【作者】 王海燕；

【导师】 高崧； 刘秀梵；

【作者基本信息】 扬州大学 ， 预防兽医学， 2008， 博士

【摘要】 猪圆环病毒（porcine circovirus,PCV）属于圆环病毒科,为单股环状DNA病毒。分为1型（porcine circovirus type 1,PCV1）和2型（porcinecireovirus type 2,PCV2）,其中PCV1不致病,PCV2具有致病性,能引起猪的多种病症,如断奶仔猪多系统衰竭综合征（post-weaning multisystemicwasting syndrome,PMWS）、猪皮炎与肾病综合征（porcine dermatitis andnephropathy syndrome,PDNS）及繁殖障碍等。自加拿大1991年首次报道发生PMWS以来,现在全世界大多数养猪国家均有该病发生。PCV2感染能造成免疫抑制,易继发或并发其它病原体感染,对养猪业造成很大的经济损失。因此,寻找一些新的方法防制该病已成为一项紧迫任务。对该病的防制,一方面在于研制新型疫苗,如DNA疫苗和亚单位苗;另一方面,一些新技术的应用,如RNA干扰（RNA interference,RNAi）技术。RNA干扰是近年来发展起来的一种新技术,可通过对特定基因转录出的mRNA进行降解,对病原体的复制产生抑制,从而防止疾病的发生。本研究首先从临床上疑为PMWS的病例中分离到了10株PCV2,测定其全基因组序列,并对基因序列进行了遗传分析,获得了基因组的变异信息。对一株PCV2参考毒株的开放阅读框2进行了克隆,鉴定后构建了表达PCV2衣壳蛋白（capsid,Cap）的重组杆状病毒,对其表达的Cap蛋白进行了免疫生物学特性研究,同时进行了杆状病毒表达的Cap蛋白对小鼠的免疫保护试验。对测定的和网上登录的PCV基因组序列进行了同源性分析,应用相关软件和网上资源设计了6个PCV特异性小干扰RNA（small interference RNA,siRNA）片段,构建了siRNA表达质粒,鉴定出了相应的插入序列正确的克隆,对PCV1和PCV2毒株在Dulac细胞及PCV2毒株在小鼠体内复制的抑制作用进行了初步研究。1.江苏部分地区10株猪圆环病毒2型分离株的分子流行病学从PMWS病例中分离到10株PCV2,对其全基因组序列进行了测定和分析,并将10株PCV2毒株基因组序列提交到GenBank。分析结果表明,分离到的10株PCV2全长均为1767个核苷酸,分属于3个不同的基因亚群,其中苏州0511毒株在一个较远的分支上,扬州0705与其它8个毒株分布在不同分支上,遗传上有一定差异,但总体上遗传关系较近,基因组同源性在98.3-99.5%之间,同处于PCV2基因1群中。其中一株分离自家养野猪的PCV2和其它7株遗传关系非常近,可能是同一个起源。这些分离株的cap基因变异程度不大,可作为亚单位苗或DNA疫苗的良好候选基因。2.表达猪圆环病毒2型衣壳蛋白的重组杆状病毒对小鼠的免疫保护作用通过PCR方法扩增出PCV2 XSC株cap基因,克隆到pGEM-T^?easy载体,测序验证后亚克隆到转座载体pFastBac^TM1,经再次测序验证后将含有PCV2衣壳蛋白基因的表达盒转座到穿梭载体Bacmid中。将获得的重组质粒Bacmid-Cap转染Sf9细胞,获得了重组杆状病毒,病毒扩增后重组杆状病毒的滴度达到5×10⁸pfu/ml,以10⁵pfu的reBac-Cap感染Sf9细胞,经SDS-PAGE和Western blot证实Cap蛋白已表达,分子量约28kDa,与天然衣壳蛋白分子量大小一致。SDS-PAGE证实,PCV2 Cap蛋白非分泌性、可溶性地表达于Sf9细胞胞浆内。取感染reBac-Cap的Sf9细胞进行免疫荧光试验,能与猪抗PCV2高免血清发生特异性反应,显示重组核衣壳蛋白具有良好的抗原性。重组杆状病毒免疫小鼠,应用间接免疫荧光方法对接种后7d、14d、21d、28d、35d、42d、49d、56d的小鼠进行血清抗体检测。结果显示从接种后21d开始小鼠血清产生了针对PCV2 Cap蛋白的特异性抗体,攻毒后不同时间应用建立的TaqMan探针定量PCR方法检测血清中PCV2病毒拷贝数,结果发现表达PCV2 Cap的重组杆状病毒免疫后对病毒在小鼠体内的复制具有一定的抑制作用。3.质粒表达的siRNA片段对猪圆环病毒在Dulac细胞中复制的抑制作用在对PCV2基因组进行分析的基础上设计了siRNA片段,合成后构建了表达载体。对GenBank中登录和实验室测定的PCV基因组序列进行分析,将PCV毒株基因保守区序列提交到Ambion公司提供的siRNA查找工具,产生候选siRNA片段,将候选siRNA片段在internet上进行同源比对,将与其它物种同源性高的siRNA片段剔除。结合已发表文献中的siRNA片段序列,最终确定了6个siRNA片段,其中4个是针对猪圆环病毒rep基因的,命名为SH1（S50-68）、SH2（S554-572）、SH3（S653-671）、SH4（S624-642）;2个针对PCV2 cap基因,命名为SH5（R1429-1411）、SH6（R1307-1289）。根据所用表达载体RNAi-Ready pSIREN-RetroQ ZsGreen的要求设计了6对67个碱基的DNA片段,该序列可在转染细胞中产生发夹结构RNA（shorthairpin RNA,shRNA）分子。人工合成相应DNA片段,退火后形成双链,与载体试剂盒中的针对萤光素酶（Luciferase,Luc）的67个碱基的siRNA相应DNA片段及阴性对照片段（46个碱基）分别连接到载体鼠源U6启动子下游,转化大肠杆菌DH5α,挑取阳性克隆,测序鉴定,得到了正确的克隆,分别命名为Retro-SH1、Retro-SH2、Retro-SH3、Retro-SH4、Retro-SH5、Retro-SH6、Retro-Luc和Retro-NC。首先对siRNA表达质粒Retro-SH1、Retro-SH2、Retro-SH3、Retro-SH4、Retro-SH5、Retro-SH6的抑制作用进行检测,筛选出抑制作用较强的siRNA。重悬后的对数生长期Dulac细胞0.5ml/孔（2.0×10⁵个细胞）置于24孔细胞板中进行培养,6h后加入梭华-Sofast^TM基因转染试剂及0.6μg的siRNA表达质粒Retro-SH1、Retro-SH2、Retro-SH3、Retro-SH4、Retro-SH5、Retro-SH6、Retro-Luc和对照质粒复合物,12h、24h后分别进行荧光观察。24h后每孔加入0.1ml含有10⁵ TCID₅₀的PCV2 XSC株病毒液感染上述细胞,感染后72h收集上清和细胞,应用TaqMan探针Real-time PCR法对PCV2病毒拷贝数进行检测。结果发现,细胞在转染上述质粒后12h开始出现绿色荧光,24h后荧光较强。观察到荧光表明转染成功,siRNA片段可能已经表达。siRNA表达质粒Retro-SH1,Retro-SH4,Retro-SH6转染细胞后病毒基因拷贝数与未转染质粒细胞组和转染其它质粒对照组差异显著,说明Retro-SH1,Retro-SH4,Retro-SH6表达的siRNA片段作用较强。用筛选出的3个对病毒复制抑制作用较强的siRNA表达质粒进行以下试验。转染质粒后不同时间对后续接种的病毒复制的干扰作用进行了比较,转染不同质粒及三种质粒的混合体24h后加0.1ml含10⁵TCID₅₀的PCV2 XSC参考株感染细胞,分别于感染后24h、36h、48h、60h、72h、84h、96h、120h收集上清和细胞,应用TaqMan探针real-time PCR法对PCV2病毒拷贝数进行检测。结果发现,感染60h后载体表达的特异性siRNA片段对PCV2病毒的复制表现出很强的抑制作用,可以持续到96h以后,不同的siRNA片段抑制作用有一定差异,其中表达质粒Retro-SH4的干扰作用最强,而3个siRNA表达质粒混合体对PCV2病毒的干扰作用与单独作用最强的处理组的干扰作用没有明显差异,并未显示出协同作用。对不同浓度的质粒产生的干扰作用进行了比较,转染50ng、100ng,500ng、1000ng、1500ng、2500ng表达siRNA质粒的Dulac细胞在感染PCV2后60h收集上清和细胞,检测其病毒在细胞内的拷贝数,发现不同浓度的表达质粒对PCV2 XSC株的复制均有抑制作用,且500ng-2500ng浓度的质粒其干扰作用无明显差别。综合考虑质粒对细胞的干扰强度相当且节省质粒等原则,随后在细胞中的抑制试验均选用500ng的质粒。应用流式细胞术对500ng的Retro-SH1,Retro-SH4,Retro-SH6表达的siRNA片段对病毒复制的抑制作用进行了分析,结果表明三个质粒表达的siRNA对PCV2的复制均有一定的抑制作用,其中Retro-SH4作用最强。Dulae细胞感染PCV2 XSC株后12h、24h、36h、48h和60h分别转染表达siRNAs的质粒,运用定量PCR方法进行检测,发现细胞感染12h后转染表达PCV2特异的siRNAs质粒对PCV2毒株的复制有很强的抑制作用,细胞感染PCV2 36h后再转染质粒其抑制作用降低,而混合质粒对PCV2病毒的干扰与单独抑制能力最强的质粒的作用没有明显差异,并未显示协同作用,Retro-Luc和对照质粒组则没有抑制作用。以上结果说明,细胞感染PCV2后,再转染表达的特异性siRNA的质粒能有效抑制病毒的繁殖,但以细胞感染PCV2 12h内即转染干扰质粒其抑制效果最好。siRNA对临床分离毒株在Dulac细胞上复制的抑制作用进行了比较试验,500ng siRNA表达质粒Retro-SH1,Retro-SH4,Retro-SH6分别转染Dulac细胞,置于24孔细胞板上培养,24h后加入10⁵个TCID₅₀的不同PCV2分离株的细胞培养物,结果表明,500ng的质粒Retro-SH1,Retro-SH4,Retro-SH6对不同PCV2分离株在细胞中的复制均有一定抑制作用,不同的siRNA分子作用有差异,但不同毒株之间差异不大。500ng siRNA表达质粒Retro-SH1,Retro-SH4,Retro-SH6分别转染Dulac细胞,培养24h后加入10⁵个TCID₅₀的PCV1毒株的细胞培养物,结果表明,Retro-SH1,Retro-SH4对PCV1复制有很强的抑制作用,而Retro-SH6对PCV1在细胞中的复制没有明显抑制作用。4.表达siRNA的质粒对PCV2在小鼠体内复制的抑制作用将72只8周龄BALB/c小鼠随机分为12组,每组6只,第1-4组每只分别肌肉注射10μg表达的siRNA质粒Retro-SH1,Retro-SH4,Retro-SH6,Retro-NC,第5组肌肉注射10μg Retro-SH1,Retro-SH4和Retro-SH6混合质粒。24h后每只按10⁵TCID₅₀的量口服及腹腔注射0.1ml的PCV2病毒。第6-10组先用10⁵TCID₅₀ PCV2攻毒,24h后每只分别肌肉注射10μg表达的siRNA质粒Retro-SH1,Retro-SH4,Retro-SH6或它们的混合体及Retro-NC。第11组为攻毒对照组,第12组为健康对照组。分别于感染后第5天及第11天进行扑杀采样,每次采集3只小鼠血清和脾脏,应用real-timePCR方法检测血清中PCV2病毒拷贝数,通过免疫组化和组织学方法检测组织中病毒抗原与病理损伤。结果表明10μg siRNA表达质粒对PCV2攻毒的小鼠有很好的保护力,但不同的siRNA分子保护力有差异。3个siRNA表达质粒混合体与单独作用最强的质粒组的保护力没有明显差异,并未显示协同作用。而对已感染PCV2的BALB/c小鼠进行治疗时发现,载体表达的PCV2特异性的siRNA分子对小鼠也有一定的保护作用。综上所述,本研究从临床上出现PMWS症状的病猪中分离出10株PCV2,测定了它们的基因组序列,遗传发生分析结果表明,10个毒株分属于3个遗传分支,但总体上遗传距离很近,核苷酸的同源性在98.9%以上,尤其是cap基因很保守。通过PCR扩增出了cap基因,构建了表达该基因的重组杆状病毒,动物实验证明,重组病毒免疫后能够抑制PCV2在小鼠体内的复制。设计出了6个针对猪圆环病毒的siRNA片段,构建了shRNA分子的质粒表达载体,筛选出了3个作用较强的siRNA片段（Retro-SH1,Retro-SH4,Retro-SH6）,证实了这3个表达的siRNA片段在Dulac细胞和小鼠体内都能够对PCV2的复制产生抑制作用,其中2个在Dulac细胞中对PCV1也有抑制作用,以上siRNA表达质粒可能成为控制猪圆环病毒2型感染的新工具之一。更多还原

【Abstract】 Porcine circovirus type 2（PCV2） infection has been associated with post-weaning multisystemic wasting syndrome（PMWS）,porcine dermatitis and nephropathy syndrome（PDNS）,porcine respiratory disease complex and reproductive disorders.Since the first case was reported in Canada in 1991, PCV2 infections have been confirmed in many countries of the world.The pigs infected with PCV2 were in an immunosuppressive status which could trigger co-infection with other pathogens.It is not an easy task to get the epidemics under control or eradication because of wide spread of the virus.It is necessary to explore some novel approaches for better prevention and control of the disease. Within the scope of the development of new vaccines against PCV2 infection, subunit vaccines based on the baculovirus expression system should be a putative prophylactic tool.RNA interference（RNAi） is a recently developed technology in which double-stranded RNA（dsRNA） directs sequence-specific degradation of messenger RNA（mRNA） and serves as a potential therapeutic strategy for the inhibition of virulence-associated genes of some pathogens.In this study,10 isolates of PCV2 were obtained from the clinical cases infected with PWMS in Jiangsu Province.Complete genomes of these PCV2 strains were sequenced and analyzed.The cap gene of PCV2 strain XSC was expressed by the recombinant baculovirus reBac-Cap and its immunogenicity was evaluated in mice.Several small interference RNAs（siRNAs） specific to porcine circovirus type 2 were designed,synthesized and expressed by the plasmid vector. Their inhibition effect on the replication of PCV1 and PCV2 by these siRNAs was evaluated in both Dulac cells and mice.1.Molecular characteristics of ten isolates of PCV2 from Jiangsu ProvinceTen isolates of PCV2 were obtained from the clinical cases infected with PMWS by passaging in Dulac cells.Complete genomes of these PCV2 strains were amplified by over-lapping PCR and sequenced directly.The whole genomic sequences of all these isolates were spliced successfully and all of them were 1767 nucleotides long,which have been deposited in GenBank.Sequence analysis showed that these PCV2 isolates exhibited high level of nucleotide similarity and were classified into group 1 of PCV2 based on their genomic sequences.The phylogenetic tree analysis and multiple alignments of nucleotide sequences present that the isolate Suzhou 0511 and Yangzhou 0705 were in other cluster departed from other 8 isolates.All genomics of these isolates exhibited 98.3-99.5%nucleotide sequence identities and their cap genes were also in higher similarity.2.The immunogenicity of the PCV2 capsid protein expressed by the recombinant baculovirusThe capsid protein gene of PCV2 strain XSC was amplified by PCR and cloned into pGEM-T（?） easy vector,and subcloned into pFastBac^TM1 donor plasmid,then inserted into baculovirus shuttle vector Bacmid by site-specific transposition.The recombinant baculovirus,designated as reBac-Cap,was constructed when the recombinant shuttle vector Bacmid-Cap was transfected into Sf9 cells.To evaluate the expression level of capsid protein gene,a viral stock with a high-titer up to 5×10⁸ pfu/ml was obtained and determined by the plaque assay.Infected Sf9 cells with 10⁵ pfu reBac-Cap were harvested at 72 h post infection,the expressed capsid protein with molecular weight of 28 kDa,the same size as the natural capsid protein,was identified to be expressed in cytoplasm in soluble,non-secreted form.Furthermore,the expression product was observed to react with the pig antisera specific to PCV2 virus.Sera samples collected from all control and vaccinated animals at 0,7,14,21,28,35,42 49,56 day post-innoculation（dpi） were assayed for anti-PCV2 capsid antibodies by IFA.The results indicated that the recombinant baculovirus reBac-Cap induced specific antibodies against the pathogenic PCV2 capsid antigen from 21dpi.The sera were detected by real-time PCR.The results indicated that the recombinant baculovirus reBac-Cap was experienced the highest replication inhibition rate of PCV2.3.Inhibition of the replication of the porcine circovirus type 2 in the Dulac cells by the siRNAs expressed by the plasmidsSequences of PCV capsid protein genes and replicase genes determined in this study or from the GenBank were submitted to the website of Ambion company for target sequences searching,and some candidate siRNA sequences were generated.The candidate siRNA sequences were blasted,those shared any identity with other known organism genes were exclued.Six siRNAs were chosen to be evaluated for their selected inhibition effect on the replication of PCVs,four of these were specific to the rep gene of PCVs,designated as SH1（S50-68）、SH2（S554-572）、SH3（S653-671）、SH4（S624-642）,and the others were specific to the cap gene of PCV2,designated as SH5（R1429-1411） and SH6（R1307-1289）. Six DNA fragments with 67 nucleotides producing short hairpin RNA（shRNA） were designed based on the plasmid vector.The corresponding DNA fragments were synthesized,annealed and ligated to the downstream of the mouse originated U6 promoter of the RNAi-Ready pSIREN-RetroQ ZsGreen vector.As controls, the siRNAs specific to Luciferase gene within the vector and negative fragments for any organisms were also included.Recombinant plasmids were transformed into the host bacteria DH5αand positive clones were selected.Positive clones carried the expected insertions were identified by sequencing and designated as Retro-SH1、Retro-SH2、Retro-SH3、Retro-SH4、Retro-SH5、Retro-SH6、Retro-Luc and Retro-NC,respectively.To test whether siRNAs expressed by these six plasmids inhibit the PCV2 replication,we first examined their effect on the replication of the PCV2 virus in the Dulac cells.The plamids were introduced into the Dulac cells using Sofast^TM transfection reagent.Each samples had triplication.As controls,Retro-Luc and Retro-NC were similarly introduced into the same cells,followed by PCV2 virus infection.The cells were collected at 72 h after infection.Virus loads was detected by real-time PCR assay established in our lab.The results showed that virus loads strongly reduced in cells transfected by Retro-SH1,Retro-SH4 and Retro-SH6,while did not reduce in the cells transfected by other plasmids and the controls.These three siRNAs expressed plasmids were further evaluated in the following experiments.The kinetic effect of siRNAs expressed by plasmids on PCV2 replication in Dulac cells was determined according to the method mentioned above.Three kinds of siRNA expressing plasmids and their mixture were transfected into the cells,and then infected by PCV2 at 24 h after transfection.Collected cell cultures at 24 h,36 h,48 h,60 h,72 h,96 h,120 h after infection were determined for the copies of genomic DNA of PCV2 by real-time PCR.The results showed that there was strong inhibition effect on PCV2 replication from 60 h post its infection,and the effect lasted at least 48 h.As the dose of plasmids transfected,a positive dose-dependent effect was confirmed.500 ng of each plasmid was optimal.The inhibition effect of the Retro-SH4 was the strongest among three plasmids,and no synergism was observed in the cells transfected by the mixture of the three plasmids.The results were also confirmed by the FACS technique.When Dulac cells were infected with PCV2 XSC strain,then each of three plasmids and their mixture were transfected into above cells at different intervals.The results indicated that the siRNAs expressing plasmids should be tranfected as early as possible in order to get strong inhibitions.Transfection of the plasmids at 36 h after virus infection could not inhibit the ongoing replication of the PCV2 effectively.For ten of PCV2 field isolates,the siRNAs expressed by Retro-SH1, Retro-SH4,Retro-SH6 could inhibit the replication of all isolates in Dulae cells. For PCV1,the siRNAs expressed by Retro-SH1,Retro-SH4 specific to the rep gene of PCV exhibited inhibition effect on the replication of PCV1 in Dulac cells while Retro-SH6 did not.4.Inhibition of the replication of the porcine circovirus type 2 in mice by the siRNAs expressed by the plasmidsSeventy-two of 8-week-old BALB/c mice were divided randomly into 12 groups,6 mice for each.Mice in four groups received 10μg of Retro-SH1, Retro-SH4,Retro-SH6 and Retro-NC,respectively,another group were intramuscularly injected with the mixture of Retro-SH1,Retro-SH4 and Retro-SH6,mice in these five groups were challenged by oral administration and intraperitoneal injection with 10⁵ TCID₅₀ of PCV2.Mice in other five groups were oral administration and intraperitoneal injected with 10⁵ TCID₅₀ of PCV2 and then intramuscularly introduced by Retro-SH1,Retro-SH4,Retro-SH6 or their mixture and Retro-NC at 24 h after PCV2 infection.One group was treated as challenge control and the other as a blank control group.All of the mice were slaughted for sera and tissues collection at 5 d and 11 d post challenge.The sera were detected by real-time PCR and the tissues were examined by histopathological and immunohistochemical techniques.Both of the prophylactic and therapy experiments showed that siRNAs expressed by Retro-SH1, Retro-SH4,Retro-SH6 could offer some protection against PCV2 challenge, Retro-SH4 inoculation group experienced the highest replication inhibition rate up to 99.8%.No synergism was observed in the group transfected by the mixture of the three plasmids.In summary,10 isolates of PCV2 were obtained from the clinical cases infected with PMWS in Jiangsu Province.Genomic sequences of these isolates were in high level of homology.The cap gene of PCV2 was expressed by the recombinant baculovirus reBac-Cap and its immunogenicity was evaluated in mice. The mice immunized with the recombinant baculovirus could elicit specific antibody against PCV2.Six of siRNAs specific for PCV2 genomic fragments were designed and the plasmid-based system for generating unimolecular,hairpin siRNAs were constructed successfully and three of them（Retro-SH1,Retro-SH4, Retro-SH6） were selected.These siRNAs expressed by the plasmids could inhibit the replication of the PCVs in the Dulac cells and provide some protection against PCV2 infection in mice.These data indicated that siRNA technique should be a potential useful tool for developing novel strategy to prevent and control PCV2 infection.更多还原