本文關鍵詞：Mid基因簇miRNAs在馬立克病病毒致病中的潛在功能研究　出處：《甘肅農(nóng)業(yè)大學》2017年博士論文　論文類型：學位論文

  更多相關文章： 馬立克病病毒 miRNA Mid基因簇 BAC 基因缺失 致病性 致瘤性

【摘要】：馬立克病(Marek’s disease,MD)是由馬立克病病毒(Marek’s disease virus,MDV)早期感染雛雞引發(fā)的一種重要的免疫抑制病及腫瘤病。近年來由于MDV毒力的持續(xù)增強,MD疫情在全球范圍內仍不斷暴發(fā)。該病是第一個可用病毒疫苗成功預防的腫瘤病,為深入研究皰疹病毒致瘤的生物學、遺傳學及免疫學提供了極好的動物模型。MicroRNA(miRNA)是一類非編碼小分子RNA,在許多生物學過程中發(fā)揮重要的基因調控功能。MDV-1編碼的26個成熟miRNA來自于14個miRNA前體,在基因組中形成3個明顯的miRNA基因簇,分別命名為Meq基因簇、LAT基因簇和Mid基因簇,其中Meq基因簇和Mid基因簇的轉錄和表達受同一個啟動子調控。多項研究發(fā)現(xiàn),Meq基因簇miRNA在調控MDV-1致瘤過程中發(fā)揮關鍵作用,而位于其下游的Mid基因簇miRNA的潛在調控功能尚不清楚。因此,本研究利用細菌人工染色體(BAC)技術和Rec E/T同源重組技術,構建Mid基因簇中的miRNA單基因和基因簇缺失MDV-1毒株,通過動物實驗分析Mid基因簇內編碼的病毒miRNA與MDV-1致病性的關系,篩選并鑒定該基因簇中潛在調控MDV-1致病或致瘤表型的病毒miRNA,為進一步闡明它們的分子調控機制奠定重要基礎。首先,利用MDV-1 GX0101-BAC質粒為親本載體,利用Rec E/T系統(tǒng)進行Mid基因簇miRNAs基因同源重組敲除,構建miR-M1、miR-M11和miR-M31單基因及整個Mid基因簇缺失的候選BAC克隆,并對其進行PCR擴增鑒定、DNA測序以及限制性片段長度多態(tài)性(RFLP)分析。結果表明,經(jīng)過2輪Rec E/T同源重組,成功獲得了3個miRNA前體基因缺失和Mid基因簇缺失的BAC克隆質粒,分別命名為GXΔmiR-M1-BAC、GXΔmiR-M11-BAC、GXΔmiR-M31-BAC和GXΔMid-mi Rs-BAC。然后,將4個BAC重組質粒分別轉染CEF細胞,進行病毒miRNA基因缺失感染性BAC克隆的拯救,并同上對拯救毒株的基因組DNA進行PCR擴增和DNA測序鑒定。同時提取各miRNA基因缺失毒株感染CEF細胞的總RNA,利用q RT-PCR分別進行相應miRNA的表達以及相關基因的表達鑒定。提取GX0101親本毒株和各miRNA基因缺失毒株感染CEF細胞后不同時間細胞毒總DNA,利用qPCR對相應DNA的meq和gB基因進行定量檢測,測定病毒增殖曲線。結果表明:成功拯救獲得4個Mid基因簇miRNAs的單基因或基因簇的雙等位基因缺失毒株,分別命名為GXΔmi R-M1、GXΔmiR-M11、GXΔmiR-M31和GXΔMid-mi Rs;在各miRNA基因缺失毒株中,相鄰miRNA的表達并未受到影響,且相鄰基因meq、RLORF6、RLORF5a和RLORF4的表達也與預期相符;各miRNA基因缺失毒株與GX0101親本毒株具有相似的體外病毒增殖曲線。最后,用GX0101親本毒株、GXΔmi R-M1、GXΔmi R-M11、GXΔmiR-M31和GXΔMid-mi Rs基因缺失毒株分別感染1日齡SPF雞,以等劑量CEF正常細胞為陰性對照,對各毒株的體內復制能力(病毒血癥)、感染雞生長性能(體重)、免疫器官指數(shù)(胸腺/體重、法氏囊/體重指數(shù))、致死率及致瘤率等進行分析。結果表明:與GX0101親本毒株相比,各miRNA基因缺失毒株在感染雞體內復制曲線稍有不同;從14 dpi開始,各組感染雞體重均顯著或不同程度低于CEF細胞陰性對照組,14~30 dpi各組感染雞的囊重比指數(shù)和胸腺重比指數(shù)顯著低于CEF細胞陰性對照組;14 dpi以后除CEF細胞對照組雞未發(fā)生死亡外,各攻毒組雞群均發(fā)生不同程度的雞只死亡,其中GXΔMid-miRs感染組至75 dpi時全部死亡,死亡率為100%,肉眼腫瘤率為21.0%;至90 dpi實驗周期結束時,GXΔmi R-M1、GXΔmiR-M11和GXΔmiR-M31感染組的死亡率分別為83.3%、71.8%和69.2%,肉眼腫瘤率分別為31.7%、45.0%和31.7%;而親本毒GX0101感染組的死亡率和腫瘤發(fā)生率分別為79.4%和40.0%。上述研究結果表明,Mid基因簇編碼的miRNAs是MDV-1復制的非必須基因,與病毒感染導致的免疫抑制關系不大,但這些miRNAs的基因缺失不同程度的改變了MDV-1的致死率或致瘤率。與GX0101親本毒株相比,整個Mid基因簇的缺失顯著增強了MDV-1的致死率;miR-M1的單基因缺失增強了MDV-1的致死率,miR-M11的單基因缺失則增強了MDV-1的致瘤率,而miR-M31的基因缺失則同時降低了MDV-1的致死率和致瘤率。這些結果提示,Mid基因簇編碼的miRNAs在MDV-1致病和致瘤過程中可能扮演原癌基因或抑癌基因不同角色,為今后深入研究這些病毒miRNA的分子調控機制提供了重要線索。
[Abstract]:Marek's disease (Marek 's disease, MD) by Marek's disease virus (Marek' s disease virus, MDV) an important early infection in chickens caused by inhibition of immune disease and tumor disease. In recent years due to the increasing virulence of MDV, MD is still epidemic outbreaks continue in the global scope of the disease is the first. Available vaccine successfully prevent tumor disease, for further study of herpes simplex virus induced tumor biology, genetics and Immunology provides an excellent animal model of.MicroRNA (miRNA) is a class of small molecule non encoding RNA, play the 26 mature miRNA gene encoding the important function of.MDV-1 from the 14 miRNA precursor in many biological in the process of forming 3 distinct miRNA gene cluster in the genome, named Meq gene cluster, LAT gene cluster and Mid gene cluster, the transcription and expression of Meq gene cluster and Mid gene cluster from the same promoter. Study found that the Meq gene cluster miRNA to play a key role in the process of tumor regulation of MDV-1, Mid and miRNA genes located downstream of the potential regulatory function remains unclear. Therefore, this study using bacterial artificial chromosome (BAC) and Rec E/T homologous recombination technology, construction of Mid gene cluster in miRNA gene deletion of MDV-1 gene cluster and strain analysis of the relationship between Mid gene cluster encoding viral miRNA and pathogenicity of MDV-1 through animal experiment, screening and identification of the potential regulation of MDV-1 gene cluster in the pathogenic or tumorigenic phenotype of virus miRNA, to further elucidate the molecular mechanism of them lay an important foundation. First of all, the use of MDV-1 plasmid GX0101-BAC the parent carrier, Mid gene cluster miRNAs gene homologous recombination knockout, constructing miR-M1 using Rec E/T system, BAC miR-M11 and miR-M31 single candidate gene and the deletion of Mid gene cluster humancloning, and The PCR amplification, DNA sequencing and restriction fragment length polymorphism (RFLP) analysis. The results showed that after 2 rounds of Rec E/T homologous recombination, successfully obtained BAC plasmid 3 miRNA precursor gene deletion and deletion of Mid gene cluster, named GX GX, Delta miR-M1-BAC, Delta miR-M11-BAC, Delta GX miR-M31-BAC and GX Mid-mi Rs-BAC. and 4 BAC recombinant plasmids were transfected into CEF cells, were rescued virus miRNA gene deletion infectious BAC clone, and the genomic DNA of rescue strain were amplified by PCR and the same DNA sequencing. At the same time, total RNA was extracted from CEF cells infected by miRNA gene deletion strains, using Q RT-PCR the expression of miRNA were identified and the expression of the corresponding genes. CEF extracted from GX0101 cells infected with the parent virus and miRNA gene deletion strains at different time after cytotoxic DNA, using qPCR mEq and gB based on DNA For quantitative detection, determination of virus proliferation curve. The results show that the successful rescue won 4 Mid gene cluster miRNAs single gene or gene cluster biallelic deletion strains, which were named as GX mi R-M1 GX GX, Delta miR-M11, Delta miR-M31 and delta Mid-mi GX Rs; the miRNA gene deletion strains in in the expression of adjacent miRNA was not affected, and the adjacent meq gene, RLORF6, expression of RLORF5a and RLORF4 are also in line with expectations; the miRNA gene deletion strains and parental strain GX0101 with in vitro virus proliferation curve similarity. Finally, using GX0101 GX mi R-M1 parental strains, Delta GX, delta mi R-M11, GX miR-M31 GX Mid-mi and Rs gene deletion strains were infected with 1 day old SPF chickens, with the dose of CEF normal cells as negative control, replication of the virus (viremia), infected chicken growth performance (body weight), index of immune organs (thymus / body weight, bursa / BMI The number), the fatality rate and tumorigenicity were analyzed. The results showed that: compared with the parental strain GX0101, the miRNA gene deletion strains in chickens in vivo replication curve is slightly different; from the beginning of the 14 DPI groups, infected chickens were significantly lower than the weight or CEF cells in the negative control group, 14~30 group of DPI infected chickens capsule weight ratio index and thymus weight index was significantly lower in CEF cells than the negative control group; 14 DPI after addition of CEF cells in the control group without the occurrence of chicken deaths, the chickens challenged groups had different degrees of chicken deaths, of which GX Mid-miRs infection group at 75 DPI of all deaths, the mortality rate was 100%, the naked eye the tumor rate was 21%; to the end of 90 DPI experimental period, GX mi R-M1, GX GX and delta miR-M11 Delta miR-M31 infection group, the mortality rates were 83.3%, 71.8% and 69.2%, gross tumor rates were 31.7%, 45% and 31.7%; and the parental virus GX0101 infection group, mortality And the incidence of tumor was 79.4% and 40.0%. respectively. The results showed that the Mid gene cluster encoding miRNAs MDV-1 replication non essential gene, immune and viral infection leads to inhibition of the relationship is not, but the lack of these miRNAs genes are changed in different degrees of MDV-1 mortality or tumorigenic rate. Compared with the parental strain GX0101. The deletion of Mid gene cluster significantly enhanced the lethality of MDV-1; single gene deletion of miR-M1 increased death rate of MDV-1, single gene deletion miR-M11 enhances the tumorigenic rate of MDV-1, and the miR-M31 gene deletion and reduce the death rate of MDV-1 and tumorigenic rate. These results suggest that Mid gene cluster encoding miRNAs may play oncogenes or tumor suppressor genes in different roles in the pathogenesis of MDV-1 and tumorigenesis, provides important clues for further study of these virus molecular regulatory mechanisms of miRNA.

【學位授予單位】：甘肅農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：S852.65

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