪中与该基因同源的基因。
为了达到上述目的，本发明采取以下技术措施：
抑制猪Kxd1基因表达的制剂在制备抗PRRSV感染药物中的应用；所述的Kxd1基因包括猪中的NCBI登录号为NC_010444.4的Kxd1基因或猪中与该基因同源的Kxd1基因，例如绿猴的Kxd1基因，绿猴Kxd1基因编码的氨基酸序列为SEQ ID NO.4所示。
以上所述的应用中，优选的，所述的抑制方法包括但不限于针对猪Kxd1基因的CRISPR/Cas9敲除技术、siRNA干扰技术。
本发明的保护内容还包括：通过敲除或者沉默猪Kxd1基因来制备抗PRRSV感染的细胞模型。
与现有技术相比，本发明具有以下优点：
申请人分别通过CRISPR敲除及基因干扰技术在PK15-CD163、Marc-145细胞中靶向敲除/抑制Kxd1的表达，首次发现敲除/抑制Kxd1的表达的细胞可以通过影响PRRSV的复制从而抵抗PRRSV的感染，该基因可作为PRRSV的新型抗病靶点。本发明为研制有效安全防治PRRS的新型疫苗药物提供了新的靶标，也为抗PRRS育种研究提供了新型候选基因，具有广泛的应用前景。
附图说明
图1：PK15-Cas9-CD163单克隆细胞系的构建及验证；
其中：(A)扩增猪CD163基因的CDS序列；
(B)Western Blot检测PK15-Cas9-CD163细胞中CD163蛋白的表达；
(C)RT-PCR检测NSP2在PRRSV感染48h后的转录，泳道1：Marc145细胞(PRRSV感染)；2：Marc45细胞(未感染PRRSV)；3：PK15-Cas9-CD16细胞(PRRSV感染)；4：PK15-Cas9-CD163细胞(未感染PRRSV)；
(D)qPCR检测PK15-Cas9-CD163细胞被PRRSV感染后ORF7不同时间点的相对表达量，β-actin为内参基因。
图2：KO-Kxd1单克隆细胞系的构建；
其中：(A)Western Blot检测CD163和Kxd1基因在KO-NC细胞和KO-Kxd1单克隆敲除细胞中的蛋白表达；
(B)MTT检测KO-NC细胞和KO-Kxd1细胞的活性。(ns，t-test)。
图3：PK15-CD163细胞敲除Kxd1后，qPCR检测感染12、24和48h PRRSV ORF7 mRNA的表达水平。
ns P>0.05，**P<0.01，t-test。
图4：Marc-145细胞干扰Kxd1后，抑制PRRSV增殖；
其中(A)转染Kxd1干扰片段到Marc-145细胞48h后，qPCR检测Kxd1的相对表达量；
(B)敲低Kxd1基因后，PRRSV(MOI＝1)感染48h，q-PCR检测检测ORF7的相对表达；
(C)敲低Kxd1基因后，PRRSV(MOI＝1)感染48h，TCID50检测PRRSV滴度。(*P<0.05，**P<0.01，***P<0.001，t-test)。
具体实施方式
本发明所述技术方案，如未特别说明，均为本领域的常规方案；所述试剂或材料，如未特别说明，均来源于商业渠道。
实施例1：
可用于PRRSV感染的猪PK15-Cas9-CD163细胞的构建和验证
1.PK15-Cas9-CD163细胞的构建
(1)按照Omega公司总RNA提取试剂盒(R6834-01)的操作说明书提取PK15-CD163细胞(该细胞是在PK15细胞中稳定表达猪源CD163蛋白的细胞系，对PRRSV感染敏感，由华中农业大学肖少波老师馈赠，Wang et al,2018)RNA并利用反转录试剂盒(Thermo Scientific，00238582)将RNA反转录成第一链cDNA。
(2)以适量cDNA为模版，引物CD163-F：ctagtctagaATGGTGCTACTTGAAGACTCTG和CD163-R：cgcggatccTCATTGTACTTCAGAGTGGTCTCC，使用Q5超保真扩增酶(NEB，E0555S)将猪CD163基因CDS区域全长扩增，并在两端加上XbaI和BamHI限制性酶切位点。PCR结果经凝胶电泳检测成功后(图1中A)，使用凝胶回收试剂盒(Sangon Biotech，B110092)回收纯化猪CD163基因的CDS片段。
(3)将猪CD163基因的CDS片段和pCDH-EF1-copGFP-T2A-Puro质粒(Addgene，72263)使用XbaI(Thermo fisher，FD0684)和BamHI(Thermo fisher，FD0054)限制性内切酶双酶切，使用凝胶回收试剂盒(Sangon Biotech，B110092)回收纯化。
(4)使用T4连接酶(Thermo fisher，15224041)将猪CD163基因CDS区连入pCDH-EF1-copGFP-T2A-Puro。转化入DH5α菌株(全式金，CD201-01)，涂LB(含AMP)平板。37℃培养16h后，挑单克隆并测序。
(5)按NEOFECT公司的DNA转染试剂(TF201201)的操作说明书以辅助质粒psPax2(Addgene，12260)、pMD2.G(Addgene，12259)，核心质粒pCDH-EF1-copGFP-T2A-Puro(步骤4中测序验证CD163已成功连入的质粒)包装慢病毒。
(6)使用适量此慢病毒感染PK15-Cas9细胞系(该细胞是在PK15细胞中稳定表达Cas9蛋白的细胞系，可用于转入gRNA后构建基因敲除细胞，由华中农业大学赵书红老师馈赠，Zhao et al,2020)。感染48h后，细胞经过流式细胞分选GFP阳性细胞后扩大培养。
(7)经流式分选仪分选的细胞经过有限梯度法分选出稳定表达猪CD163蛋白和Cas9蛋白的单克隆细胞系PK15-Cas9-CD163。
2.PK15-Cas9-CD163单克隆细胞系的验证
(1)利用Western Blot方法检测PK15-Cas9-CD163细胞中CD163的表达
1)蛋白样品的制备
a)PK15-Cas9-CD163单克隆细胞及野生型PK15细胞长满后，吸出培养液，用预冷的PBS洗1-2次。
b)每个30mm平皿中加入200μL蛋白裂解液(每100μL RIPA裂解液中，加入1μL蛋白酶抑制剂蛋白酶抑制剂Cocktail(Sigma，P8340)、磷酸化蛋白酶抑制剂PPEi(Sigma，G2007)，在冰上裂解，并用细胞刮板刮取细胞。
c)吸取裂解产物至1.5mL离心管中，反复吹打至清亮。
d)加入相应体积的5×SDS-PAGE上样缓冲液，沸水煮10min，-80℃储存。
2)SDS-PAGE电泳
3)Western Blot检测分析：
a)一抗分别为CD163兔多克隆抗体(博士德公司，A00812-1)，β-actin鼠单克隆抗体(Proteintech，66009-1-Ig)
b)二抗孵育：HRP偶联抗鼠IgG(Proteintech，SA00001-1)，HRP偶联抗兔IgG(Proteintech，SA00001-2)；
c)蛋白检测：采用ECL发光法。
Western Blot分别检测PK15-Cas9-CD163细胞和野生型PK15细胞中CD163基因表达。PK15-Cas9-CD163细胞中表达猪源CD163蛋白(图1中B右边泳道)，而阴性对照PK15细胞则没有CD163基因的表达(图1中B左边泳道)，说明PK15-Cas9-CD163细胞系构建成功。
(2)PK15-Cas9-CD163细胞系可被PRRSV感染的验证
1)利用RT-PCR技术检测PRRSV基因NSP2在感染后的转录
a)将相同数量及适量的实验组细胞(PK15-Cas9-CD163)和对照组细胞(Marc-145)接种到6孔板中，5％CO2、37℃培养箱培养。24h后弃培养基并用DMEM洗2遍，加入PRRSV(MOI＝1，WUH3毒株，GenBank accession NO.HM853673)混匀，37℃孵育1h后，再加入1mL DMEM+10％FBS完全培养基，继续培养。
b)感染后48h收集细胞并提取细胞总RNA。提取RNA方法按Omega公司总RNA提取试剂盒(R6834-01)的操作说明书操作。
c)测得RNA浓度后，利用反转录试剂盒(Thermo Scientific，00238582)将RNA反转录成第一链cDNA。反转录反应体系见表1。
d)PCR扩增cDNA，扩增使用宝生物工程(大连)有限公司“LA Taq”(RR02MQ)，所用引物为NSP2-F:TGATGGGCGACAATGTCC和NSP2-R：CGCAGACAAATCCAGAGG。
e)PCR产物通过琼脂糖凝胶电泳检测。
结果如图1中C所示，从图1中C可以看出，PRRSV感染的PK15-Cas9-CD163细胞和阳性对照Marc145细胞中检测到230bp的NSP2特异片段，而阴性对照并没有检测到，说明PRRSV可以感染PK15-Cas9-CD163细胞系。
2)利用qPCR检测PRRSV可以在PK15-Cas9-CD163细胞中增殖
a)将相同数量及适量的PK15-Cas9-CD163细胞接种到6孔板中，5％CO2、37℃培养箱培养。24h后弃培养基并用DMEM洗2遍，加入PRRSV(MOI＝0.5，WUH3毒株，GenBankaccession NO.HM853673)混匀，37℃孵育1h后，再加入1mL DMEM+10％FBS完全培养基，继续培养。
b)感染后不同时间点收集细胞并提取细胞总RNA。提取RNA方法按Omega公司总RNA提取试剂盒(R6834-01)的操作说明书操作。
c)测得RNA浓度后，利用反转录试剂盒(Thermo Scientific，00238582)将RNA反转录成第一链cDNA。
d)cDNA经RNase Free H2O 5倍稀释。
e)qPCR检测PRRSV保守基因ORF7 mRNA表达水平。
cDNA样本通过Bio-Rad CFX96荧光定量PCR仪进行扩增，检测不同样本中特定基因表达的差异，同时设置内参基因，每个样本设置3个复孔。三步法的扩增反应条件为：预变性95℃4min，循环反应为变性95℃5sec，退火60℃20sec，延伸72℃20sec，并收集荧光信号，共进行40个循环，最后延伸72℃2min。65℃-95℃升温，升温速度0.5℃/5sec以检测融解曲线。目的基因的相对表达量按照2-△△Ct进行计算，所使用的体系和引物如下表所示。
SYBR扩增体系
qPCR引物
从图1中D可见，随PRRSV感染时间的增加，ORF7相对表达量也随之升高，表明PK15-Cas9-CD163细胞可以被PRRSV感染并使其在细胞内增殖。
实施例2：
Kxd1敲除细胞系的构建及验证：
目的基因Kxd1基因序列的NCBI号为NC_010444.4，具体为SEQ ID NO.1所示。
1.gRNA设计及表达载体的构建
(1)gRNA的设计：选择需要gRNA靶向基因位置的部分序列，使用http://crispr.mit.edu/，http://crispr.cos.uni-heidelberg.de/index.html和http://www.rgenome.net/cas-database/网站进行设计。敲除gRNA的序列为5'-CCCCCACAGGACACTGAAA-3'。
(2)将公司合成的oligo gRNA稀释为100nM后，互补序列各取5μL混合。
(3)将混合液上PCR仪器，退火得到双链gRNA。程序如下：95℃，10min，65℃，40min，10℃，10min。
(4)将pLH-sgRNA1载体(Addgene，75388)使用BbsI或BsmbI酶切回收后，使用T4连接酶将gRNA与载体连接。
(5)转化载体到DH5α，涂平板，挑单克隆菌落送往测序验证。
(6)阳性菌落测序鉴定正确后，扩繁菌液提取质粒。
2.质粒提取，按TIANGEN公司的质粒小提试剂盒(DP103)的操作说明书操作。
3.慢病毒gRNA包装，按NEOFECT公司的DNA转染试剂(TF201201)的操作说明书操作。
4.构建Kxd1敲除单克隆细胞系
慢病毒gRNA感染PK15-Cas9-CD163细胞
1)将PK15-Cas9-CD163细胞接种到6孔细胞培养板的2个孔，一孔作为实验组，另一孔作为对照组，用含有10％FBS，1％双抗的DMEM高糖培养基培养。
2)待细胞汇合度达到60％～70％时，进行感染(MOI～1)。用含有10％FBS，1％双抗的DMEM高糖培养基稀释步骤3制备的慢病毒，并加入终浓度为8μg/mL的polybrene。
3)感染1d后，细胞传代至10cm细胞培养皿，继续培养。
4)培养2d后，加含有潮霉素的培养基(10％FBS，1％双抗的DMEM高糖培养基)，潮霉素在该培养基中的终浓度为300μg/mL(以下简称潮霉素培养基)。
5)每天更换新鲜的潮霉素培养基，待对照组细胞全部死完，实验组有细胞存活时，将实验组细胞接种到10cm细胞培养皿，使其形成单个细胞，加入潮霉素培养基，终浓度为300μg/mL。
6)继续培养7d左右，待单个细胞长成单克隆细胞团，用玻璃针刮下来放置24孔培养板中，加入潮霉素培养基获得KO-Kxd1单克隆细胞系。
5.验证KO-Kxd1细胞系中的Kxd1的表达和细胞活性
(1)利用Western Blot方法检测KO-Kxd1细胞中Kxd1和CD163的表达；以KO-NC细胞(即PK15-Cas9-CD163)为对照；
1)蛋白样品的制备
a)KO-Kxd1单克隆细胞系长满后，吸出培养液，用预冷的PBS洗1-2次。
b)每个30mm平皿中加入200μL蛋白裂解液(每100μL RIPA裂解液中，加入1μL蛋白酶抑制剂蛋白酶抑制剂Cocktail(Sigma，P8340)、磷酸化蛋白酶抑制剂PPEi(Sigma，G2007)，在冰上裂解，并用细胞刮板刮取细胞。
c)吸取裂解产物至1.5mL离心管中，反复吹打至清亮。
d)加入相应体积的5×SDS-PAGE上样缓冲液，沸水煮10min，-80℃储存。
2)SDS-PAGE电泳
3)Western Blot检测分析：
一抗分别为CD163兔多克隆抗体(博士德公司，A00812-1)，Kxd1兔多克隆抗体(USBiological，032800)，GAPDH鼠单克隆抗体(Proteintech，60004-1-Ig)；
e)二抗孵育：HRP偶联抗鼠IgG(Proteintech，SA00001-1)，HRP偶联抗兔IgG(Proteintech，SA00001-2)；
f)蛋白检测：采用ECL发光法。
Western Blot检测发现KO-Kxd1单克隆细胞系不表达KXD1蛋白，同时检测了CD163蛋白的表达以确保该细胞系能够感染PRRSV(图2中A)，结果发现CD163蛋白能够表达，说明该细胞系已成功敲除Kxd1基因且并未影响CD163的表达，若后期验证发现该细胞系能够抑制PRRSV的增殖，那么不是CD163丢失引起的，而是Kxd1基因被敲除发挥了抗PRRSV的作用。
(2)扩增Kxd1基因敲除位点附近序列，测序确认敲除情况
1)Kxd1敲除单克隆细胞系及对照组PK15-Cas9-CD163细胞(简称KO-NC细胞)DNA的提取，按TIANGEN公司的血液/细胞/组织基因组DNA提取试剂盒(DP304)的操作说明书操作。
2)PCR扩增Kxd1基因敲除位点附近序列并送公司测序，扩增使用宝生物工程(大连)有限公司“LA Taq”(RR02MQ)，引物为：Kxd1(pig)-F：TCCTGGAAGGCTGTCCTGGTA和Kxd1(pig)-R：CAATGCTGGGATCTTTGACCTG。
设定PCR程序如下：预变性95℃10min，循环反应为变性95℃30sec，退火60℃30sec，延伸72℃1min，共进行35个循环，最后延伸72℃5min。PCR产物送公司进行测序。
测序发现由于KO-Kxd1单克隆细胞系缺失10对碱基而导致了Kxd1基因的移码突变，从而不产生KXD1蛋白(图2中A)。敲除前的KO-NC细胞(即PK15-Cas9-CD163)扩增序列是SEQ ID NO.2所示；而在敲除后的KO-Kxd1单克隆细胞系中的扩增序列为SEQ ID NO.3所示。
(3)利用MTT法检测KO-Kxd1单克隆细胞系活性
1)将相同数量及适量的实验组细胞和对照组细胞接种到96孔板中，每组3个技术重复。5％CO2、37℃培养箱继续培养48h。
2)取出96孔板，每孔加入20μL MTT溶液，避光继续培养4h。
3)吸弃MTT溶液，每孔加入150μL DMSO。避光反应5-10min。
4)使用酶标仪测每孔450nm的吸光值。
利用MTT实验检测Kxd1基因敲除后对细胞活性的影响，结果发现Kxd1敲除前后，细胞的MTT值并无显著差异，说明敲除Kxd1基因后对细胞活性并无显著影响(图2中B)。
实施例3：
利用qPCR检测PK15-CD163细胞敲除KXD1对PRRSV ORF7 mRNA的抑制效果。
将相同数量及适量的实验组细胞(KO-Kxd1)和对照组细胞(KO-NC)接种到6孔板中，5％CO2、37℃培养箱培养。24h后弃培养基并用DMEM洗2遍，加入PRRSV(MOI＝1，WUH3毒株，GenBank accession NO.HM853673)混匀，37℃孵育1h后，再加入1mL DMEM+10％FBS完全培养基，继续培养。随后的收样及qPCR操作步骤与实施例1相同。
结果发现，在12h和24h时，对照组和实验组的ORF7表达并无显著差异，但在48h时，ORF7在两组间的相对表达出现极显著差异(图3)。说明在PK15-CD163细胞中敲除Kxd1基因后显著抑制了PRRSV的增殖。
实施例4：
利用qPCR及TCID50检测Marc-145细胞干扰Kxd1表达对PRRSV的抑制效果。
1.干扰片段的转染及干扰效率的检测
(1)绿猴的KXD1蛋白(UniProtKB-A0A0D9QZ58)如SEQ ID NO.4所示，根据其mRNA序列选择特异干扰片段，该干扰片段序列(5'-GGACCCUGGUAGAGAUGAAUU-3')由苏州吉玛公司合成。
(2)转染前一天将生长良好的Marc145细胞以1×105个/孔的量加入6孔细胞培养板，在37℃5％CO2培养箱中培养，待细胞汇合度达到50％-60％时准备转染；具体细胞转染步骤参考见Lipofectamine)3000(ThermoFisher scientific,USA)转染试剂盒上说明书。
(3)利用qPCR方法检测siKxd1的干扰效率，具体操作步骤与实施例1相同，所用引物如下表所示。
结果如图4中A所示，siKxd1的干扰效率超过80％。
2.利用qPCR检测干扰Kxd1对PRRSV ORF7 mRNA的抑制效果。
转染方法同步骤1，检测在Marc-145细胞中干扰Kxd1对PRRSV ORF7 mRNA的抑制效果，PRRSV基因ORF7的引物如实施例1所述，Marc-145细胞基因β-actin的引物序列如上表所示。
结果如图4中B所示，Marc-145细胞中敲低Kxd1后，PRRSVORF7相对表达量显著下降，说明在Marc-145细胞中干扰该基因表达可抑制PRRSV增殖。
3.利用TCID50检测干扰Kxd1对PRRSV的抑制效果。
按本实施例步骤1转染干扰片段，24h后感染PRRSV(MOI＝1)。感染48h后，TCID50检测PRRSV滴度，具体方法如下：
(1)将病毒作10倍连续稀释，从10-1到10-10。
(2)将长满单层的Marc145细胞消化后加入细胞生长液，制备成细胞悬液。然后按照每孔加入100μL细胞悬液铺满96孔板。
(3)按每孔100μL的量将稀释好的病毒液加入96孔培养板中，每一个稀释度有8个孔。另外有16孔细胞中加入100μL的细胞生长液，作为空白对照。
(4)将96孔板放在37℃、5％CO2培养箱中培养，每天进行观察，3d-5d后统计出现CPE的孔数。
(5)按Reed-Muench两氏法计算，具体方法如下：距离比例＝(高于50％病变率的百分数－50％)/(高于病变率的百分数－低于病变率的百分数)，lgTCID50＝距离此例×稀释度对数之间的差+高于50％病变率的稀释度的对数。
结果如图4中C所示，Marc-145细胞中敲低Kxd1后，病毒滴度显著下降，同样说明在Marc-145细胞中干扰该基因表达可抑制PRRSV增殖。
序列表
<110> 华中农业大学
<120> 敲除或沉默猪的Kxd1基因在提高猪抗猪繁殖与呼吸综合征病毒中的应用
<160> 20
<170> SIPOSequenceListing 1.0
<210> 1
<211> 8593
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
aaacagtaac tgcgtctaag tcccacctct tcctgccgcg cctccacgca cgcaagtcgg 60
ccccgggcac gttgcggaca cgcgcgctag cactcatggt acgtgatgac gttgaggcgg 120
cactggcgcc atcccggaag cgcgagcaag gccgccagat gtgcaggtgc tgctgccacc 180
gacgccgggg ccgagttcag ggtggggctg gggacgcagg ggcttctggg cagctgggcc 240
gggcgggagc agtccccggg ctgtcgcggg gtggggcgcg ccgctggcaa catctggaca 300
ccctgcgcac aggcctcagt ttccccggct gcgcagtgga cgcgcggacg gcgccgtggg 360
ctggatccgg atccggggcg ccagagttgg gggcgtctat gttgaagctg tgctatatca 420
cccagaaccg ggcttttccc tgcttaggcc tcagtttccc ggcttcacag gggcggggct 480
tgaggggctt tgtgagcccc gccgcactgg ggggttttgc agagctgggg tagcccagtt 540
aaattctgtt aaatgatcta ggtggagtcg cggcgtctct ctgcctaggt gtgtgacctt 600
gggcaactga gcccacctct ctagccttgg ttgccttcgg aaatgatagt gtcagtctat 660
ctgcctctca ggggtgatta gggattaaat gaagtgtttt acaagtgttc gtttgtattc 720
ttggaagcac tggggagacc ctcattagaa tagtgattct tggcccagtc ctaggttttc 780
ttaacactcc cctcggagaa actgcctgct accagtccac tgtctcatca ctattggttt 840
cattacccaa agatagtgtt gtgagattct gattctttgt tattcctagc ttctggatga 900
gaaggaccca ggaggtgagg tgactttcct aaggtcacgg agcctccctc tctgcgtgtt 960
tcccccataa atatatttgg agctgggact agtgcctata ctggatcatt tttcaccctg 1020
tatatgggtc agaccgatac ttagtcaggg aaggcttccc gtgagaagca gctggtgagg 1080
gagtatgcat tgaatctctc agccctcaca tctgggagcc agagacagga aagagcctta 1140
ctttgaagca gaaccatttc ccggggtcca aatgttttgc cctccaggcc tagttgtggg 1200
gttcctgcca catgcctctg tcctttgaga gcccaagtat cctctttata tatgatcttt 1260
ggaaatgtgg aggctgtgag cgtccatacc tggcacacag tcagaacaaa ataaacatgg 1320
tgattattgg gggcggggga ggggggtttc atcaggtgtg gcagttcatc tccagaaggc 1380
acataaggct gcagacccta gctagattca gagcacaaat agatatgtac tctcagcctc 1440
tatagctttg gggaaaaaaa aaaaaaaaaa aagtgttagt tgctaacatt tgtaaatgag 1500
gcgatttcat aaggggaaaa atatatgttt cagtttgttt gttttggcca tgctgcagca 1560
tccgaaagct cccaggccag ggatcaagct gctgcaatga caacacagat ccttaaccta 1620
ctgtgccact aaagaactca gctttttttc tggaaaaaat cagaagtctg gcaggaatca 1680
gctgctgtaa gaagcagttg ttctgtcaca ggtggatact gaacatctgt ccagagtttg 1740
tggatcctgt ggaccgtggc atttaagttg tgactcaagt tcactggcta tgtgacagtg 1800
gctcagttct tcagtgtctg agactcagtt ttgtcatata gaaataagga tgataacaat 1860
tgccagtgtt tgaagttggt gtcctgttta gcatacagta ggtaacaaag aggggagtag 1920
agttagcagg ctctgaaagc ccttcctgat cctgtgtgtg aatataatca attgcatgtg 1980
ggcctttctg tgggccatta aatgtgcagt ccctagagcc aagctgtgtc aggattctgg 2040
gtttttcctg aaccccctca ggaaggtcag tcctgaagct gtggccccct cctgccctgg 2100
catggtccag gaagtcttca cctcctggga tggcatctgt gtgtcaagga agctggaggc 2160
taccaagcca tctgagttgc tgggacccac agcctggctg ccagtgcccc aagttcttgc 2220
ctttaacttc tttttttttt ttttttttaa attttatttt attttattgt tattattatt 2280
tttttgtctt tttgccattt ttagggccac tcccgtggca tatggaggtt cccaggctag 2340
gggtgtaatc ggagctgtag ccactggcct acaccagagc catagcaact cagaatccga 2400
gccatgtctg caacctacac cacagctcac ggcaacgccg gattgttaac ccactgagca 2460
aggccaggga tcgaacccgc aacctcatgg ttcctagtcg gattcgttaa ccactgagcc 2520
atgaagggaa ctcccgttct tgcctttaac ttctaatcca gcttcagagc tgggtttgaa 2580
agggtccgtt tgcctccacc aaactaattt tcccaatcct tggtgtgaac caggttctgg 2640
gccaggctct ggagacagag cagtgagcac aaacaacatg cctctgctct gttgcagctg 2700
acgttgctat aggggtgaca ggcaaatggg tcgactgata acttcagaaa gagatgtgcc 2760
agtgcagcag ttaaaacaag gtgaagtgat aagagtgact agagtagggg tagctatcag 2820
aaacctcctg ggcgagaagg cattggaggg ggccataatt tgagggtcag ccatgtatgt 2880
tctaccagtg gggacagcag gtgcaaaggc cccgtggtgg caaaggcagg gggatgtggg 2940
aggagggcac atggacgggg gagctgtgca ggtgggagaa atgagagtct gatgtgctgg 3000
ggtacaagtt aaaaatatga atccctgtag ttctcaaaac attcctgaat tcctgggatg 3060
acaaagctta ggctcaggtg ccttgaccaa tttatggtcc tgcagggttc ccagcccctc 3120
ctctccactt cccccacttc ctggtacaga ctgaagactc tttcttcctg cctgtttcag 3180
gcagcggagg aggaggaaga gatggacccc ccggactcgg cctcgagggt cttctgcagc 3240
cgcatcctga gtatggtgaa tgcagacgat gtcaatgcca ttatcctggc ccagaaaaac 3300
atgtgagtga tggccaaggc ctgggccctt ggcgtgggaa ggaatccact tgctggccag 3360
cattcttgcc ctgcccgcct ctacctcggg cccacactgg tgataaaatg aaggaaatga 3420
gcaaatccgc tggctggctc caaagtaaat tccttcctgg cccggaggcg aggccttggc 3480
acaggaagcg gggctctggg actgttttgc agctgcgggc agtttggcgg gggcagcagc 3540
agctcctgtt ctgagacctg gattggggag actcgggata aggaaagggc tcctaaggga 3600
tttaagggag ccagtctgct cacttccgat tgggaacctg ttctcttaga gccttggctg 3660
tctccacagg gcacttgtgg tcatttggaa ggatagggaa agagggcttt tgccctcctt 3720
ggggcttggc actgtggcca ctgcagcctc accttaaatc tggcctgccc tgtggggagt 3780
aacagctgac acaatcttgg gtggaaggaa gcatagtctc ttggggcttc tttttatggc 3840
tgcacctgtg gcatatggaa gttcttggac caagggttga attggagctg tggctgaggt 3900
ctacaccata gcctcggcaa caccatatat gagctatatc tatgaactgc cctgcagctt 3960
gcaccaacac cggatcgtta ccccactgag caagaccaga acccacatcc tctctaagac 4020
aacactgggt ccttaacctg ctgagccaca acaggaactc ctcctggggc tttatttttg 4080
tgactttaag cccaaagctc ttccttactt cttgctttat aacatagcac aaaccttgta 4140
tcttaccttt cttgccctgt gacaggggtg ctggatatct ttaggtctca gcaatacaca 4200
tctcccttct aattctttaa gtcagattca cacttggccc tggggaactc ccagactgag 4260
aggaggcaaa atcagacttg aaccttcctg gcctcatgat gtcaggtctg ggctagagaa 4320
acaacctggg agctacagga atcagaagaa tcaaagaatc tgcttgagga gttcccattg 4380
tggcgcagcg gaaatgaatc cgactagtaa ccatgaggtt gcggctttga tccctggcct 4440
cactctgtag gctaaggatc cagtgttgtc atgggctgtg gtgtaggtca cagacttggc 4500
ttggatccag tgttggctgt ggcgtaggca ggcagctatt gctctgattg gacccctagc 4560
ctgggaatct ccatatactg caggtgcagc cctaaaaagc aaaaaaaaaa aagaatctgc 4620
ttgagggaga gaatgaaggc tacccaagaa gggcacacat gattagggtt ttgaaagttg 4680
aataggagtt tgctaggttg ctcaaacacc aggtggagct ccataggaca agggaatggt 4740
ccctggaggc tgaacagagg agttggtgtc ctctttctgc cccaggctgg accgctttga 4800
gaagaccaat gagatgctat tgaacttcaa caacttgtca agtgcccgct tgcaacagat 4860
gagtgagcgc ttcctgcacc acacaaggac cctggtggaa atgaaacggg acctggacag 4920
catctttcgc aggatcaggt gggtgcttgg ccctccaact tctgcctccc catccaagcc 4980
tcaggttact ctcctcttca acgagggcca ttctctccac ataacagggc agtaggggga 5040
tttagctcat ttgttttatg agtcttcaat gtatacttag tattggagaa gatctgaaaa 5100
ctttagactt actgaattta aattttactt cttttttgaa aatatagcat agcaccagac 5160
ccaggatgca tcaggcaggc tggtgcagac aatgaaaggc tagcctcctg tcctccttgg 5220
cctttgccca ggtcttttgt caggaggcaa atgagaatgc caggttttgg gggtactttt 5280
tttttttttt ttttttggtc ctttgagggc cgcacccacg gcatatggag gttcccaggc 5340
taggggtcta atcggagctg ttgctgccag cctacgccag agccacagca atgccagatc 5400
tgagccatgt ttgccaccta ccccacagct tgaggcaacg ccagatcctt aactcactga 5460
gtgaagccag ggatcgaacc tgcaacctca tggtttttag attcgtttcc gctgcgccac 5520
aacaggaact ccaggggtac ttcttaagtt ggcgtccctg aggacaggga cagatgatgt 5580
ccaatccgca gctacacaga gtcctggaca gtacctggag ctcgcatcat cactgctgcc 5640
gttaacaact gctgctgggg cacccaggtc tggaacaagc tggcactgca gctgtatttc 5700
tgcccacttg tgccacttcc tctgttccag gggcagctgc acctgctggc agtgcaggtg 5760
ccacaaattg aaggcaccct gtttccttga ccaagctgtg tgcagaaatc acaagacaag 5820
agttcccgtt gtggctcagc agcagtgaac ctgactagta tccatgagga tgtagtttca 5880
atccctggcc tcatccagtg ggttaaggat ctggcattgc tgtgggttgt ggtgtaggtt 5940
gcagacacag cttggatcct gcattgctgt ggctgtggct gtgagggcgg caagtggcag 6000
cactggtact tcagggccgg gggtggggtg ggggtggggg tgctggttgc ccatagtcag 6060
gggtgagaga ttgctctgag gacccttttg tacttatttg taaacagaac tgtgttactt 6120
caggtaaaaa ggaaaaacac cagcaggttc aagatggaag caggtgcttc tgtttcgtgg 6180
gtgcccatat tcagtatttg gggtttggac agtcagcact cttggccaca cacagacatc 6240
ctccccatag ggatggagag acagtctggg acgaaagtca ccccaaggag aacttattga 6300
agagggggca agggaggctt cctggaaggc tgtcctggta gaagagggat agacataagg 6360
cactgtgggc agaaccagtg ctgagggcac tcagaagcca cgtgacagct gggtagctgc 6420
cacccatcct gggggtggga ggtccgcagg gctgagtgcc tgggcctccc tcctgcctgg 6480
agcagagtgt ggcaagcagt cagggggagg tctgcccact cctacctgcc cagagctggg 6540
agcaggggtt catggcctgc accaggtgca gccagtccac tcagcaggtt cagccctgtg 6600
tgccttccct tcccccacag gacactgaaa gggaagctgg ccaggcagca cccggaggcc 6660
ttcagccgta agtgtcaccc agagctcttg catcctgctt ctgggctctg cgcccttgga 6720
tgctgatgcc ctcagcccag ggctggcttc cagaaggtca ccgcaggctg acaccaagcc 6780
caggccctgg tgggcagtgg gtccagagaa gctgggggca ggggcggggg tgccggcctg 6840
gtctcagcaa cctgctgaac aggtgttggg accagagtct cctccctcat gggaggtcac 6900
atgaggggat gaggacaaga atgatgacag tcctgggagt gccctggtgg ctcagcaggt 6960
caaagatccc agcattgtca ctgctgtggc tcaggtagat ccctggcctg agaatttctg 7020
catgccacgg gtgtggccaa aaaagaagaa tgatgatagt ccttacccca gtgaatgtca 7080
tgtacttcta ggggccaggc tatgaggtgt aggctgtgcc cagaggagca gtgacttgtc 7140
agagggaggg cagcatcctc catgctgagc ctctgtccac cagtctgctg aatgggccaa 7200
cagctcccca ccccagggcc acaaaggcca caggcacaaa taaccatagg aacataacca 7260
caggcatgaa agcaatgcca tcattcccag ttcctagagc agggagctga ggcatgggca 7320
ggggaggagc agacccaggt gacacagtca agagggcaga gccaggccca gatactttac 7380
cccagggccc atctaggcct gaccccagag aacctgagtc cttaacctca ccttgaagag 7440
ggacctctca tgcactcagg cctctataaa atgaaccagg tcccatgggg ttgggggagc 7500
catcaggggt catgggccca gttaattcct aatcacagtg acttagcatg gtgactaagc 7560
caattcctgg gcctccttcc cccagacatc ccggaggcgt ccctcctgga agacgaggat 7620
gaagacccca tccctcccag caccacaaca accattgcca cctcggaaca gagcacaggc 7680
tcatgtgaca ccagccctga cacagtctcg ccctccctca gccctggctt cgaggacctg 7740
tcccatgtcc ggcctggctc ccctgccatc aatggccaca gccatacaga tgacgaggag 7800
acaccaggcg agtagctctc ctcctgggag ctccaagggg tctcagagca gcggcagcac 7860
caaccccaca tgtctgaggt ggcagcagat agccctgccc catgatggtc agctctgcct 7920
ccctattctg tcatttgggg ccccctgggg gacaaggctc cctctctgga atatggaatt 7980
cctggggatc tttcattccc accccttcct cactgagaat atctctcctc tgtagacccc 8040
tctgccaggc caggggacaa gcagcccagc tggagtcatt gggttgggct caaggaaact 8100
tccagagcca ggcctgtgat ctgttctgaa caacactcag attcccagag accagatcca 8160
gatgtcccct gccccagcac cctggtcagg acctcctcaa ggcggccaag cactgtcatg 8220
tctgagttca tcctagccca tccctgcatc tacaaattct tgtctccttg ctcctccctc 8280
caatgaaagt attcagtact ttcttcaatc attagctcaa ggtttctgag acagttgtgg 8340
aaggttcgag acagttgtgg aaggttcaag gcagagatta gccatccact tggcctcgaa 8400
cctggtaagg cccatgtttc tctgaccaga ggtgtggact ctgaggggcc agcagggctc 8460
tttcggggcc tctgtggagc aagccgagcc accatggaaa acagagttaa gcagaatatt 8520
tttgtacccg atgtttacag atgctgttgg gaagttatca ataaaaagac tctgttacaa 8580
agggaagact gta 8593
<210> 2
<211> 658
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
tcctggaagg ctgtcctggt agaagaggga tagacataag gcactgtggg cagaaccagt 60
gctgagggca ctcagaagcc acgtgacagc tgggtagctg ccacccatcc tgggggtggg 120
aggtccgcag ggctgagtgc ctgggcctcc ctcctgcctg gagcagagtg tggcaagcag 180
tcagggggag gtctgcccac tcctacctgc ccagagctgg gagcaggggt tcatggcctg 240
caccaggtgc agccagtcca ctcagcaggt tcagccctgt gtgccttccc ttcccccaca 300
ggacactgaa agggaagctg gccaggcagc acccggaggc cttcagccgt aagtgtcacc 360
cagagctctt gcatcctgct tctgggctct gcgcccttgg atgctgatgc cctcagccca 420
gggctggctt ccagaaggtc accgcaggct gacaccaagc ccaggccctg gtgggcagtg 480
ggtccagaga agctgggggc aggggcgggg gtgccggcct ggtctcagca acctgctgaa 540
caggtgttgg gaccagagtc tcctccctca tgggaggtca catgagggga tgaggacaag 600
aatgatgaca gtcctgggag tgccctggtg gctcagcagg tcaaagatcc cagcattg 658
<210> 3
<211> 648
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
tcctggaagg ctgtcctggt agaagaggga tagacataag gcactgtggg cagaaccagt 60
gctgagggca ctcagaagcc acgtgacagc tgggtagctg ccacccatcc tgggggtggg 120
aggtccgcag ggctgagtgc ctgggcctcc ctcctgcctg gagcagagtg tggcaagcag 180
tcagggggag gtctgcccac tcctacctgc ccagagctgg gagcaggggt tcatggcctg 240
caccaggtgc agccagtcca ctcagcaggt tcagccctgt gtgccttccc ttcccccctt 300
cgggaagctg gccaggcagc acccggaggc cttcagccgt aagtgtcacc cagagctctt 360
gcatcctgct tctgggctct gcgcccttgg atgctgatgc cctcagccca gggctggctt 420
ccagaaggtc accgcaggct gacaccaagc ccaggccctg gtgggcagtg ggtccagaga 480
agctgggggc aggggcgggg gtgccggcct ggtctcagca acctgctgaa caggtgttgg 540
gaccagagtc tcctccctca tgggaggtca catgagggga tgaggacaag aatgatgaca 600
gtcctgggag tgccctggtg gctcagcagg tcaaagatcc cagcattg 648
<210> 4
<211> 176
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Met Asp Leu Pro Asp Ser Ala Ser Arg Val Phe Cys Gly Arg Ile Leu
1 5 10 15
Ser Met Val Asn Thr Asp Asp Val Asn Ala Ile Ile Leu Ala Gln Lys
20 25 30
Asn Met Leu Asp Arg Phe Glu Lys Thr Asn Glu Met Leu Leu Asn Phe
35 40 45
Asn Asn Leu Ser Ser Ala Arg Leu Gln Gln Met Ser Glu Arg Phe Leu
50 55 60
His His Thr Arg Thr Leu Val Glu Met Lys Arg Asp Leu Asp Ser Ile
65 70 75 80
Phe Arg Arg Ile Arg Thr Leu Lys Gly Lys Leu Ala Arg Gln His Pro
85 90 95
Glu Ala Phe Ser His Ile Pro Glu Ala Ser Phe Leu Glu Glu Glu Asp
100 105 110
Glu Asp Pro Ile Pro Pro Ser Thr Thr Thr Thr Ile Ala Thr Ser Glu
115 120 125
Gln Ser Thr Gly Ser Cys Asp Thr Ser Pro Asp Thr Val Ser Pro Ser
130 135 140
Leu Ser Pro Gly Phe Glu Asp Leu Ser His Val Gln Pro Gly Ser Pro
145 150 155 160
Ala Ile Asn Gly Arg Ser Gln Thr Asp Asp Glu Glu Thr Thr Gly Glu
165 170 175
<210> 5
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
ctagtctaga atggtgctac ttgaagactc tg 32
<210> 6
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
cgcggatcct cattgtactt cagagtggtc tcc 33
<210> 7
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
tgatgggcga caatgtcc 18
<210> 8
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
cgcagacaaa tccagagg 18
<210> 9
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
tcagctgtgc caaatgctgg 20
<210> 10
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
aaatggggct tctccgggtt tt 22
<210> 11
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
tggcaccaca ccttctaca 19
<210> 12
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
atcttctcac ggttggcttt g 21
<210> 13
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
cccccacagg acactgaaa 19
<210> 14
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
tcctggaagg ctgtcctggt a 21
<210> 15
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
caatgctggg atctttgacc tg 22
<210> 16
<211> 21
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 16
ggacccuggu agagaugaau u 21
<210> 17
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
tgcagcagat gagcgagcg 19
<210> 18
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
cccgtgctct gttctgagg 19
<210> 19
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
agcaagcagg agtatgacga gt 22
<210> 20
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
caagaaaggg tgtaacgcaa ct 22
Claims (2)
1.抑制Kxd1基因表达的制剂在制备抗PRRSV感染药物中的应用，所述的Kxd1基因为猪中的NCBI登录号为NC_010444.4的Kxd1基因或编码氨基酸序列为SEQ ID NO.4所示的绿猴Kxd1基因；抑制猪中的NCBI登录号为NC_010444.4的Kxd1基因的制剂为gRNA5'-CCCCCACAGGACACTGAAA-3'，抑制编码氨基酸序列为SEQ ID NO.4所示的绿猴Kxd1基因的制剂为siRNA：5'-GGACCCUGGUAGAGAUGAAUU-3'。
2.敲除或者沉默Kxd1基因制备的抗PRRSV感染的细胞模型，所述的Kxd1基因为猪中的NCBI登录号为NC_010444.4的Kxd1基因或编码氨基酸序列为SEQ ID NO.4所示的绿猴Kxd1基因。
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