MDV流行毒株分离鉴定及GX0101感染宿主的病毒动态转录组学研究

发布时间：2018-01-21 11:02

本文关键词： 马立克病病毒病毒分离 GX0101 致病阶段转录组基因表达实时荧光定量PCR　出处：《河南科技大学》2017年硕士论文　论文类型：学位论文

【摘要】：鸡马立克病(Marek’s disease,MD)是由马立克病病毒(Marek’s disease virus,MDV)感染鸡引发的一种重要的免疫抑制病及肿瘤病。近年来,随着鸡群MD疫苗长期而广泛的应用,在免疫压力选择下MDV对疫苗的免疫耐受不断加大,毒力越来越强,流行毒株不断发生进化。在我国,MDV的流行也导致了MD疫情的频繁爆发,给家禽养殖业造成巨大经济损失。河南作为中国最大的家禽养殖基地,了解河南鸡群MDV的最新流行情况具有重要现实意义。另外,MDV基因组庞大,编码上百种蛋白,很多蛋白的功能目前仍不清楚,进一步深入研究未知基因功能、挖掘潜在致病因子的任务仍十分艰巨。为了解当前MDV分子进化特征并为MD防控提供有效参考,本研究首先应用PCR扩增和DNA测序分析对河南焦作土鸡群MD疑似病例进行了确诊,并通过CEF细胞盲传培养和间接免疫荧光实验(IFA)分离鉴定了3个流行毒株,分别命名为HNJZ101、HNJZ102和HNJZ103。进一步研究结果显示,3个MDV分离株均为MDV-1毒株、未发生REV共感染,并且132-bp重复序列均为双拷贝。基于meq、gE和gI基因进行了系统发生树分析,结果提示,3个分离株HNJZ101、HNJZ102和HNJZ103很可能是当前河南土鸡群中流行的致病性MDV-1毒株,其确切的致病型及导致免疫失败的原因仍有待进一步深入研究。为了给后续开展MDV感染及致病致瘤机制等相关研究奠定基础,本研究用vvMDV-1毒株GX0101接种1日龄SPF鸡,建立了动物感染模型,然后用qPCR分析10个具有代表性的MDV蛋白编码基因的时空表达谱。并结合此前研究鉴定了GX0101感染宿主的致病阶段:早期增殖性-限制性感染期为1~7 d,潜伏感染期约为10 d前后,晚期溶细胞性感染及免疫抑制期发生于14~21 d,而T细胞转化及淋巴瘤形成阶段可能在14 d前后就已经开始。为进一步研究MDV编码基因的转录组学,本研究应用RNA-Seq技术,对GX0101感染宿主后3、7、14、21、30和60 d的脾脏样本进行了转录组高通量测序。测序结果显示,GX0101的182个病毒编码基因均可被检测到,并最终得到了MDV编码基因在不同致病阶段的动态表达谱;以3 dpi为对照,采用DEGseq进行基因差异表达分析,分别从7、14、21、30和60 dpi筛选出差异表达基因57、33、70、68和33个,其中上调基因分别为33、28、37、29和15个,下调基因分别为24、5、33、39和18个,进一步的qPCR验证也充分证明了DEGseq结果的可靠性。本研究从RNA-Seq数据分析中获得了大量GX0101编码基因的转录组信息,为后续进一步挖掘潜在致病、致瘤基因的研究奠定了重要基础。
[Abstract]:Marekhos disease (MDD) of chicken Marek disease is caused by Marekhos disease virus of Marek disease virus. MDV) infection is an important immunosuppressive disease and oncology in chickens. In recent years, with the long-term and extensive application of MDV vaccine in chickens, the immune tolerance of MDV to the vaccine has been increasing under the selection of immune pressure. The prevalence of MDV in China also led to the frequent outbreak of MD. Henan is the largest poultry breeding base in China. It is of great practical significance to know the latest epidemic situation of MDV in Henan chicken herd. In addition, the genome of Henan chicken population is huge. It encodes hundreds of proteins, and the functions of many proteins are still unclear, so the function of unknown genes is further studied. The task of excavating potential pathogenic factors is still very arduous. It provides an effective reference for understanding the evolutionary characteristics of MDV molecules and preventing and controlling MD. In this study, PCR amplification and DNA sequencing were used to confirm the suspected MD cases of Jiaozuo Tujiao chickens in Henan Province. Three prevalent strains were isolated and identified by blind transmission culture of CEF cells and indirect immunofluorescence assay (IFA), respectively named HNJZ101. HNJZ102 and HNJZ103.The results of further study showed that the three MDV isolates were all MDV-1 strains, and no co-infection of REV occurred. And the 132-BP repeats were all double copies. Phylogenetic tree analysis based on MEQ GE and GI genes indicated that three isolates were HNJZ101. HNJZ102 and HNJZ103 are probably the most prevalent pathogenic MDV-1 strains in Henan native chicken herd at present. The exact pathotypes and the causes of immune failure still need to be further studied in order to lay a foundation for further research on MDV infection and pathogenicity. In this study, vvMDV-1 strain GX0101 was inoculated into 1-day-old SPF chicken, and the animal model of infection was established. Then qPCR was used to analyze the temporal and spatial expression profiles of 10 representative MDV protein coding genes. The pathogenicity of GX0101 infected host was identified by previous studies. The period of early proliferation-restrictive infection was 1 ~ 7 days. The latent infection period was about 10 days, and the late cytolytic infection and immunosuppressive period occurred at 14 to 21 days. The transformation of T cells and the formation of lymphoma may have started around 14 days. In order to further study the transcriptome of MDV coding gene, RNA-Seq technique was used in this study. The high throughput transcriptional sequencing of spleen samples from 30 and 60 days after GX0101 infection was carried out. The 182 viral coding genes of GX0101 could be detected, and the dynamic expression profiles of MDV coding genes were obtained at different stages of pathogenicity. Using 3 dpi as control, DEGseq was used to analyze the differentially expressed genes, and the differentially expressed genes (57n33N70) were screened out from 30 and 60 dpi respectively. 68 and 33, among them, the up-regulated genes were 33An 2837N 29 and 15, respectively, and down-regulated genes were 245G 339 and 18, respectively. The reliability of DEGseq results was also fully demonstrated by further qPCR validation. In this study, a large number of transcriptional information of GX0101 coding genes were obtained from RNA-Seq data analysis. It lays an important foundation for the further study of tumorigenic genes.
【学位授予单位】：河南科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.31

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