CN114940978A - 表达盖他病毒e2蛋白的重组猪繁殖与呼吸综合征病毒的构建方法 - Google Patents
表达盖他病毒e2蛋白的重组猪繁殖与呼吸综合征病毒的构建方法 Download PDFInfo
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Abstract
本发明公开了一种表达盖他病毒E2蛋白的重组猪繁殖与呼吸综合征病毒及其构建方法。发明人通过在PRRSV传代致弱株的全长感染性克隆骨架上,插入GETV的E2蛋白的核苷酸序列,获得了能够稳定表达GETV E2蛋白的重组PRRS病毒。研究表明,该重组病毒rGX‑12BS‑GE2遗传稳定,能够稳定高效地表达GETV的E2蛋白,为研制重组猪繁殖与呼吸综合征病毒基因工程疫苗奠定了基础。该疫苗的成功研制将会对GETV的防控起到积极重要的作用,并且此重组病毒的骨架为高致病性PRRSV传代致弱株,有望今后作为一种新型的基因工程活载体疫苗用于猪繁殖与呼吸综合征和猪盖他病的免疫保护,可以起到“一针防两病”的效果。
Description
技术领域
本发明属于病毒蛋白表达技术领域,尤其涉及一种表达盖他病毒E2蛋白的重组猪繁殖与呼吸综合征病毒的构建方法。
背景技术
猪繁殖与呼吸综合症病毒(Porcine reproductive and respiratory syndromevirus,PRR SV)可以引起猪繁殖与呼吸综合症,俗称“蓝耳病”,主要表现为怀孕母猪流产,尤其是怀孕后期母猪流产、产死胎、木乃伊胎、弱仔,以及各阶段猪呼吸道疾病症状。由于其病原(PRRSV)一直处于不断的变异及进化过程,为其防控带来一定困难,给世界的养猪业带来了巨大的经济损失。
盖他病毒(Getah virus,GETV)最早于1955年从马来西亚雪背库蚊(Culexgelidus)中分离到。血清学检测结果显示,在欧洲、亚洲和大洋洲的猪、马、牛及袋鼠等多种动物体内检测到GETV抗体。该病毒可通过蚊子传播感染脊椎动物,主要侵害猪和马,如造成母猪繁殖障碍,妊娠母猪流产、产死胎或弱仔,引起马的发热、皮疹、后腿浮肿、淋巴结肿大等。近年来,GETV迅速传播,已广泛分布于中国西南部到华北地区的多个省份,目前国内已在哺乳动物(猪、狐狸、马和牛)中分离到多株GETV毒株,结合全球流行趋势和我国的流行情况,该病毒近年在我国存在爆发的可能性。GETV为单链正股RNA病毒,属于披膜病毒科甲病毒属成员。由E基因编码的E蛋白是盖他病毒体最大的结构蛋白和主要包膜糖蛋白,构成病毒粒子表面的突起。该蛋白是盖他病毒吸附靶细胞、致病及诱导免疫保护的关键性蛋白,在盖他病毒的致病机理(ADE效应)及诱发机体的抗感染免疫中发挥重要的作用。E2基因具有很好的保守性,且中和位点位于E2基因,E2蛋白的抗原决定簇可以与多种单克隆抗体结合,决定病毒与宿主间的抗原关系。因此,E2是重要的抗原蛋白,可以用于GETV的疫苗研制。
反向遗传操作被广泛应用于研究病毒的生物学特性、致病机理、毒力决定因子以及不断变异的分子机制等方面。反向遗传系统也为研究PRRSV作为表达载体提供了技术支撑。 Nsp2是PRRSV中变异程度最大的蛋白之一。在PRRSV nsp2的高可变区(HVR)可以自然自发缺失和插入氨基酸。因而nsp2的HVR也可以用于插入外源基因或删除特定的个体免疫显性表位,以开发重组标记PRRSV疫苗。然而,在大多数重组病毒中,nsp2的HVR中的外源基因的融合并不总是保持合成的重组病毒的稳定性,或者有时在MARC-145细胞中连续传代后,靶基因的生物活性丧失。
发明内容
本发明要解决的技术问题是提供一种设计科学合理、表达高效稳定的表达盖他病毒E2 蛋白的重组猪繁殖与呼吸综合征病毒的构建方法。
为解决上述技术问题,本发明采用以下技术方案:
表达盖他病毒E2蛋白的重组猪繁殖与呼吸综合征病毒,该病毒中在ORF1b和ORF2a间插入猪盖他病毒E2蛋白基因。
上述表达盖他病毒E2蛋白的重组猪繁殖与呼吸综合征病毒的构建方法,将猪繁殖与呼吸综合征病毒rGXAM作为亲本毒株,利用基因工程的方法,在ORF1b和ORF2a间插入猪盖他病毒E2蛋白基因,获得重组PRRSV病毒rGX-12BS-GE2。
亲本毒株为高致病性PRRSV传代致弱毒株GXNN1396-P96;猪盖他病毒E2蛋白基因源于猪盖他病毒GETV-GX201808。
基因工程的方法是:在PRRSV感染性克隆中,利用SOE-PCR方法在ORF1b和ORF2a 间引入BstBI和Sbf I两个限制性酶切位点以及转录调控序列TRS,在此基础上,通过人为引入的酶切位点BstBI和SbfI将GETV E2蛋白基因插入pGX-12BS-TRS中,得到重组质粒pGX-12BS-GE2,再经病毒拯救获得重组PRRSV病毒rGX-12BS-GE2。
转录调控序列TRS具有序列表SEQ.NO.ID.1的碱基序列,重组质粒pGX-12BS-GE2全长序列为序列表SEQ.NO.ID.2的碱基序列。
重组质粒pGX-12BS-GE2按以下操作进行制备:以PRRSV的全长感染性克隆pGXAM为模板,使用突变引物GF11706-1F/12BS-1R和12BS-TRS-2F/JX14402Mlu I-2R分别扩增出包含有BstBI和SbfI限制性酶切位点及TRS的PCR产物1与PCR产物2,以此两段 PCR产物为模板,使用外侧引物GF11706-1F和JX14402Mlu I-2R进行第二轮融合PCR扩增,将融合PCR产物使用AscI和Mlu I酶切,同时酶切pGXAM,将含有酶切位点和TRS 的酶切片段(融合PCR产物)通过T4 DNA连接酶连至线性化的pGXAM中,获得重组质粒pGX-12BS-TRS;使用GETV E2基因的扩增引物GE2-BstBI-F和GE2-SbfI-R扩增GETV E2基因,然后使用BstBI和SbfI同时酶切PCR产物和pGX-12BS-TRS,将GETV E2基因通过T4 DNA连接酶克隆至pGX-12BS-TRS中,即得重组质粒pGX-12BS-GE2。
突变引物GF11706-1F/12BS-1R和12BS-TRS-2F/JX14402Mlu I-2R、扩增引物GE2-BstBI-F和GE2-SbfI-R分别具有序列序列表SEQ.NO.ID.3至SEQ.NO.ID.8的碱基序列。
上述构建方法得到的重组质粒。
上述表达盖他病毒E2蛋白的重组猪繁殖与呼吸综合征病毒和重组质粒在制备基因工程疫苗中的应用。
猪繁殖与呼吸综合征病毒和盖他病毒均可以在临床上引起母猪的繁殖障碍,且盖他病毒近几年曾经在国内猪场爆发,导致母猪的流产及仔猪的死亡,具有潜在爆发的隐患。发明人通过在PRRSV传代致弱株的全长感染性克隆骨架上,插入GETV的E2蛋白的核苷酸序列,获得了能够稳定表达GETV E2蛋白的重组PRRS病毒,并对其进行一系列病毒特性分析及遗传稳定性检测,还在病毒复制、转录、翻译水平对其与亲本病毒进行比较分析。使用本发明所构建的重组质粒pGX-12BS-GE2,转染BHK-21细胞后所拯救出来的病毒具有和亲本病毒rGXAM相似的病毒生物学特性。并且在至少连续6代次的传代过程中能够保持遗传稳定性。同时,基于上述重组质粒,转染BHK-21细胞后,将转染后的上清接种 MARC-145细胞可以拯救出的一株重组PRRS病毒:rGX-12BS-GE2,能与PRRSV N蛋白的单抗以及GETV E2的兔源多克隆抗体发生抗原抗体反应,出现特异性的免疫荧光和预期大小的Western blot条带。说明该重组病毒rGX-12BS-GE2遗传稳定,能够稳定高效地表达GETV的E2蛋白。
本发明是对GETV疫苗研发的一种新的尝试,其外源蛋白表达高效稳定,为研制重组猪繁殖与呼吸综合征病毒基因工程疫苗奠定了基础。该疫苗的成功研制将会对GETV的防控起到积极重要的作用,并且此重组病毒的骨架为高致病性PRRSV传代致弱株,有望今后作为一种新型的基因工程活载体疫苗用于猪繁殖与呼吸综合征和猪盖他病的免疫保护,可以起到“一针防两病”的效果。
附图说明:
图1是rGX-12BS-GE2的构建示意图。
图2是重组质粒pGX-12BS-GE2酶切鉴定电泳结果图,图中:泳道1:Marker 15,000;泳道2:pGX-12BS-GE2.
图3是细胞病变图,图中:A:rGXAM;B:rGX-12BS-GE2;C:阴性对照。
图4是拯救病毒的N蛋白和GETV E2蛋白的免疫荧光图,图中:A:一抗为抗PRRSV N蛋白;B:一抗为抗GETV E2蛋白。
图5是重组病毒病毒和亲本毒株的生长曲线图。
图6是重组病毒病毒和亲本毒株的空斑形态图,图中:A:rGXAM;B:rGX-12BS-GE2。
图7是重组病毒病毒和亲本毒株的Western Blot检测图,图中:泳道1:rGX-12BS-GE2、泳道2:rGXAM;泳道3:阴性对照。
图8是重组病毒遗传稳定性检测结果图,图中:M是Marker 2,000;P3、P6、P9、 P12、P15分别以P3、P6、P9、P12、P15代重组病毒为模板;1:pGXAM为模板;2: pGX-12BS-GE2为模板。
保藏信息说明
1.猪盖他病毒GETV-GX201808,保藏编号为CCTCC NO:V 202069,保藏日期:2020年10月13日,保藏地址为:中国湖北省武汉市武昌区珞珈山路16号武汉大学,邮编430072,保藏单位:中国典型培养物保藏中心。
2.猪繁殖与呼吸综合征病毒Gxnn1396-P96,保藏编号为CCTCC NO:V 202020,保藏日期:2020年01月16日,保藏地址为:中国湖北省武汉市武昌区珞珈山路16号武汉大学,邮编430072,保藏单位:中国典型培养物保藏中心。
具体实施方式
以下具体实施方式中未注明具体条件的实验方法,通常按照常规条件,如《分子克隆: 实验室手册》(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件进行。
1.材料与方法
1.1病毒、细胞、菌株及参考序列
GETV-GX201808毒株(GenBank accession numbers:MT269657)由申请人分离保存。 BHK-21细胞(乳仓鼠肾细胞)、MARC-145细胞(非洲绿猴肾细胞)、pGXAM由申请人保存,感受态细胞菌DH5α购自康为试剂有限公司。
其中,pGXAM系高致病性猪繁殖与呼吸综合征病毒传代致弱毒株GXNN1396-P96的感染性克隆pGXAM,是申请人参考以下文献构建并保存备用的:Wang Y,He W,Li Q,et al.Generation of a porcine reproductive and respiratory syndrome virusexpressing a marker gene inserted between ORF4 and ORF5a[J].Archives ofVirology,2020,:1-11。
1.2主要试剂和仪器
RNA酶抑制剂(RRI,ThermoFisher)、dNTP Mixture、ReverseTranscriptase M-MLV、RNase H-、DNA Marker(TaKaRa)、Green Taq Mix(Vazyme)、限制性内切酶、T4 DNA Ligase(NEB)、Plasmid Mini Kit、Gel Extraction Kit、Viral RNA Kit(OMEGA)、 Lipfectamine 2000(Invitrogen)、CO2恒温培养箱(Thermo Fisher)、倒置显微镜(Olympus CKX41)、核酸电泳仪(北京君意东方电泳设备)、Goat Anti-Rabbit IgG(H+L)(全式金);PRRSV N蛋白单克隆抗体SDOW-17(Rural Technologies)、SDS-PAGE凝胶制备试剂盒、蛋白Marker、RIPA细胞裂解液(康为世纪有限公司);HRP-山羊抗兔IgG(Abcam)。
1.3引物设计及目的基因扩增
(1)引物设计
在ORF1b和ORF2a间插入SbfI和Sbf I两个限制性酶切位点及TRS基因序列
GF11706-1F:GCGTTTCGGGCGCGCCAGAAAGG(序列表SEQ.NO.ID.3)
12BS-1R:CCTGCAGGTACCTTCGAATTCAATTCAGGCCTAAAGTTGTTC(序列表SEQ.NO.ID.4)
12BS-TRS-2F:GAACCAACTTAGGCCTGAATTGAATTCGAAGGTACCTGCAGGATGGTT CCGCGGCAACCCCTTTAACCAGAGTTTCAGCGGAACAATATGAAATGAAATGGGTCT ATGCAAAGCCT(序列表SEQ.NO.ID.5)
JX14402MluI-2R:ACGCCGGACGACAAACGCGTGGTTATCA(序列表SEQ.NO.ID.6)
(2)根据GETV-GX201808的基因组碱基序列,设计扩增E2蛋白引物。
GE2-BstBI-F:GAGTTCGAAAGTGTGACGGAACACTT(SEQ.NO.ID.7)
GE2-SbfI-R:TATCCTGCAGGTTAGGCATGCGCTCGTGGCG(SEQ.NO.ID.8)
(3)设计检测外源基因的检测引物,PCR扩增部位为外源基因及其两侧载体的片段。
TF11804:ATTTGAATCGGATACAGCGTATCTG(SEQ.NO.ID.9)
PR12186:CAGGGTGAACGGTAGAGC(SEQ.NO.ID.10)
上述引物由上海杰李生物有限公司合成。
以PRRSV的全长感染性克隆pGXAM为模板,使用引物GF11706-1F/12BS-1R以及引物12BS-TRS-2F/JX14402MluI-2R分别扩增出包含有MluI和SbfI限制性酶切位点及TRS 的PCR产物1(SEQ.NO.ID.11)与PCR产物2(SEQ.NO.ID.12),以此两段PCR产物为模板,使用外侧引物GF11706-1F和JX14402MluI-2R进行SOE-PCR,胶回收PCR产物。根据Viral RNA Kit试剂盒的说明书,提取GX201808病毒RNA,反转录成cDNA,以此为模板,使用设计的引物扩增GETVE2基因。
PCR体系:cDNA模板3μL,上游引物/下游引物各1μL,PrimeSTAR 25μL,ddH2O 补足至50μL。PCR反应程序条件如下:98℃预变性3min;98℃变性10s,55℃退火5s, 72℃延伸5s,共35个循环;72℃延伸10min。进行SOE-PCR时,以PCR产物1与PCR 产物2摩尔比1:1的比例为模板,分别50ng/孔,先不加两侧引物,PCR运行6个循环后,加入引物GF11706-1F/JX14402MluI-2R,按照常规程序运行。待PCR反应程序结束后,将PCR产物用1%的琼脂糖凝胶进行电泳,紫外灯照射下切取正确大小的条带于1.5mL的 EP管中,使用OMEGA的胶回收试剂盒进行回收。
1.4在PRRSV ORF1b和ORF2a间表达GETV E2蛋白的重组克隆pGX-12BS-GE2的构建
使用AscI和MluI同时酶切SOE-PCR回收产物及pGXAM,含有BstBI和Sbf I限制性酶切位点及TRS的PCR产物的通过T4 DNA连接酶连接至线性化的pGXAM,获得 pGX-12BS-TRS。使用BstBI和SbfI同时酶切胶回收的GETV E2基因和pGX-12BS-TRS,将GETV E2基因插入pGX-12BS-TRS中,获得重组质粒pGX-12BS-GE2,构建策略如图 1所示。
酶切具体步骤如下:目的基因/质粒30μL/15μL,BstbⅠ与SbfⅠ各1μL,CutSmartBuffer 5μL,ddH2O补至50μL,反应条件如下:加入BstbⅠ后在65℃水浴锅中酶切3h,加入SbfⅠ置于37℃水浴锅中酶切3h。将酶切产物使用OMEGA的反应液回收试剂盒进行回收。
将酶切回收后的片段以摩尔比3:1的比例,用T4 DNA连接酶连接,转化DH5α感受态细胞,挑取单个的菌落进行培养,提取质粒DNA。
将重组质粒pGX-12BS-GE2,用BstbⅠ与SbfⅠ进行酶切后,1%凝胶电泳,筛选阳性克隆,将酶切初步鉴定正确的质粒,如图2所示,进行测序。
1.5重组病毒的拯救
按照lipfectamine 2000说明书,将构建好的重组质粒转染到生长良好的6孔板中的 BHK-21细胞上,转染前将6孔板中的培养基更换为opti-MEM,转染剂量为2μg,转染后将细胞板放置于含有5%CO2的37℃培养箱中培养,6h后更换为2mL的含有2%胎牛血清的MEM培养基维持液,48h后收取细胞上清,置于-80℃保存。
1.6重组病毒的传代
将48h后收取的细胞上清记为P0代病毒,取300μL接种于生长良好的MARC-145 细胞上,孵育1h后,更换成含有2%胎牛血清的MEM培养基维持液,将6孔板放置于含有5%CO2的37℃培养箱中培养,每天观察细胞病变,当细胞出现明显CPE,如图3 所示时,收取上清,离心后于-80℃保存,记为P1代病毒。将P1代病毒100倍稀释后,取300μL接种到生长良好的MARC-145细胞上,待细胞脱落60%时收取细胞上清,记为P2病毒,将收取的上清以同样的方式再次接种到生长良好的MARC-145细胞上,依次循环,持续传代15代。将拯救出的病毒名命rGX-12BS-GE2。若无出现明显病变,盲传3 代。
1.7间接免疫荧光(IFA)鉴定重组病毒
将6孔板内生长良好的MARC-145细胞,以0.1MOI的剂量接种P3代的重组病毒,接种完成后,将细胞板置于在37℃、5%CO2的培养箱中培养36h后,弃去细胞上清,PBS 洗涤3次,使用预冷的甲醇于4℃固定细胞30min,然后用1.0%BSA室温封闭30min,封闭完成后,分别加入500μL PRRSV的单克隆抗体SDOW(1:2000稀释)和GETV E2的兔源特异性多克隆抗体(1:1000稀释),4℃过夜孵育,孵育完成后,每孔加入500μL (1:500)稀释的FITC标记的羊抗鼠或者羊抗兔的二抗,室温避光孵育1h,孵育完成后,加入DAPI进行核染,放置于荧光倒置显微镜下观察荧光。
结果如图4所示,使用PRRSV N蛋白抗体作为一抗时,亲本病毒和重组病毒感染的细胞内都可以观察到特异性荧光,而使用GETV E2蛋白多克隆抗体时,只有重组病毒感染的细胞内可以观察到特异性荧光,阴性对照始终都观察不到特异性荧光,表明重组病毒表达了GETV E2蛋白,并且可以被多克隆抗体识别。
1.8病毒细胞半数感染量(TCID50)测定
用96孔细胞培养板的方法进行感染性滴度的测定。取P3代病毒,用2%MEM作10 倍系列稀释后,将10-1-10-9连续稀释的病毒接种96孔细胞培养板上单层培养的MARC-145 细胞,每稀释度接种8孔,每孔0.1mL,设阴性对照(即用维持液代替病毒液),置含有5% CO2的37℃培养箱中培养,2-4天后观察感染细胞,记录出现细胞病变的孔数,并按 Reed-Muench法计算TCID50。
1.9病毒多步生长曲线测定
以低剂量(0.01MOI)病毒感染MARC-145细胞,分别在感染后的12h、24h、48h、 72h、96h、120h收取细胞培养上清,将收取的每个时间点病毒用TCID50计算病毒滴度,并根据不同时间点病毒的滴度,绘制病毒多步生长曲线。
结果如图5所示,结果说明利用反向遗传操作技术,获得的嵌合GETV E2蛋白基因的全长重组质粒拯救出来的病毒和亲本毒株在病毒增殖各个时间点的病毒滴度,指数增长期,平台期等生长曲线方面具有相似的生物学活性,只是病毒滴度略低于亲本病毒 rGXAM。
1.10病毒空斑表型测定
取rGXAM与rGX-12BS-GE2 P3代病毒液,倍比稀释成100TCID50、1000TCID50、10000TCID50等梯度,取200μL稀释好的病毒液接种到长满的6孔板中的MARC-145细胞上,置于37℃、5%CO2的培养箱中吸附1h,吸附结束后,弃掉上清,将MEM(含有4%FBS)和2%Agarose等体积混匀,每孔加入3mL,室温凝固后,然后倒置于37℃、5%CO2的培养箱中培养。出现明显噬斑后,用1%福尔马林固定6h,弃掉将培养基,用0.5%的结晶紫溶液染色30min,流水冲洗后观察。
结果如图6所示,重组病毒在MARC-145细胞上可以形成和亲本病毒相似的空斑形态。
1.11重组病毒rGX-12GETV-E2表达GETV E2蛋白的Western Blot鉴定
将rGX-12BS-GE2与rGXAM接种于MARC-145细胞,同时设置为不接种病毒的细胞孔作为阴性对照,36h收样,进行SDS-PAGE电泳,采用半干转膜仪将蛋白转至PVDF膜上,以5%脱脂奶粉封闭,一抗分别为GETV E2蛋白兔多克隆抗体以及Antiβ-Tubulin Mouse,二抗分别为HRP-山羊抗兔IgG以及HRP标记山羊抗小鼠IgG。在PVDF膜上加入DAB工作液充分接触混匀,在暗室条件下显色5min,立即通过蛋白印迹成像仪进行观察并拍照保存。
结果如图7所示,rGX-12BS-GE 2的感染孔蛋白样品中出现了明显的特异性杂交条带,而rGXAM感染孔及未感染病毒的对照孔样品均无相应大小的特异性条带。表明重组病毒表达了GETV E2蛋白,兔抗GETV E2的多克隆抗体能够特异性识别GETV的E2蛋白。
1.12重组病毒遗传稳定性测定
将传代获得P1至P15代病毒液,然后按照Viral RNA Kit(OMEGA)说明书中的操作方法分别提取P3,P6和P10代病毒液RNA,反转录成cDNA,使用引物TF11804/P R12186,采用RT-PCR检测外源报告蛋白在重组病毒内的遗传稳定性,同时设置亲本毒株及pGX-12BS-GE2作为对照。
结果如图8所示,GETV E2蛋白基因随着重组病毒至少在细胞上传代6次后,稳定存在于PRRSV基因组中。
综上,本发明在以PRRSV的全长感染性克隆pGXAM为骨架,利用SOE-PCR及酶切、 T4DNA连接酶连接的方法,在ORF1b和ORF2a间插入猪盖他病毒E2蛋白基因序列,获得表达猪盖他病毒E2蛋白的重组PRRSV全长感染性克隆质粒pGX-12BS-GE2,经病毒拯救,获得了活病毒rGX-12BS-GE2,经RT-PCR鉴定,嵌合重组病毒rGX-12BS-GE2能够稳定传代6次而不发生缺失,重组病毒具有和亲本病毒相似的病毒学特性。因此,rGX-12 BS-GE2有望可作为新型GETV的PRRSV活载体疫苗,并能够同时防治猪场的GETV和P RRSV感染。
序列表
<110> 广西大学
<120> 表达盖他病毒E2蛋白的重组猪繁殖与呼吸综合征病毒的构建方法
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atggttccgc ggcaacccct ttaaccagag tttcagcgga acaatatgaa 50
<210> 2
<211> 20450
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 60
attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 120
gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc 180
caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc 240
ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag 300
cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa 360
agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac 420
cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg 480
caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg 540
gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg 600
taaaacgacg gccagtgagg gccggccatt taaattaatt aaggacattg attattgaca 660
tgttattaat agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc 720
gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg 780
acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa 840
tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca 900
agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac 960
atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc 1020
atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga 1080
tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg 1140
gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta 1200
cggtgggagg tctatataag cagagctcgt tatataaacc gtcatgacgt ataggtgttg 1260
gctctatgcc acggcatttg tattgtcagg agctgtgacc attggcacag cccaaaactt 1320
gctgcacggg aacaccctcc tgtgacagct ctcttcaggg ggattagggg tctgtcccta 1380
acaccttgct tccggagttg cactgcttta cggtctctcc actcctttaa ccatgtccgg 1440
gatacttgat cggtgcacgt gtacccccaa tgccagggtg tttgtggcgg agggccaggt 1500
ctactgcaca cgatgtctca gtgcacggtc tctccttcct ctgaatctcc aagtctctga 1560
gcttggggtg ctgggtctat tttataggcc cgaagagcca ctccggtgga cgttgccacg 1620
tgcattcccc actgtcgagt gctcccccgc cggggcctgc tggctttctg cgatctttcc 1680
gattgcacga atgactagtg gaaacctgaa ctttcaacaa agaatggtgc gggttgcagc 1740
tgaaatctat agagccggcc aactcacccc tacagttcta aagactctac aagtttatga 1800
gcggggttgt cgttggtacc ccattgtcgg gcccgtccct ggggtaggcg ttttcgccaa 1860
ctccctgcat gtaagtgaca aacctttccc gggagcaact catgtgttaa ccaatttgcc 1920
gctcccgcag aggcccaaac ctgaggactt ttgccctttt gagtgtgcta tggctgacgt 1980
ctatgacatt ggtcgtggcg ccgtcatgta tgtggccgga ggaaaggtct cttgggcccc 2040
tcgtggtggg aatgaagtga aattcaaacc tgtccccaag gagttgaagt tggttgcgaa 2100
ccgactccac gcctccttcc cgccccatca cgcagtggac atgtccagat ttaccttctt 2160
gacccctggg agtggtgtct ccatgcgggt tgagtaccaa tacggctgcc tccccgccga 2220
cactgtcccc gaaggaaact gctggtggcg cttgtttgac tcgctcccac cggaagttca 2280
atacgaagaa attcgccatg ctaaccaatt tggctatcaa accaagcatg gtgtccctgg 2340
caagtatcta cagcggaggc tgcaagctaa tggtcttcga gcagtgaccg acacacatgg 2400
acctatcgtc atacagtact tctctgttaa ggagagttgg atccgccacc tgaagttggt 2460
ggatgaaccc agcctccccg ggtttaagga tctcctcaga atcagggttg agcccaacac 2520
gtcgccactg gccggaaagg atgagaagat tttccggttt ggcagttata agtggtacgg 2580
tgccgggaag agagcaagga aaacacgctc tggcgcgact actacggtca ctcatcacgc 2640
ttcgtccgct cataaaaccc ggcaggccac gaagcacgag ggtgccagcg ccgacaaagc 2700
tgaacatctc aatcgctact ctccgcctgc cgaggggaac tgcggttggc actgcatttc 2760
cgccattgcc aaccggatgg tgaattccaa ctttgagacc acccttcctg agagagtaag 2820
gccttcagaa gactgggcca ctgatgagga tcttgtgaac accatccaaa ttcttaggct 2880
ccctgcggcc ttggatagga acggcgcttg cggtagcgcc aagtacgtgc ttaaactgga 2940
gggtgagcat tggactgtct ctgtgatccc tgggatgtcc cctactttgc tcccccttga 3000
atgcgttcag ggttgttgtg agcataaggg cggtcttgtt tccccggatg cggtcgaaat 3060
ttccggattt gatcctgcct gccttgaccg actggctaag gttatgcact tgcctagcag 3120
taccatccca gccgctctgg ccgaattgtc cgatgactcc aaccgcccga tttccccggc 3180
cgctaccacg tggactgttt cgcagttcta tgctcgtcat agggaaggag atcatcatga 3240
ccaggtgtac ttagggaaaa tcatcagcct ttgtcaagtt attgaggatt gctgctgcca 3300
tcagaataaa accaaccggg ctactccgga agaggtcgcg gcaaagattg atcagtacct 3360
ccgtggcgca acaagtcttg aggaatgctt ggccaaactt gagagagttt ccccgccgag 3420
cgctgcggac gcttcctttg attggaatgt tgtgctccct ggggttgagg cggcgaatca 3480
ggcaaccgaa caacttcacg tcaactcatg ctgcaccctg gtccctcccg tgactcgaga 3540
gccttcgggc aaggactcgg tccctctgac cgccttctca ctgtccaatt gttattaccc 3600
tgcacatggt gacgaggttc atcaccgtga gaggttaaat tttgtactct ctaagttgga 3660
agaggttgtc ctggaagaat atgggctcgt gtctaatgga ctaggcccgc gacccgtgct 3720
tccgcgcggg ctcgacgagc ttaaagacca gatggaggag gatctgctaa aactagccaa 3780
cacacaggca acttcagaaa tgatggcccg ggcggctgag caggtcgatt taaaagcttg 3840
ggtcaaaaac tacccgcggt ggacaccacc actccctcca ccaagagttc aacctcgaag 3900
aacaaagtct gtcaaaagct tgccagagga caagcctgtc cccgttccgc gcaggaaggt 3960
caggtccgat tgcggcagcc cggttttgat gggcgacaat gtccctaacg gttcggaaga 4020
aactgtcggt ggtcccctca attttccgac accatccgag ccgacgacac ctttgagtgg 4080
gtcggcgcca gttcccgcac cgcgtagaac tgtgacagca acgctgacgc accaggatga 4140
gcctctggat ttgtctgcgt cctcacaaac ggaatatgag gctttccccc tagcaccatc 4200
gcagaacatg ggcgtcctag aggcgggggg gcaagaagct gaggaagttc tgagtgaaat 4260
ctcggacata ctaaatgaca ccaaccctgc acctgcgtca tcgagcagct ccctgtcaaa 4320
tgttaagatc acacgcccaa aatactcagc tcaagccatc attgactctg gcgggccttg 4380
cagtgggcat ctccaaaagg aaaaagaagc atgcctcagc atcatgcgtg aggcttgtga 4440
tgcgtccaag cttggtgatc ctgccacgca ggagtggctt tctcgcatgt gggatagggt 4500
tgacatgctg acttggcgca acacgtctgc ttaccaggcg tttcgcattt taagtggcag 4560
gtttgagttt ctcccaaaga tgattctcga gacaccgccg ccccacccgt gcgggtttgt 4620
gatgttacct cgcacgcctg caccttccgt gagtgcagag agtgacctca ccattggttc 4680
agtggccacc gaggatgttc cacgcatcct cgggaaagta ggaggcactg acgagctact 4740
tgaccggggt ccctcggcgc cctccaaggg agaaccggtc tgtgaccaac ctgccaaaga 4800
tccccggatg ttgccgcggg agtctgacga gagcataata gctccgcccg cagatacagg 4860
tggtgttggc tcattcactg gtttgccgcc ttcagacggc gtggatgtgg acgggggggg 4920
gccgttaaga acggtaaaaa taaaagtagg aaggctctta gaccaactga gccaccaggt 4980
ttttagcctt gtttcccatc tccctatttt cttctcacac ctcttcaaat ctgatagtgg 5040
ttattctccg agtgattggg gttttgcagc ttttactcta ttttgcctct ttctatgtta 5100
cagttaccca ttcttcggtt ttgctcccct cttgggtgta ttttctgggt cttctcggcg 5160
tgtgcgaatg ggggtttttg gctgttggtt ggcttttgct gttggtctgt tcaaacctgt 5220
gtccgaccca gtcggcactg cttgtgagtt tgactcgcca gagtgtagga acgtccttca 5280
ttcttttgag ctcctcaaac cttgggaccc tgtccgcagc cttgttgtgg gccccgtcgg 5340
tctcggcctt gccattcttg gcaggttact gggcggggca cgctacatct ggcactttct 5400
gcttaggctt ggcattgttg cagactgtat cttggctgga gcttacgtgc tttctcaagg 5460
taggtgtaaa aagtgctggg gatcttgtgt aacaactgct cctaatgaga tcgccttcaa 5520
cgtgttccct ttcacacgcg cgaccaggtc gtcactcatc gacctgtgcg atcggttttg 5580
cgcaccaaaa agcatggacc ccatttttct cgccactggg tggcgtgggt gctggaccgg 5640
ccggagcccc attgagcaac cttctgaaaa acccatcgcg ttcgcccagc tggatgagga 5700
gaggattacg gctagaactg tggtcgctca accttatgat cccaaccagg ccgtaaagtg 5760
cttgcgggta ttacaggcgg gtggggcgat ggtggctgag gcagtcccaa aagtagtcaa 5820
agtttccgct attccattcc gagctccttt ctttcccgct ggagtgaaag ttgatcctga 5880
gtgcagaatc gtggttgatc ccgatacttt tactacagcc ctccggtctg gctattccac 5940
cgcgaatctc gtccttggta cgggggactt tgcccagctg aatggactaa agatcaggca 6000
aatttccaag ccttcagggg gaggcccaca cctcattgct gccttgcatg ttgcctgctc 6060
gatggcgtta cacatgcttg ctggtgttta tgtaactgca gtggggtcct gcggtaccgg 6120
taccaacgat ccgtggtgca ctaacccgtt tgccgtccct ggctacggac ctggctctct 6180
ttgcacgtct agattgtgca tctcccaaca cggccttacc ttgcccttga cagcacttgt 6240
ggcgggattc ggccttcaag aaattgcctt ggtcgttttg gtttttgtct ccatcggagg 6300
catggctcat aggttgagtt gtaaggctga catgttgtgc atcttactcg caattgctag 6360
ttatgtttgg gtacctttga cctggttgct ttgtgtgttt ccttgttggt tgcgctggtt 6420
ctctttgcac cccctcacca tcctgtggtt ggtgtttttc ttgatttctg taaatatacc 6480
ctcgggaatc ttggccgtgg tgttattggt ttctctctgg cttctaggtc gttatactaa 6540
cattgctggt ctcgttaccc cttatgacat tcatcattac accagtggcc cccgcggtgt 6600
tgccgccttg gccactgcac cagatggaac ctacttggct gccgtccgcc gtgctgcgct 6660
gactggtcgt accatgctgt ttaccccgtc tcagctcggg tccctccttg agggcgcttt 6720
cagaactcaa aaaccctcac taaataccgt caatgtggtc gggtcctcca tgggctctgg 6780
cggagtgttc actattgacg ggaaaatcaa gtgcgtgact gccgcacatg tccctacggg 6840
taactcagct agggtttccg gggtcggctt caatcaaatg cttgactttg atgtaaaagg 6900
ggacttcgcc atagctgatt gcccgaattg gcaaggggtt gctcccaagg cccaattctg 6960
cgaggatggg tggactggtc gcgcctattg gttgacatcc tctggcgttg aacccggtgt 7020
tattgggaat gggttcgcct tctgcttcac cgcgtgtggc gactctggat ccccagtgat 7080
taccgaagcc ggtgagcttg tcggcgtcca cacagggtca aacaaacaag gaggaggcat 7140
tgtcacgcgc ccctcaggcc agttttgtaa tgtgaagccc atcaagctga gcgagttgag 7200
tgaattcttc gctggaccta gggtcccgct cggtgatgtg aaaattggca gtcacataat 7260
taaagacaca tgcgaggtgc cttcagatct ttgtgccctg cttgctgcca aacccgaact 7320
ggaaggaggc ctttccacag ttcaacttct gtgtgtgttt ttcctcctgt ggagaatgat 7380
ggggcatgcc tggacgccct tggttgctgt ggggtttttc atcctgaatg agattctccc 7440
agctgttctg gtccggagtg ttttctcctt tgggatgttt gtgctatctt ggcttacacc 7500
atggtctgcg caagtcctga tgatcaggcc tctgacagca gcccttaaca gaaacagatg 7560
gtctcttgtt ttttacagcc ctggtgcgat aaccagtttt gtcgcagatc ttgcggttac 7620
tcaggggcat ccgttacagg tggtaatgaa cttaagcacc tatgccttcc tgccccggac 7680
gatggttgtg acctcgccag tcccagtgat cgcgtgtggt gttgtgcacc tccttgccat 7740
aattttgtac ttgtttaagt accgctgcct tcacaatgtc cttgttggcg atggggtgtt 7800
ctcttcggct ttctttttgc gatacttcgc cgagggaaag ttgagggaag gggtgtcgca 7860
atcctgcggg atgagtcatg agtcgctgac tggtgccctc gccatgagac tcagtgacga 7920
ggacttggat ttcctcacga aatggactga ttttaagtgc tttgtttctg cgtccaacat 7980
gaggaatgca gcgggccaat ttatcgaggc tgcttatgca aaagcactaa gaattgaact 8040
tgctcagttg gtacaggttg ataaggtccg aggcaccctg gccaaactcg aggcatttgc 8100
cgataccgtg gcaccccaac tctcgcccgg tgacattgtt gttgctcttg gccacacgcc 8160
tgttggcagc atcttcgact taaaggttgg taacaccaag cacactctcc aagccattga 8220
gactagagtc cttgccgggt ccaaaatgac tgtggcgcgt gtcgttgacc caacccccgc 8280
acccccaccc gtgcctgtgt ccatccctct cccaccgaaa gttctggaga acggtcccaa 8340
tgcctggggg gatgaggacc gtttgaacaa gaagaagagg cgcaggatgg aagccgtcgg 8400
catttttgtc atggacggga aaaagtacca gaaattttgg gacaagaatt ccggtgatgt 8460
gttttatgag gaggtccata ttagtacaga cgagtgggag tgccttagaa ctggcgaccc 8520
tgtcgacttt gatcctgaga cagggattca gtgtgggcat atcaccatcg aagataaggt 8580
ttacaatgtc ttcacctccc catctggtag gagattcttg gtccccgcca accctgagaa 8640
tagaagagct cagtgggaag ccgccaagct ctccgtagag caagcccttg gtatgatgaa 8700
cgtcgacggc gaactgactg ccaaagaact ggaaaaactg aaaagaataa ttgacaaact 8760
ccagggcctg actaaggagc agtgtttaaa ctactagccg ccagcggctt gacccgctgt 8820
ggtcgcggcg gcttagttgt tactgagaca gcggtaaaaa tagtcagatt tcacaaccgg 8880
accttcaccc taggacctgt gaacttaaaa gtggccagtg aggttgagct aaaagacgcg 8940
gttgagcaca accaacatcc ggttgccaga ccggttgatg gtggtgttgt gcttttgcgc 9000
tctgcagttc cttcgcttgt agatgtcttg atctccggcg ctgatgcatc tcctaagtta 9060
ctcgcccgcc acggaccggg aaacactggg attgatggca cgctttggga ttttgaggcc 9120
gaggctacca aagaggaagt tgcactcagt gcgcaaataa tacaggcttg tgatattagg 9180
cgcggcgacg cgcctaaaat tggtctccct tataagttgt accctgttag gggcaaccct 9240
gaacgggtaa aaggagtttt acagaataca aggtttggag acatacctta caaaacccct 9300
agtgacactg gaagcccggt gcacgcggct gcctgcctca cgcctaatgc taccccggtg 9360
actgatgggc gctccgtctt ggctacaacc atgccttctg gctttgagtt gtatgtgccg 9420
accattccag cgtccgtcct tgattatctt gattctaggc ctgactgccc taaacggtta 9480
acagagcacg gttgtgagga tgctgcactg agagacctct ccaagtatga tttgtccacc 9540
caaggctttg ttttgcctgg ggttcttcgc cttgtgcgga agtacctgtt cacccacgtg 9600
ggcaagtgcc cgcccgttca tcggccttcc acttaccctg ccaagaattc tatggccgga 9660
ataaatggga acaggtttcc aaccaaggac attcagagcg tccctgaaat tgacgttctt 9720
tgcgcacagg ctgtgcgaga aaactggcaa actgtcaccc cttgcaccct caagaaacag 9780
tactgtggga agaagaagac taggacaata cttggcacca ataacttcgt tgcgttggcc 9840
catcgggcag cgttgagtgg tgttacccag ggcttcatga aaaaagcgtt caactcgccc 9900
atcgccctcg ggaaaaacaa atttaaggag ctacaagccc cggtcctagg caggtgcctt 9960
gaagctgacc ttgcgtcctg cgatcgatcc acacctgcaa ttgtccgctg gtttgccgcc 10020
aatcttcttt atgaactcgc ctgtgctgag gagcatctac cgtcgtacgt gctgaactgc 10080
tgccacgatt tactggtcac gcagtccggc gcggtgacta agagaggtgg cctgtcgtct 10140
ggcgacccga ttacctctgt gtcgaacacc atttacagct tagtgatata tgcacagcac 10200
atggtgctca gttacttcaa aagtggtcac cctcatggct ttctgtttct gcaagaccag 10260
ctaaagtttg aggacatgct caaggttcaa cccctgatcg tctattcgga cgaccttgtg 10320
ctgtatgccg agtctccctc catgccaaac taccactggt gggttgaaca tctgaatctt 10380
atgctgggtt tccaaacaga cccaaagaag acaaccatca cagactcacc atcattccta 10440
ggttgtagga taataaatgg acgccagcta gtccctaacc gtgacaggat ccttgcggcc 10500
ctcgcctacc acatgaaggc aagtaatgtt tctgaatact acgcctcggc ggctgcaata 10560
ctcatggaca gctgtgcttg tttagagtat gatcctgaat ggtttgaaga gctcgtggtt 10620
gggatagcgc agtgcgcccg taaggacggc tacagctttc ctggcccacc gttcttcttg 10680
tccatgtggg aaaaactcag gtccaaccat gaggggaaga agtccagaat gtgcgggtac 10740
tgtggggccc cggctccgta tgccactgcc tgtggtctcg atgtctgtgt ttaccacacc 10800
cactttcacc agcattgtcc tgttataatc tggtgtggcc acccggcggg ttctggttct 10860
tgtagtgagt gcgaaccccc cctaggaaaa ggcacaagcc ctctagatga ggtgttagaa 10920
caagttccgt acaagcctcc gcgaaccgtg atcatgcatg tggagcaggg tctcacccct 10980
cttgacccag gtagatacca gactcgccgc ggattagtct ccgttaggcg tggcatcaag 11040
gggaatgaag tcgacctacc agacggtgat tacgctagta ccgccttgct ccctacttgt 11100
aaggagatca acatggtcgc tgtcgcctct aacgtgttgc gcagcaggtt tatcatcggc 11160
ccacccggtg ctgggaaaac acactggctt cttcagcaag tccaggatgg tgatgtcatt 11220
tacacgccga ctcaccagac catgctcgac atgattaggg ctttggggac gtgccggttc 11280
aacgtcccag caggtacaac gctgcaattc cctgccccct cccgtaccgg cccatgggtt 11340
cgcatcttgg ccggcggttg gtgtcctggc aagaactcct tcctagatga agcggcgtat 11400
tgcaatcacc ttgatgtctt gaggcttctc agtaaaacaa cactcacttg cctaggggac 11460
ttcaaacaac tccaccctgt gggttttgac tcccattgct atgtgtttga catcatgcca 11520
cagacccaat taaagactat ctggaggttc gggcagaata tctgtgatgc cattcaacca 11580
gattacaggg acaaacttat gtccatggtc aacacgactc gcgtgaccta cgtggaaaaa 11640
cctgtcaggt atgggcaagt cctcaccccc taccacaggg accgagagga cggcgccatt 11700
actattgact ccagtcaagg cgccacattt gatgtggtta cactgcattt gcccactaaa 11760
gattcactca atagacaaag agctcttgtt gctatcacta gggcaagaca tgccatcttc 11820
gtgtatgacc cacacaggca attgcagatc atgttccatc ttcccgcgaa aggcacaccc 11880
gtcaacctcg cagtgcaccg tgacgaacag ctgatcgtat tagacagaaa caacagagaa 11940
atcacggttg ctcaggctct aggtaatgga gataaattta gggccacaga taagcgcgtt 12000
gtagattctc tccgcgctat ttgcgcggac ctggaagggt cgagctcccc gctccccaag 12060
gtcgcgcata atttgggatt ctatttctca cctgacttga ctcagtttgc taaactccca 12120
gcagaacttg caccccactg gcccgtggtg acaacccaga ataatgaaag gtggccagat 12180
cggctggtag ccagccttcg ccctatccat aaatatagcc gcgcgtgtat tggtgccggc 12240
tatatggtgg gcccctcggt gtttttaggc acccctgggg ttgtgtcata tcatctcaca 12300
aaatttgtta gaggcgaggc tcaagtgctt ccggagacag tcttcagcac cggccgaatt 12360
gaggtagatt gtcgagagta tcttgatgat cgggagcgag aagttgctga gtccctcccg 12420
catgccttca tcggcgatgt caaaggtacc accgttgggg gatgtcatca cgtcacctcc 12480
aaataccttc cgcgcttcct tcccaaggaa tcagttgcgg tggtcggggt ttcgagcccc 12540
gggaaagccg cgaaagcagt ctgcacattg acggatgtgt acctcccaga ccttgaagcg 12600
tacctccacc cagaaaccca gtccaggtgc tggaaagtga tgttggactt taaggaggtt 12660
cgactgatgg tatggaaaga caagacggcc tattttcaac ttgaaggccg ccattttacc 12720
tggtatcaac ttgcaagcta cgcctcatac atccgagtac ctgttaattc tactgtgtac 12780
ctggacccct gtatgggccc tgctctttgc aacagaaggg ttgtcgggtc cacccattgg 12840
ggggctgatc tcgcagtcac cccttatgat tacggtgcca aaatcattct gtctagtgca 12900
taccatggtg aaatgcctcc aggttacaaa attctggcgt gtgcggagtt ctcgcttgat 12960
gatccagtaa ggtacaaaca cacctgggga tttgaatcgg atactgcgta tctgtacgag 13020
tttactggaa atggtgagga ctgggaggat tacaatgatg cgtttcgggc gcgccagaaa 13080
gggaaaattt acaaagctac tgccaccagc atgaagtttc attttccccc gggccctgtc 13140
attgaaccaa ctttaggcct gaattgaatt cgaaatgagt gtgacggaac acttcaatgt 13200
ctacaaggcc acgaaaccat atctagcgta ttgcgcggac tgcggagacg ggcagttctg 13260
ttacagcccg gtggctatag aaaaaattag ggatgaggct tccgatggca tgataaagat 13320
ccagatcgca gcgcaaatcg gcatcaacaa aggaggaaca cacgaacaca acaaaatcag 13380
gtacatcgcc gggcatgaca tgaaagaggc gaaccgggat tctttacaag tgcatacttc 13440
cggtgtgtgc gctatccgag gcacgatggg ccacttcatc gtggcctact gccctccagg 13500
ggacgaacta aaggtccagt tccaagatgc agaatcgcac acacaggcct gcaaagtgca 13560
gtacaaacac gcaccggccc cagtaggcag agaaaaattc actgtcaggc cccacttcgg 13620
tatcgaagtg ccatgcacaa cgtaccagct gactaccgca ccaacggagg aagagatcga 13680
catgcatacc ccaccggata tcccagatat aacgttgctg tcgcaacagt caggcaatgt 13740
aaagatcaca gcaggaggaa agaccatcag atacaactgc acgtgcggta gtggccatgt 13800
gggcaccacc agtagcgaca agactatcaa ttcgtgcaaa atagcgcagt gccatgctgc 13860
ggtgactaac cacgacaagt ggcagtacac ctcctcgttt gtccctagag ccgaccagtt 13920
gtctcgcaaa ggtaaagtgc acgtaccctt ccctctgacc aactccacat gcagggtgcc 13980
cgttgcacgt acaccaggtg tcacatacgg aaagagagaa ctgacagtga aactgcaccc 14040
agatcatccc acgctgttga cgtaccgaag tctaggagca gatccgcgcc cgtatgagga 14100
gtggatagac cgatacgtcg aacggaccat accggtgacc gaagatggga tcgagtacag 14160
atggggaaac aacccacccg tgcgcttgtg ggcccagctg acaactgaag gcaaacccca 14220
tggatggccg cacgagatca tactctatta ctatgggcta tacccagcag ccaccatcgc 14280
cgccgtctca gccgcgggtc tcgcagccat attatcgctg ctggcgtcat gctacatgtt 14340
cgccactgca cgccgcaagt gcttgacccc atacgccctg acccccgggg ccgtcattcc 14400
ggtaacacta ggtgtactat gctgcgcgcc acgagcgcat gcctaacctg caggatggtt 14460
ccgcggcaac ccctttaacc agagtttcag cggaacaata tgaaatgaaa tggggtctat 14520
gcaaagcctc tttaacaaga ttggccaact ttttgtggat gctttcacgg aatttctggt 14580
gtccattgtt gacatcatca tttttttggc aattttgttt ggcttcacca tcgccggctg 14640
gctggtggtc ttctgcatca gattggtttg ctccgcggta ctccgtgcgc gccctaccgt 14700
ttaccctgag caactacaga agatcttatg aggcttttct ttctcagtgt cgggttgaca 14760
ttcccgcctg gggcgtcaaa caccctttgg gggtgctttg gcaccataag gtgtcgaccc 14820
tgattgatga aatggtgtcg cgtcgaatgt accgcatcat ggaaaaagcg gggcaggctg 14880
catggaaaca ggtggtgagc gaggccacat tgtctcgcat tagtggtttg gatgtggtgg 14940
ctcactttca acatcttgcc gccattgaag ccgagacttg taaatatttg gcttcccggc 15000
tacccatgct gcataatctg cgcttgacag ggtcaaatgt aaccatagtg tataatagta 15060
ctttgaatca ggtgtttgct atcttcccaa cccctggttc ccggccgaag cttcatgatt 15120
ttcagcaatg gctagtagct gtacattcct ccatattctc ctccgttgca gcttcttgta 15180
ctctctttgt tgtgctgtgg ttgcgaattc caatgctacg ttctgttttt ggtttccgct 15240
ggttaggggc aatttttctt ttgaactcac ggtaaactac acggtgtgcc cgccttgcct 15300
cactcggcag gcagccgctg agatctacga acccggtagg tccctttggt gcaggatagg 15360
gcatgaccga tgtagtgagg gtgatcatga cgaactaggg ttcatggttc cgcctggcct 15420
ctccagcgaa ggccatttga ccagtgttta tgcctggttg gcgttcctgt cctttagcta 15480
cacggcccag ttccatcccg agatatttgg gatagggaat gtaagtcaag tttatgttga 15540
catcaagcac caactcatct gtgctgttca cgacggggat aacaccacct tgcctcgcca 15600
tgacaatata tcagccgtat ttcagaccta ctaccaacac caggtcgacg gcggtaattg 15660
gtttcacctg gaatggctgc gccctttctt ttcctcttgg ttggtgctaa atgtttcgtg 15720
ggttctcagg cgttcgcctg caagccgtgt ttcagttcga gtctttcaga catcaaaacc 15780
aacaccaccg cggcatcagg ctttgttgtc ctccaggaca tcagctgcct taggcatggc 15840
gactcgtcct ctccgacgat tcgcaaaaat tctcagtgcc gcacggcgat agggacgcca 15900
gtgtacatta ccatcactgc caatgtcaca gatgagaatt atctacattc ttctgatctc 15960
ctcatgcttt cttcttgcct tttctatgct tccgagatga gtgaaaaggg attcaaagtg 16020
gtgtttggca atgtgtcagg catcgtggct gtgtgcatca actttaccag ctacgttcaa 16080
catgtcaagg agtttaccca acgctcccta gtggtcgatc atgtgcgcct gcttcatttc 16140
atgacacctg agaccatgag gtgggcaacc gtcttagcct gtctttttgc tatcctactg 16200
gcaatttgaa tgttcaagta tgttggggaa gtgcttgacc gcgtgctgtt gctcgcgatt 16260
gctttttttg tggtgtatcg tgccgttcta tcttgctgtg ctcgtcaacg ccagcaacaa 16320
caacagctct catattcagt tgatttataa cttaacgcta tgtgagctaa atggcacgga 16380
ttggctggca caaaaatttg actgggcagt ggagactttt gtcatcttcc ccgtgttgac 16440
tcacattgtt tcctatgggg cactcaccac cagccacttc cttgacacag ttggcctggc 16500
cgctgtgtcc accgccggat attatcacga gcggtatgtc ttgagtagca tttacgcggt 16560
ctgtgctctg gctgcgctga tttgctttgt cactaggctt gcgaagaact gcatgtcctg 16620
gcgctactct tgtaccagat ataccaactt ccttctggac actaagggca gactttatcg 16680
ttggcggtca cccgtcattg tggagaaagg gggtaaggtc gaggtcgaag gtcacctgat 16740
cgacctcaag agagttgtgc ttgatggttc tgcggcaact cctttaacca gagtttcagc 16800
ggaacaatgg agtcgtctct agacgacttc tgcaatgata gcaaagctcc acaaaaggtg 16860
cttttggcgt tttccattac ctacacgcca gtaatgatat atgctctaaa ggtaagtcgc 16920
ggccgactgt tagggcttct gcaccttttg atctttctga attgtgcttt caccttcggg 16980
tacatgacat ccgtgcactt tcatagcaca aatagggtcg cgctcactat gggagcagta 17040
gttgcacttc tttggggagt gtactcagcc atagaaacct ggaaattcat cacctccagg 17100
tgccggttgt gcttgctagg ccgcaagtac attctggccc ctgcccacca cgtcgaaagt 17160
gccgcgggct ttcatccgat tgcggcaaat gataaccacg cgtttgtcgt ccggcgtccc 17220
ggctccacta cggttaacgg cacattggtg cccgggttga aaagcctcgt gttgggtggc 17280
agaaaagctg ttaagcaggg agtggtaaac cttgtcaaat atgccaaata acaacggcaa 17340
gcagcaaaag aaaaagaagg ggaatggcca gccagtcaat cagctgtgcc agatgctggg 17400
taagatcatc gcccagcaaa accagtctag aggcaaggga ccggggaaga aaagtaagaa 17460
gaaaaacccg gagaagcccc attttcctct agcgactgaa gatgacgtca ggcatcactt 17520
tacccctggt gagcggcaat tgtgtctgtc gtcaatccag actgccttca atcaaggcgc 17580
tggaacttgc gccctgtcag actcagggag gataagttac actgtggagt ttagtttgcc 17640
gacgcatcat actgtgcgcc tgatccgcgt cacagcatca ccctcaacat gatgggctgg 17700
cattctttgg cacctcagtg ttagaatttg agaatgtgtg gtgaatggca ctgattggca 17760
ctgtgcctct aagtcaccta ttcaattagg gcgaccgcat gggggtaaag tttaattggc 17820
gagaaccatg cggccgtaat taaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 17880
aaaaaaaaaa aaagggtcgg catggcatct ccacctcctc gcggtccgac ctgggcatcc 17940
gaaggaggac gcacgtccac tcggatggct aagggagggc gctcgagctg atcagcctcg 18000
actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc 18060
ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt 18120
ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat 18180
tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcggc cgccaccgcg 18240
gtggagctcc agcttttgtt ccctttagtg agggttaatt gcgcgcttgg cgtaatcatg 18300
gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc 18360
cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc 18420
gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat 18480
cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac 18540
tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt 18600
aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca 18660
gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc 18720
ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact 18780
ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct 18840
gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag 18900
ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca 18960
cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa 19020
cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc 19080
gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag 19140
aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg 19200
tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca 19260
gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc 19320
tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag 19380
gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata 19440
tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat 19500
ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg 19560
ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc 19620
tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc 19680
aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc 19740
gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc 19800
gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc 19860
ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa 19920
gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat 19980
gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata 20040
gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca 20100
tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag 20160
gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc 20220
agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc 20280
aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata 20340
ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta 20400
gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac 20450
<210> 3
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
gcgtttcggg cgcgccagaa agg 23
<210> 4
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
cctgcaggta ccttcgaatt caattcaggc ctaaagttgt tc 42
<210> 5
<211> 116
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
gaaccaactt aggcctgaat tgaattcgaa ggtacctgca ggatggttcc gcggcaaccc 60
ctttaaccag agtttcagcg gaacaatatg aaatgaaatg ggtctatgca aagcct 116
<210> 6
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
acgccggacg acaaacgcgt ggttatca 28
<210> 7
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
gagttcgaaa gtgtgacgga acactt 26
<210> 8
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
tatcctgcag gttaggcatg cgctcgtggc g 31
<210> 9
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
atttgaatcg gatacagcgt atctg 25
<210> 10
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
cagggtgaac ggtagagc 18
<210> 11
<211> 127
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
gcgtttcggg cgcgccagaa agggaaaatt tacaaagcta ctgccaccag catgaagttt 60
cattttcccc cgggccctgt cattgaacca actttaggcc tgaattgaat tcgaaggtac 120
ctgcagg 127
<210> 12
<211> 2805
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
gaaccaactt aggcctgaat tgaattcgaa ggtacctgca ggatggttcc gcggcaaccc 60
ctttaaccag agtttcagcg gaacaatatg aaatgaaatg gggtctatgc aaagcctctt 120
taacaagatt ggccaacttt ttgtggatgc tttcacggaa tttctggtgt ccattgttga 180
catcatcatt tttttggcaa ttttgtttgg cttcaccatc gccggctggc tggtggtctt 240
ctgcatcaga ttggtttgct ccgcggtact ccgtgcgcgc cctaccgttt accctgagca 300
actacagaag atcttatgag gcttttcttt ctcagtgtcg ggttgacatt cccgcctggg 360
gcgtcaaaca ccctttgggg gtgctttggc accataaggt gtcgaccctg attgatgaaa 420
tggtgtcgcg tcgaatgtac cgcatcatgg aaaaagcggg gcaggctgca tggaaacagg 480
tggtgagcga ggccacattg tctcgcatta gtggtttgga tgtggtggct cactttcaac 540
atcttgccgc cattgaagcc gagacttgta aatatttggc ttcccggcta cccatgctgc 600
ataatctgcg cttgacaggg tcaaatgtaa ccatagtgta taatagtact ttgaatcagg 660
tgtttgctat cttcccaacc cctggttccc ggccgaagct tcatgatttt cagcaatggc 720
tagtagctgt acattcctcc atattctcct ccgttgcagc ttcttgtact ctctttgttg 780
tgctgtggtt gcgaattcca atgctacgtt ctgtttttgg tttccgctgg ttaggggcaa 840
tttttctttt gaactcacgg taaactacac ggtgtgcccg ccttgcctca ctcggcaggc 900
agccgctgag atctacgaac ccggtaggtc cctttggtgc aggatagggc atgaccgatg 960
tagtgagggt gatcatgacg aactagggtt catggttccg cctggcctct ccagcgaagg 1020
ccatttgacc agtgtttatg cctggttggc gttcctgtcc tttagctaca cggcccagtt 1080
ccatcccgag atatttggga tagggaatgt aagtcaagtt tatgttgaca tcaagcacca 1140
actcatctgt gctgttcacg acggggataa caccaccttg cctcgccatg acaatatatc 1200
agccgtattt cagacctact accaacacca ggtcgacggc ggtaattggt ttcacctgga 1260
atggctgcgc cctttctttt cctcttggtt ggtgctaaat gtttcgtggg ttctcaggcg 1320
ttcgcctgca agccgtgttt cagttcgagt ctttcagaca tcaaaaccaa caccaccgcg 1380
gcatcaggct ttgttgtcct ccaggacatc agctgcctta ggcatggcga ctcgtcctct 1440
ccgacgattc gcaaaaattc tcagtgccgc acggcgatag ggacgccagt gtacattacc 1500
atcactgcca atgtcacaga tgagaattat ctacattctt ctgatctcct catgctttct 1560
tcttgccttt tctatgcttc cgagatgagt gaaaagggat tcaaagtggt gtttggcaat 1620
gtgtcaggca tcgtggctgt gtgcatcaac tttaccagct acgttcaaca tgtcaaggag 1680
tttacccaac gctccctagt ggtcgatcat gtgcgcctgc ttcatttcat gacacctgag 1740
accatgaggt gggcaaccgt cttagcctgt ctttttgcta tcctactggc aatttgaatg 1800
ttcaagtatg ttggggaagt gcttgaccgc gtgctgttgc tcgcgattgc tttttttgtg 1860
gtgtatcgtg ccgttctatc ttgctgtgct cgtcaacgcc agcaacaaca acagctctca 1920
tattcagttg atttataact taacgctatg tgagctaaat ggcacggatt ggctggcaca 1980
aaaatttgac tgggcagtgg agacttttgt catcttcccc gtgttgactc acattgtttc 2040
ctatggggca ctcaccacca gccacttcct tgacacagtt ggcctggccg ctgtgtccac 2100
cgccggatat tatcacgagc ggtatgtctt gagtagcatt tacgcggtct gtgctctggc 2160
tgcgctgatt tgctttgtca ctaggcttgc gaagaactgc atgtcctggc gctactcttg 2220
taccagatat accaacttcc ttctggacac taagggcaga ctttatcgtt ggcggtcacc 2280
cgtcattgtg gagaaagggg gtaaggtcga ggtcgaaggt cacctgatcg acctcaagag 2340
agttgtgctt gatggttctg cggcaactcc tttaaccaga gtttcagcgg aacaatggag 2400
tcgtctctag acgacttctg caatgatagc aaagctccac aaaaggtgct tttggcgttt 2460
tccattacct acacgccagt aatgatatat gctctaaagg taagtcgcgg ccgactgtta 2520
gggcttctgc accttttgat ctttctgaat tgtgctttca ccttcgggta catgacatcc 2580
gtgcactttc atagcacaaa tagggtcgcg ctcactatgg gagcagtagt tgcacttctt 2640
tggggagtgt actcagccat agaaacctgg aaattcatca cctccaggtg ccggttgtgc 2700
ttgctaggcc gcaagtacat tctggcccct gcccaccacg tcgaaagtgc cgcgggcttt 2760
catccgattg cggcaaatga taaccacgcg tttgtcgtcc ggcgt 2805
Claims (9)
1.一种表达盖他病毒E2蛋白的重组猪繁殖与呼吸综合征病毒,其特征在于该病毒中在ORF1b和ORF2a间插入猪盖他病毒E2蛋白基因。
2.权利要求1所述表达盖他病毒E2蛋白的重组猪繁殖与呼吸综合征病毒的构建方法,其特征在于:将猪繁殖与呼吸综合征病毒rGXAM作为亲本毒株,利用基因工程的方法,在ORF1b和ORF2a间插入猪盖他病毒E2蛋白基因,获得重组PRRSV病毒rGX-12BS-GE2。
3.根据权利要求2所述的构建方法,其特征在于:所述亲本毒株为高致病性PRRSV传代致弱毒株GXNN1396-P96;所述猪盖他病毒E2蛋白基因源于猪盖他病毒GETV-GX201808。
4.根据权利要求2所述的构建方法,其特征在于所述基因工程的方法是:在PRRSV感染性克隆中,利用SOE-PCR方法在ORF1b和ORF2a间引入BstBI和Sbf I两个限制性酶切位点以及转录调控序列TRS,在此基础上,通过人为引入的酶切位点BstBI和SbfI将GETV E2蛋白基因插入pGX-12BS-TRS中,得到重组质粒pGX-12BS-GE2,再经病毒拯救获得重组PRRSV病毒rGX-12BS-GE2。
5.根据权利要求3所述的构建方法,其特征在于:所述转录调控序列TRS具有序列表SEQ.NO.ID.1的碱基序列,所述重组质粒pGX-12BS-GE2全长序列为序列表SEQ.NO.ID.2的碱基序列。
6.根据权利要求4所述的构建方法,其特征在于所述重组质粒pGX-12BS-GE2按以下操作进行制备:以PRRSV的全长感染性克隆pGXAM为模板,使用突变引物GF11706-1F/12BS-1R和12BS-TRS-2F/JX14402Mlu I-2R分别扩增出包含有BstBI和SbfI限制性酶切位点及TRS的PCR产物1与PCR产物2,以此两段PCR产物为模板,使用外侧引物GF11706-1F和JX14402MluI-2R进行第二轮融合PCR扩增,将融合PCR产物使用AscI和Mlu I酶切,同时酶切pGXAM,将含有酶切位点和TRS的酶切片段通过T4 DNA连接酶连至线性化的pGXAM中,获得重组质粒pGX-12BS-TRS;使用GETV E2基因的扩增引物GE2-BstBI-F和GE2-SbfI-R扩增GETV E2基因,然后使用BstBI和SbfI同时酶切PCR产物和pGX-12BS-TRS,将GETV E2基因通过T4 DNA连接酶克隆至pGX-12BS-TRS中,即得重组质粒pGX-12BS-GE2。
7.根据权利要求4所述的构建方法,其特征在于:所述突变引物GF11706-1F/12BS-1R和12BS-TRS-2F/JX14402Mlu I-2R、扩增引物GE2-BstBI-F和GE2-SbfI-R分别具有序列序列表SEQ.NO.ID.3至SEQ.NO.ID.8的碱基序列。
8.权利要求3至6所述构建方法得到的重组质粒。
9.权利要求1所述表达盖他病毒E2蛋白的重组猪繁殖与呼吸综合征病毒和权利要7所述重组质粒在制备基因工程疫苗中的应用。
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Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2007033436A1 (en) * | 2005-09-26 | 2007-03-29 | The University Of Queensland | Modulation of gene expression and agents useful for same |
CN104561092A (zh) * | 2014-12-19 | 2015-04-29 | 中国农业科学院上海兽医研究所 | 表达猪瘟病毒e2蛋白重组prrs病毒基因工程疫苗的构建方法和应用 |
CN112458064A (zh) * | 2020-11-20 | 2021-03-09 | 广西大学 | 盖他病毒全长感染性克隆、复制子系统及其制备和应用 |
CN113215192A (zh) * | 2020-02-06 | 2021-08-06 | 广西大学 | 猪繁殖与呼吸综合征病毒双荧光标记基因重组毒株的构建方法 |
-
2022
- 2022-04-08 CN CN202210367608.2A patent/CN114940978B/zh active Active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2007033436A1 (en) * | 2005-09-26 | 2007-03-29 | The University Of Queensland | Modulation of gene expression and agents useful for same |
CN104561092A (zh) * | 2014-12-19 | 2015-04-29 | 中国农业科学院上海兽医研究所 | 表达猪瘟病毒e2蛋白重组prrs病毒基因工程疫苗的构建方法和应用 |
CN113215192A (zh) * | 2020-02-06 | 2021-08-06 | 广西大学 | 猪繁殖与呼吸综合征病毒双荧光标记基因重组毒株的构建方法 |
CN112458064A (zh) * | 2020-11-20 | 2021-03-09 | 广西大学 | 盖他病毒全长感染性克隆、复制子系统及其制备和应用 |
Non-Patent Citations (4)
Title |
---|
HAIHONG ZHENG等: "Recombinant PRRSV expressing porcine circovirus sequence reveals novel aspect of transcriptional control of porcine arterivirus", 《VIRUS RESEARCH》 * |
HAO WANG等: "Generation of a Recombinant Porcine Reproductive and Respiratory Syndrome Virus Stably Expressing Two Marker Genes", 《FRONTIERS IN VETERINARY SCIENCE》 * |
YUXU WANG等: "Generation of a porcine reproductive and respiratory syndrome virus expressing a marker gene inserted between ORF4 and ORF5a", 《ARCHIVES OF VIROLOGY》 * |
李清清等: "以PRRSV为载体表达外源基因的研究进展", 《中国动物传染病学报》 * |
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