【作者】 孙爱军；

【导师】 崔治中；

【作者基本信息】 山东农业大学 ， 预防兽医学， 2009， 博士

【摘要】 马立克氏病病毒(Marek’s disease virus,MDV)是α类疱疹病毒,其基因组为174kb的双股DNA。对于未经免疫的鸡群,有很高的传染性和致肿瘤性。鸡在感染MDV后除了造成死亡外,耐过鸡可终身带毒并长期排毒。因而,在鸡场里和周围环境中该病毒长期存在。禽网状内皮增生症病毒(Reticuloendotheliosis,REV)是一类C型反转录病毒,基因组是8kb的单股正链RNA,其基因组结构类似于一个完整的转座子,这是鸡的另一种致肿瘤病毒。张志等(2004)等首次从患鸡的抗凝血中分离到整合进REV-LTR片段的天然MDV野毒株GX0101和GD0202,这表明在自然感染的条件下,REV除了能与MDV共感染同一只家禽外,还能整合到MDV的基因组中,本研究将进一步阐明MDV这种重组野毒株致病性是否发生变化,特别是这种重组作用是否会增强MDV的传染性或在体内的复制能力,外源片段的的插入对病毒的致病性,特别是其生物学特性产生了怎样的影响。在以上背景下,本研究比较了天然重组野毒株GX0101对鸡的致病性,并将其构建成感染性细菌人工染色体。在此基础上将插入片段REV-LTR敲除再进一步研究其生物学特性。同时,对MDV的两个致瘤基因1.8kb mRNA及ICP4基因进行了相关研究。不仅积累了大量的数据而且提出并验证了新的观点。1天然重组野毒株GX0101的致病性研究分别选用海蓝褐公鸡和SPF鸡进行GX0101毒株的致病性测定。1日龄免疫HVT疫苗后于5日龄分别接种野毒株GX0101及超强毒株rMd5,每天观察记录死亡并逐只剖检记录发生肿瘤情况。试验结果显示,GX0101株病毒对海蓝褐公鸡的生长抑制作用要比rMd5株弱。而且GX0101株病毒对已经有HVT疫苗免疫的海蓝褐公鸡的致死率只有44%要显著低于rMd5株的70%(P<0.01),而且它的致肿瘤率只有16%显著低于rMd5株的36%(P<0.05), GX0101株病毒对已免有HVT疫苗的SPF鸡的致死率只有28.24%要显著低于rMd5株的64.63%(P<0.01),而且它的致肿瘤率只有7.06%显著低于rMd5株的19.51%(P<0.05),该结果表明GX0101株病毒的致病性要低于强毒株rMd5株。张志(2004)和庄国庆(2006)分别在SPF鸡中做了人工感染试验,结果显示GX0101毒株的致病性要强于强毒株GA株。因此,GX0101株的致病性应是介于强毒GA与超强毒rMd5间的一株病毒。由此看来,GX0101的致病性并不是使其成为流行毒株的竞争优势。2带有REV-LTR片段的MDV野毒株GX0101的BAC感染性克隆的构建及其致病性比较2.1带有MDV基因组US2片段作为同源臂的转基因BAC载体的构建MDV的US2区两侧的同源臂用于同源重组时将BAC载体插入MDV基因组,其中2.1kb的同源臂来自质粒pDS-pHAI,4.0kb同源臂来自质粒pUS2-Partial-F,通过限制性内切酶HindⅢ和XhoⅠ的双酶切作用,及T4DNA Ligation连接,成功构建重组转基因载体质粒pDS-pHAI-US2。2.2 GX0101 BAC感染性克隆的构建及其拯救利用磷酸钙或脂质体转染技术,将转移载体pDSpHAI-US2与MDV GX0101感染CEF细胞的总DNA共感染鸡胚成纤维细胞。待出现细胞病变时,将其传至已加有霉酚酸-黄嘌呤-次黄嘌呤选择培养基的原代CEF上,经四轮筛选、富集重组病毒后,提取重组病毒感染CEF细胞的总DNA,电转化大肠杆菌DH10B。次日,挑取阳性克隆。经酶切及PCR鉴定后,提取BAC DNA转染CEF细胞,再次启动了病毒感染,产生了MDV特异性病毒蚀斑,拯救出了重组病毒;进一步研究表明重组病毒bac-GX0101与亲本毒GX0101在体外CEF单层上的生物学特性相似。2.3 GX0101感染性克隆的致病性比较将BAC克隆毒bac-GX0101与亲本毒GX0101接种1日龄SPF鸡,动物实验结果表明bac-GX0101毒株与亲本GX0101毒株对机体的生长性能等方面的影响没有差异,而且bac-GX0101毒株仍然保留着很好的免疫抑制功能及致肿瘤能力。该感染性克隆bac-GX0101为研究该重组野毒株中REV-LTR插入片段及其他基因的生物学功能提供了有用的技术平台。3敲除REV-LTR的感染性克隆的构建及其生物学特性比较3. 1 MDV天然重组野毒株GX0101中LTR序列的敲除MDV和REV间的自然重组的机率是很低的,在我们近几年分离到的十多个野毒株中,却有两个是重组病毒,说明这一重组过程一定有某种选择竞争性优势。为了阐明天然重组插入的REV-LTR究竟能给原始的MDV带来什么生物学特性变化,对基因组做相应的突变是必经的步骤。将GX0101构建成感染性细菌人工染色体,由于我们可以比较方便地对感染性克隆质粒GX0101-BAC基因组进行修饰,如定点敲除重组野毒株中某个基因或基因片段。这就可以用其作为研究该株MDV的特定基因或基因片段与致病性或其他生物学活性关系的一个新的技术平台。本研究利用Red/ET介导的突变技术,用卡那霉素抗性基因代替要敲除的LTR序列。设计引物,以质粒PKD13为模板,扩增卡那霉素抗性基因。这对引物3’端20个碱基来源于卡那霉素抗性基因,用来扩增该抗性基因,5’端50个碱基来源于MDV,用来同源重组,上下游引物5’端的这50个碱基来源于MDV基因组,分别位于要敲除的LTR序列的两端。卡那霉素抗性基因两端FRT位点是重组酶Flpe的识别位点。卡那霉素抗性基因取代LTR序列后,用0.1%的阿拉伯糖诱导表达Flpe重组酶,从而将卡那霉素抗性基因去除。利用脂质体转染技术,拯出重组病毒,命名为bac-GX0101△LTR。3.2整合进GX0101基因组中的REV-LTR序列的生物学特性研究在5日龄时,分别接种bac-GX0101及bac-GX0101△LTR于1日龄免疫HVT疫苗的SPF鸡,无论是bac-GX0101还是bac-GX0101△LTR,感染SPF鸡后,对生长性能和对NDV、AIV(H9-)灭活疫苗免疫后HI抗体反应均呈现出不同程度的抑制作用。相对而言,bac-GX0101△LTR对SPF鸡的致病作用低于bac-GX0101;斑点杂交检测羽毛囊中的MDV基因组DNA说明,与接种bac-GX0101毒株的同笼饲养的未攻毒鸡的MDV基因组的检出时间和接种bac-GX0101△LTR毒株的同笼饲养的未攻毒鸡的MDV基因组的检出时间相比要早一周,并且检出率也要高。接种bac-GX0101毒株和bac-GX0101△LTR毒株的鸡群在100天内的死亡率分别是28.13%和43.75%;出现肿瘤的比率是9.3%和12.5%。这充分说明REV-LTR的插入不是增强GX0101毒株的致病性和致肿瘤能力而是增强了GX0101毒株的横向传播能力,从而使GX0101毒株具有一定的竞争优势,成为流行毒株。4 MDV中1.8-kb mRNA基因的潜在阅读框A和C的敲除及其功能研究为了研究马立克氏病毒(MDV)1.8kb mRNA中潜在阅读框的功能,利用Red/ET技术将1.8kb mRNA中潜在阅读框(A+C)敲除,并成功转染出病毒bac-GX0101△(A+C),同时将bac-GX0101△(A+C)与bac-GX0101分别感染CEF后,转染包含MDV上游双向启动子的质粒pP(pp38)-CAT和pP(1.8-kb)-CAT,48h后,通过测定转染细胞裂解液中氯霉素乙酰转移酶(CAT)的活性确定MDV 1.8-kb mRNA中潜在阅读框对双向启动子活性的影响。结果显示,1.8kb mRNA中潜在阅读框(A+C)的缺失可以使其上游双向启动子两个方向的活性均显著下降(P<0.01)。本研究结果证明了1.8-kb mRNA对其上游双向启动子活性具有增强作用。将bac-GX0101△(A+C)和bac-GX0101毒株感染SPF鸡,动物试验结果表明,GX0101毒株缺失1.8kbmRNA后,对鸡群的增重影响较轻。且对NDV和AIV灭活疫苗免疫后HI抗体滴度均高于bac-GX0101感染组, bac-GX0101△(A+C)感染的鸡只肿瘤出现时间比bac-GX0101晚22d,说明仅仅敲掉开放阅读框A和C并不能消除其致瘤性。5 MDV中ICP4基因的敲除及其致病性比较本文利用Red/ET介导的突变技术,将ICP4基因敲除,拯救出重组病毒bac-GX0101△ICP4。分别将bac-GX0101△ICP4和bac-GX0101接种SPF鸡,动物试验结果表明,GX0101毒株缺失ICP4基因后,对NDV和AIV灭活疫苗免疫后HI抗体滴度低于对照组,且与bac-GX0101的差异不显著(P>0.05),且鸡群在感染bac-GX0101△ICP4毒株后58d检测到内脏肿瘤,说明ICP4基因并不直接参与肿瘤的形成。更多还原

【Abstract】 Marek’s disease virus (MDV) is a member of the Alphaherpesvirinae subfamily of the Herpesviridae, and the genome of MDV(174kb) is double-stranded DNA, MDV can induce highly infectiousness and oncogenicity on non-vaccine chickens. The chickens infected with MDV can cause to death and the survival birds can releasing virus long term, so the virus exsited permantly in the surrounding environment. Reticuloendotheliosis virus(REV) is a“C”retrovirus and the genome of REV (8kb)is single-stranded RNA, the structure of the genome is analogous to a integrity transposon. REV is another tumor virus of chicken.Zhang et al(2004) first isolated the MDV strain integrated with REV-LTR from anticoagulated blood of tumor chickens naturally infected with MDV, named GX0101,GD0202, which demonstrated besides it can infected the same poultry with MDV simultaneously, REV can integrated into the MDV genome. This study further clarified the pathogenicity of the MDV recombination virus, particularly if the transmissibility or replication capacity in vivo can be enhanced due to the recombination. Based on above backgrounds, we Comparatived pathogenicity of the recombinant strain GX0101 and cloned it as an infectious bacterial artificial chromosome, on the base of it, we deleted the REV-LTR insertion to study the bionomics. Simultaneous, correlated investagation were carried out on two tumorigenesis gene (1.8kb mRNA、ICP4). Not only accumulated a large amount of data and make and verify a new point of view.1 To determination virulence of recombination field strain GX0101 on different species chickensThe determination pathogenicity of recombination field strain GX0101 were carried on Hy-line brown layer and SPF chickens. At 1 day of age these birds were vaccined with HVT and at 5 days of age these chickens were infected with either field strain GX0101 virus or vv vorulent rMd5 virus. The chickens were inspected daily and all the chickens that died during the experiment were evaluated for gross and histological lesions by necropsy. Experimental results show rMd5 infection more strongly inhibited growth rates of infected Hy-line brown layer than GX0101. There were 44.0% and 70.0% of mortality in Hy-line brown layer groups inoculated with GX0101 and rMd5(P<0.01). Also, 16.0% and 36.0% of birds demonstrated tumors in different tissues or organs in groups infected with GX0101 and rMd5(P<0.05). Both the mortality and oncogenicity of GX0101 strain were lower than the vvMDV strain rMd5. There were 28.24% and 64.63% of mortality in SPF chickens groups inoculated with GX0101 and rMd5(P<0.01). Also, 7.06% and 19.51% of SPF chickens demonstrated tumors in different tissues or organs in groups infected with GX0101 and rMd5(P<0.05). This thoroughly demonstrated that the virulence of GX0101 strain is lower than rMd5 strain. Zhang(2004) and Zhuang(2006) carried out artificial challenge experiment on SPF chickens, found that virulence of GX0101 strain is higher than GA strain. So the pathogenicity of GX0101 is between GA and rMd5. In view of this, the pathogenicity of GX0101 is not the competitive advantage to make it become a popular strain.2 Cloning of Marek’s Disease Virus field strain With REV-LTR integration as an infectious BAC Clone and the Comparasion of pathogenicity2.1 Construction of Transfer Plasmid pDS-pHAI-US2For constructing the BAC transfer vector, two fragments (2.1 kb and 4.0 kb) on either side of the MDV US2 gene were used as the left- and right-sided homology arms. The left 2.1 kb homology arm was obtained from the plasmid pDS-pHAI by digesting with HindⅢand XhoⅠ. The right 4.0 kb homology arms came from plasmid pUS2-Partial-F by digesting with HindⅢand XhoⅠ. The transfer plasmid pDS-pHAI-US2 was constructed by ligating the two fragments.2.2 Cloning of GX0101 as an infectious BAC Clone and rescuing the recombinant virusTransfer vector pDSpHAI-US2 and GX0101 Total-DNA were cotransfected into CEF by LipfectamineTM2000 or calcium phosphate. Virus-containing cells were passaged four rounds in selection medium containing xanthine﹑hypoxanthine and mycophenolic acid to obtain puried recombinant viruses when the cells showed the CPE. Recombinant viral genomes was extracted and electroporated into E.coli DH10B. The second days, single colonies were picked and verified by digested with BamHI restriction enzyme or PCR ,recombinant virus were reconstituted from GX0101 BAC successfully. Further experiments indicated BAC-derived viruses had similar growth kinetics on CEF to the parental virus GX0101 in vitro.2.3 Comparison of the Pathogenicity of the GX0101 infectious BAC CloneAnimal experiments were carried out in 1-day-old specific-pathogen-free chicken.Further experiments indicated that there was no difference in growth ability and pathogenicity to chickens between the BAC derived virus and its parental virus. The BAC derived virus also maintained its oncogenicity and immunosuppressive effects. With the powerful BAC manipulation system, the infectious clone will provide a useful tool for further understanding the functional roles of the inserted REV-LTR sequence in the GX0101 strain of MDV.3 The Construction of infectious cloning of deleting REV-LTR and Comparisons of biological properties3.1 Deletion REV-LTR inserts from recombinant MDV strain GX0101In order to illustrate the change of biological properties caused by REV-LTR, gene mutation is a must procedure. Cloned GX0101 as infectious bacterial artificial chromosome. All the manipulations including DNA fragment deletion or insertion and point mutations can be carried out accurately in E. coli conveniently, and the mutated DNA can be used directly to reconstitute mutant viruses in transfected host cells. It would accelerate the understanding of the MDV gene functions and the relationships with pathogenesis and other biology properties.In this study, we constructed an identical REV-LTR deletion mutant of GX0101 BAC by Red/ET mutagenesis (Yu et al., 2000; Lee et al., 2001) .Using KanR cassette to instead of the REV-LTR inserts. KanR cassette flanked by FRT sites was amplified using primers LTR-kanaR-a and LTR-kanaR-b from the plasmid pKD13. KanaR selectable marker can be removed by using flanking Flpe target recognition sites(FRT). The recombinant virus was successfully rescued by transfection of the recombinant BAC DNA into primary chicken embryo fibroblast (CEF) by LipfectamineTM2000,named bac-GX0101△LTR.3.2 Characterization of Marek’s Disease Virus GX0101 that lack REV-LTR inserts: REV-LTR inserts can increased horizontal transmission ability of MDVOn days 5 of age, SPF chickens vaccined HVT on day 1 of age were infected intra-abdominally with 1000 p.f.u. either bac-GX0101△LTR virus or bac-GX0101 virus. When SPF chickens were infected with bac-GX0101△LTR virus or bac-GX0101 virus, HI antibody titers against NDV, H5-AIV or H9-AIV after vaccination or growth ability were decreased at different levels compared to the control,relatively speaking, the pathogenicity of bac-GX0101△LTR was less than bac-GX0101.The result of detected MDV genome from feather tips by Dot-blot demonstrated, the time of detection MDV genome from non-inoculated chickens breeded with chickens inoculated bac-GX0101 in one cage was one week earlier than the time of detection MDV genome from non-inoculated chickens breeded with chickens inoculated bac-GX0101△LTR in one cage, and also with a higher detection ration. During 100 days after challenged at 1d of age with two MDVs, there were 28.13% and 43.75% of mortality in groups inoculated with bac-GX0101 and bac-GX0101△LTR respectively while no mortality in the control group. Also, 9.3% and 12.5% of chickens demonstrated tumors in different tissues or organs in groups infected with GX0101 and bac-GX0101.The result of animal experiment demonstrated the insertion of REV-LTR enhanced the horizontal transmission of GX0101 strain but not the virulence and the oncogenicity which made the GX0101 strain have some advantages in competitive selection and become an epidemic strain.4 The Deletion ORF A and C of MDV 1.8kbmRNA and Study of gene functionIn order to study the ORF’s function of 1.8kbmRNA, we deleted the ORF(A+C) by Red/ET, and got the recovered virus bac-GX0101△(A+C) successfully.`Plasmid pP(pp38)-CAT and pP(1.8-kb)-CAT contained MDV upstream’s bidirectional promoter were transfected after bac-GX0101△(A+C) and bac-GX0101 infected CEF respectly.48 hours later, in order to ascertain the influence to bidirectional promoter active of MDV 1.8-kb mRNA’s ORF, we measured the CAT active in CEF lysate. The result demonstrated that the active of bidirectional promoter from two direction backdown significantly (P<0.01)because of the deleting of ORF A and C. The result certificated that 1.8kb mRNA could enhance the active of bidirectional promoter.SPF chickens were infected with bac-GX0101△(A+C) and bac-GX0101 strain respectively. The result of animal experiment demonstrated that the influence to weight was slight when deleteing ORF A and C of 1.8kbmRNA and the HI titre to NDV and AIV was higher than the group infected bac-GX0101. The time of tumor occurred on the chickens infected with bac-GX0101△(A+C) was 22 days later than infected with bac-GX0101,demonstrated the oncogenicity couldn’t disappear only deleted the ORF A and C.5 The Deletion of MDV ICP4 gene and the Comparison of the PathogenicityIn this article,ICP4 gene were deleted and the recombinant virus bac-GX0101△ICP4 recovered sucessfully. SPF chickens were infected with bac-GX0101△ICP4 and bac-GX0101 respectively. The result of animal experiment demonstrated that the HI titre to NDV and AIV after deleting ICP4 gene was lower than control group, but there were no difference between bac-GX0101△ICP4 and bac-GX0101(P>0.05). The chickens infected with bac-GX0101△ICP4 can be detected with tumors 58 days after challenged which demonstrated ICP4 gene didn’t participated in the forming tumor. But cell multiplication experiment demonstrated that ICP4 gene was correlation with the replication of viruses.更多还原