新型水貂阿留申病毒感染性克隆的构建及其生物学特性鉴定

发布时间：2018-06-01 01:18

本文选题：水貂阿留申病毒 + 末端发夹结构　；参考：《中国农业大学》2016年博士论文

【摘要】：水貂阿留申病(Aleutian disease of mink, AD)是由水貂阿留申病毒(Aleutian mink disease virus, AMDV)感染而引起的重要疫病。该病以浆细胞弥漫性增生和慢性持续性感染为主要特点,常可导致免疫系统紊乱,母貂产仔数减少,仔貂大量死亡,给养貂业造成了极大的危害。由于AMDV基因组的3’-端和5’-端分别存在Y-型和U-型结构,给全基因组序列测定造成一定的困难。目前,仅AMDV弱毒株ADV-G完成了全基因组测序,而野病毒株序列均为基因组中间部分。另外,弱毒株ADV-G能够在克兰德尔猫肾细胞系(Crandell feline kidney cells, CRFK)细胞系中连续培养,而野病毒株均无法在体外持续传代。上述特点使阐明ADV-G与野毒株基因组间的差异无法进行,更无法阐明其分子致病机理,给该病的病原学、免疫学研究造成了严重的阻碍。本研究首先设计AMDV基因组末端特殊克隆方法,成功完成了一野毒株BJ的全基因测序(GenBank KT329428)。通过BJ与ADV-G的各部分基因的同源比对,预测可能影响病毒体外复制和致病力的功能基序。以成功构建的ADV-G感染性克隆为基础,在其主要结构蛋白VP2中引入系列突变,通过检测突变体克隆在CRFK细胞中生长水平的差异,定位影响ADV-G体外增殖的功能残基。进而将ADV-G中的个别氨基酸残基或部分片段替换成BJ中的对应氨基酸残基或部分片段,构建出嵌合体感染性克隆。成功实现体外拯救的感染性克隆对水貂的感染,为AMDV致病机理的研究奠定了坚实基础。主要研究结果和结论如下：(1)通过设计特殊末端重复序列(Inverted terminal repeat, ITR)克隆方法,完成了一野毒株BJ的全基因组测序。经序列比对分析发现,BJ的结构蛋白2(Viral protein 2, VP2)基因中存在所有高致病毒株保守的氨基酸残基,证实BJ为高致病毒株。通过与ADV-G进行同源比较,发现两毒株5'U型ITR结构存在明显差异。并证实AMDV毒株非结构蛋白3 (Non-structural protein 3,NS3)开放阅读框终止密码子的位置并不固定,这个现象在其它细小病毒中十分罕见。(2)参照BJ和ADV-G的同源比对结果,选择人工合成非结构蛋白1 (Non-structural protein 1, NS1)a.a.36-52和VP2 a.a.428-446两个保守的亲水肽段,免疫小鼠制备单克隆抗体,成功筛选数个杂交瘤细胞株,产生的细胞培养上清液经检测与拯救毒株rADV-G反应灵敏且特异,可用于拯救病毒的检测。(3)参照NCBI上公布的ADV-G全序列,人工合成基因组3’和5’端难以克隆的区域,再利用PCR克隆中间非发夹序列,利用In-Fusion技术将各片段连接起来,构建成ADV-G全长基因组克隆质粒pADV-G,并对基因组进行无义突变(A3875C)引入Sac I酶切位点作为遗传标记。将pADV-G转入CRFK细胞,连续传代,从基因组复制、转录和翻译水平证明成功拯救病毒rADV-G,对盲传13代rADV-G的cDNA进行遗传标记检测,证实rADV-G能够在CRFK细胞中稳定遗传。(4)利用从国内不同地区分离鉴定的AMDV毒株(BJ, SD, HLJ和HB)基因序列与其他国内外报道的17株AMDV序列进行同源比对,发现野毒株与ADV-G在VP2上存在4个保守的氨基酸位点差异,即92、94、115和116位。在pADV-G引入上述突变,通过突变体克隆在细胞中转录和翻译水平的检测,证实位于VP2衣壳表面三重对称轴附近的92H和94Q氨基酸残基对于ADV-G在CRFK细胞中的持续增殖起着决定性作用。(5)将BJ高致病毒株编码VP2 455-590位氨基酸区段的核苷酸序列替换pADV-G感染性克隆中的对应序列,再引入VP2 I352V氨基酸突变,构建出两个嵌合体全基因克隆,并成功拯救出病毒。水貂接种实验证明,嵌合体病毒能够感染水貂,在接种10天后产生短暂的病毒血症,接种30天仍能检测到抗AMDV的抗体,但组织切片显示未产生典型的AD。以上结果从全病毒的角度定位了AMDV衣壳蛋白VP2上存在的决定ADV-G体外复制能力的关键功能残基,并且通过构建嵌合体病毒的方式证明了ADV-G VP2部分基序的改变可以使病毒获得感染水貂的能力。同时在全基因水平比较野毒株BJ同ADV-G间的差异,为AMDV致病性的研究提供了新的方向。
[Abstract]:Mink Aleutian disease (Aleutian disease of mink, AD) is an important epidemic disease caused by the sable Aleutian virus (Aleutian mink disease virus, AMDV). The disease is characterized by diffuse proliferation of plasma cells and chronic persistent infection, which often leads to the disorder of the immune system, the decrease of the number of mother mink, the death of mink, and the feeding of mink. Because the 3 '- end and 5' - terminal of the AMDV genome has Y- and U- type structure respectively, it is difficult to determine the whole genome sequence. At present, only the AMDV weak strain ADV-G has completed the whole genome sequencing, and the wild virus strains are all the middle part of the genome. In addition, the weak strain ADV-G can be in the klander cat. The Crandell feline kidney cells (CRFK) cell line was continuously cultured and the wild virus strains were unable to continue to be passaged in vitro. The above characteristics made it impossible to elucidate the differences between the genome of ADV-G and the wild strains, and could not elucidate the molecular pathogenicity of the virus, and caused serious obstruction to the disease origin and immunology research. First, the special cloning method of the AMDV genome terminal was designed, and the whole gene sequencing of the wild virus strain BJ was successfully completed (GenBank KT329428). Through the homologous comparison of various parts of the genes of BJ and ADV-G, the functional sequence that could affect the replication and pathogenicity of the virus in vitro was predicted. Based on the successful construction of ADV-G infectious clones, the main structural eggs were found. In white VP2, a series of mutations are introduced. By detecting the difference in the growth level of the mutant clones in CRFK cells, the functional residues that affect the proliferation of ADV-G in vitro are located. Then the individual amino acid residues or segments in the ADV-G are replaced with the corresponding amino acid residues or segments in BJ, and a chimeric infection clone is constructed. The infectious clone of the foreign rescue laid a solid foundation for the study of the pathogenic mechanism of AMDV. The main results and conclusions were as follows: (1) the whole genome of BJ was sequenced by the design of the special terminal repeat sequence (Inverted terminal repeat, ITR), and the structure of BJ was found by sequence alignment analysis. In the protein 2 (Viral protein 2, VP2) gene, there was a conservative amino acid residue in all high virus strains and confirmed that BJ was a high virus strain. By homologous comparison with ADV-G, it was found that there was a significant difference in the ITR structure of the 5'U type of two strains. The unstructured protein 3 of the AMDV virus (Non-structural protein 3, NS3) open reading frame terminated the codon The location is not fixed, and this phenomenon is rare in other parvovirus. (2) according to the homologous comparison between BJ and ADV-G, two conservative hydrophilic peptide segments were synthesized by artificial synthesis of non structural protein 1 (Non-structural protein 1, NS1) a.a.36-52 and VP2 a.a.428-446, and monoclonal antibodies were prepared by immunization mice, and several hybridoma cells were successfully screened. The cell culture supernatant is sensitive and specific to save the virus rADV-G and can be used to save the virus detection. (3) refer to the ADV-G full sequence published on the NCBI, the region which is difficult to clone the 3 'and 5' ends of the genome, and then clone the middle non hairpin sequence by PCR, and use In-Fusion technology to connect the fragments. The ADV-G full-length genome clone plasmid pADV-G was constructed, and the non sense mutation (A3875C) of the genome was introduced into the Sac I enzyme cut site as a genetic marker. PADV-G was transferred into CRFK cells for continuous generation, and the virus rADV-G was successfully saved from the genome replication, transcription and translation level, and the genetic markers were detected for the 13 generation rADV-G cDNA of the blind transmission. The real rADV-G can be stable in CRFK cells. (4) using the homologous comparison between the AMDV strains (BJ, SD, HLJ and HB) isolated from different regions of China and other 17 AMDV sequences reported at home and abroad, it is found that there are 4 conserved amino acid loci differences between the wild and ADV-G in VP2, namely 92,94115 and 116. In this mutation, the 92H and 94Q amino acid residues located near the three symmetrical axis of the VP2 capsid on the surface of the capsid of the capsid on the surface of the capsid of the VP2 capsid play a decisive role in the continuous proliferation of ADV-G in CRFK cells. (5) the nucleotide sequence of the BJ high virus strain coding VP2 455-590 - bit amino acid section By changing the corresponding sequence of pADV-G infectious clones and introducing VP2 I352V amino acid mutation, two chimerism full gene clones were constructed and the virus was successfully saved. The inoculation experiment of mink showed that the chimera virus could infect mink, produce transient viremia after 10 days of inoculation, and still detect anti AMDV antibody for 30 days, but the tissue can still be detected. The slice showed that no typical AD. results were found to be a key functional residue on the AMDV capsid protein VP2 that determines the ability of ADV-G in vitro replication from the whole virus point of view, and by constructing a chimera virus, it was proved that the changes in the partial sequence of ADV-G VP2 could enable the virus to acquire the ability to infect mink. At the same time, the whole gene was in the whole gene. The difference between horizontal comparison wild strain BJ and ADV-G provides a new direction for the pathogenicity study of AMDV.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S852.65

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