Mid基因簇miRNAs在马立克病病毒致病中的潜在功能研究

发布时间：2018-01-04 11:00

本文关键词：Mid基因簇miRNAs在马立克病病毒致病中的潜在功能研究　出处：《甘肃农业大学》2017年博士论文　论文类型：学位论文

【摘要】：马立克病(Marek’s disease,MD)是由马立克病病毒(Marek’s disease virus,MDV)早期感染雏鸡引发的一种重要的免疫抑制病及肿瘤病。近年来由于MDV毒力的持续增强,MD疫情在全球范围内仍不断暴发。该病是第一个可用病毒疫苗成功预防的肿瘤病,为深入研究疱疹病毒致瘤的生物学、遗传学及免疫学提供了极好的动物模型。MicroRNA(miRNA)是一类非编码小分子RNA,在许多生物学过程中发挥重要的基因调控功能。MDV-1编码的26个成熟miRNA来自于14个miRNA前体,在基因组中形成3个明显的miRNA基因簇,分别命名为Meq基因簇、LAT基因簇和Mid基因簇,其中Meq基因簇和Mid基因簇的转录和表达受同一个启动子调控。多项研究发现,Meq基因簇miRNA在调控MDV-1致瘤过程中发挥关键作用,而位于其下游的Mid基因簇miRNA的潜在调控功能尚不清楚。因此,本研究利用细菌人工染色体(BAC)技术和Rec E/T同源重组技术,构建Mid基因簇中的miRNA单基因和基因簇缺失MDV-1毒株,通过动物实验分析Mid基因簇内编码的病毒miRNA与MDV-1致病性的关系,筛选并鉴定该基因簇中潜在调控MDV-1致病或致瘤表型的病毒miRNA,为进一步阐明它们的分子调控机制奠定重要基础。首先,利用MDV-1 GX0101-BAC质粒为亲本载体,利用Rec E/T系统进行Mid基因簇miRNAs基因同源重组敲除,构建miR-M1、miR-M11和miR-M31单基因及整个Mid基因簇缺失的候选BAC克隆,并对其进行PCR扩增鉴定、DNA测序以及限制性片段长度多态性(RFLP)分析。结果表明,经过2轮Rec E/T同源重组,成功获得了3个miRNA前体基因缺失和Mid基因簇缺失的BAC克隆质粒,分别命名为GXΔmiR-M1-BAC、GXΔmiR-M11-BAC、GXΔmiR-M31-BAC和GXΔMid-mi Rs-BAC。然后,将4个BAC重组质粒分别转染CEF细胞,进行病毒miRNA基因缺失感染性BAC克隆的拯救,并同上对拯救毒株的基因组DNA进行PCR扩增和DNA测序鉴定。同时提取各miRNA基因缺失毒株感染CEF细胞的总RNA,利用q RT-PCR分别进行相应miRNA的表达以及相关基因的表达鉴定。提取GX0101亲本毒株和各miRNA基因缺失毒株感染CEF细胞后不同时间细胞毒总DNA,利用qPCR对相应DNA的meq和gB基因进行定量检测,测定病毒增殖曲线。结果表明:成功拯救获得4个Mid基因簇miRNAs的单基因或基因簇的双等位基因缺失毒株,分别命名为GXΔmi R-M1、GXΔmiR-M11、GXΔmiR-M31和GXΔMid-mi Rs;在各miRNA基因缺失毒株中,相邻miRNA的表达并未受到影响,且相邻基因meq、RLORF6、RLORF5a和RLORF4的表达也与预期相符;各miRNA基因缺失毒株与GX0101亲本毒株具有相似的体外病毒增殖曲线。最后,用GX0101亲本毒株、GXΔmi R-M1、GXΔmi R-M11、GXΔmiR-M31和GXΔMid-mi Rs基因缺失毒株分别感染1日龄SPF鸡,以等剂量CEF正常细胞为阴性对照,对各毒株的体内复制能力(病毒血症)、感染鸡生长性能(体重)、免疫器官指数(胸腺/体重、法氏囊/体重指数)、致死率及致瘤率等进行分析。结果表明:与GX0101亲本毒株相比,各miRNA基因缺失毒株在感染鸡体内复制曲线稍有不同;从14 dpi开始,各组感染鸡体重均显著或不同程度低于CEF细胞阴性对照组,14~30 dpi各组感染鸡的囊重比指数和胸腺重比指数显著低于CEF细胞阴性对照组;14 dpi以后除CEF细胞对照组鸡未发生死亡外,各攻毒组鸡群均发生不同程度的鸡只死亡,其中GXΔMid-miRs感染组至75 dpi时全部死亡,死亡率为100%,肉眼肿瘤率为21.0%;至90 dpi实验周期结束时,GXΔmi R-M1、GXΔmiR-M11和GXΔmiR-M31感染组的死亡率分别为83.3%、71.8%和69.2%,肉眼肿瘤率分别为31.7%、45.0%和31.7%;而亲本毒GX0101感染组的死亡率和肿瘤发生率分别为79.4%和40.0%。上述研究结果表明,Mid基因簇编码的miRNAs是MDV-1复制的非必须基因,与病毒感染导致的免疫抑制关系不大,但这些miRNAs的基因缺失不同程度的改变了MDV-1的致死率或致瘤率。与GX0101亲本毒株相比,整个Mid基因簇的缺失显著增强了MDV-1的致死率;miR-M1的单基因缺失增强了MDV-1的致死率,miR-M11的单基因缺失则增强了MDV-1的致瘤率,而miR-M31的基因缺失则同时降低了MDV-1的致死率和致瘤率。这些结果提示,Mid基因簇编码的miRNAs在MDV-1致病和致瘤过程中可能扮演原癌基因或抑癌基因不同角色,为今后深入研究这些病毒miRNA的分子调控机制提供了重要线索。
[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.

【学位授予单位】：甘肃农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S852.65

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