本文關(guān)鍵詞： 馬立克病病毒 病毒分離 GX0101 致病階段 轉(zhuǎn)錄組基因表達(dá) 實(shí)時(shí)熒光定量PCR　出處：《河南科技大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：雞馬立克病(Marek’s disease,MD)是由馬立克病病毒(Marek’s disease virus,MDV)感染雞引發(fā)的一種重要的免疫抑制病及腫瘤病。近年來(lái),隨著雞群MD疫苗長(zhǎng)期而廣泛的應(yīng)用,在免疫壓力選擇下MDV對(duì)疫苗的免疫耐受不斷加大,毒力越來(lái)越強(qiáng),流行毒株不斷發(fā)生進(jìn)化。在我國(guó),MDV的流行也導(dǎo)致了MD疫情的頻繁爆發(fā),給家禽養(yǎng)殖業(yè)造成巨大經(jīng)濟(jì)損失。河南作為中國(guó)最大的家禽養(yǎng)殖基地,了解河南雞群MDV的最新流行情況具有重要現(xiàn)實(shí)意義。另外,MDV基因組龐大,編碼上百種蛋白,很多蛋白的功能目前仍不清楚,進(jìn)一步深入研究未知基因功能、挖掘潛在致病因子的任務(wù)仍十分艱巨。為了解當(dāng)前MDV分子進(jìn)化特征并為MD防控提供有效參考,本研究首先應(yīng)用PCR擴(kuò)增和DNA測(cè)序分析對(duì)河南焦作土雞群MD疑似病例進(jìn)行了確診,并通過(guò)CEF細(xì)胞盲傳培養(yǎng)和間接免疫熒光實(shí)驗(yàn)(IFA)分離鑒定了3個(gè)流行毒株,分別命名為HNJZ101、HNJZ102和HNJZ103。進(jìn)一步研究結(jié)果顯示,3個(gè)MDV分離株均為MDV-1毒株、未發(fā)生REV共感染,并且132-bp重復(fù)序列均為雙拷貝�；趍eq、gE和gI基因進(jìn)行了系統(tǒng)發(fā)生樹(shù)分析,結(jié)果提示,3個(gè)分離株HNJZ101、HNJZ102和HNJZ103很可能是當(dāng)前河南土雞群中流行的致病性MDV-1毒株,其確切的致病型及導(dǎo)致免疫失敗的原因仍有待進(jìn)一步深入研究。為了給后續(xù)開(kāi)展MDV感染及致病致瘤機(jī)制等相關(guān)研究奠定基礎(chǔ),本研究用vvMDV-1毒株GX0101接種1日齡SPF雞,建立了動(dòng)物感染模型,然后用qPCR分析10個(gè)具有代表性的MDV蛋白編碼基因的時(shí)空表達(dá)譜。并結(jié)合此前研究鑒定了GX0101感染宿主的致病階段:早期增殖性-限制性感染期為1~7 d,潛伏感染期約為10 d前后,晚期溶細(xì)胞性感染及免疫抑制期發(fā)生于14~21 d,而T細(xì)胞轉(zhuǎn)化及淋巴瘤形成階段可能在14 d前后就已經(jīng)開(kāi)始。為進(jìn)一步研究MDV編碼基因的轉(zhuǎn)錄組學(xué),本研究應(yīng)用RNA-Seq技術(shù),對(duì)GX0101感染宿主后3、7、14、21、30和60 d的脾臟樣本進(jìn)行了轉(zhuǎn)錄組高通量測(cè)序。測(cè)序結(jié)果顯示,GX0101的182個(gè)病毒編碼基因均可被檢測(cè)到,并最終得到了MDV編碼基因在不同致病階段的動(dòng)態(tài)表達(dá)譜;以3 dpi為對(duì)照,采用DEGseq進(jìn)行基因差異表達(dá)分析,分別從7、14、21、30和60 dpi篩選出差異表達(dá)基因57、33、70、68和33個(gè),其中上調(diào)基因分別為33、28、37、29和15個(gè),下調(diào)基因分別為24、5、33、39和18個(gè),進(jìn)一步的qPCR驗(yàn)證也充分證明了DEGseq結(jié)果的可靠性。本研究從RNA-Seq數(shù)據(jù)分析中獲得了大量GX0101編碼基因的轉(zhuǎn)錄組信息,為后續(xù)進(jìn)一步挖掘潛在致病、致瘤基因的研究奠定了重要基礎(chǔ)。
[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.
【學(xué)位授予單位】：河南科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S858.31

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