本文選題：水貂阿留申病毒 + 末端發(fā)夾結(jié)構(gòu)　； 參考：《中國(guó)農(nóng)業(yè)大學(xué)》2016年博士論文

【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。該病以漿細(xì)胞彌漫性增生和慢性持續(xù)性感染為主要特點(diǎn),�？蓪�(dǎo)致免疫系統(tǒng)紊亂,母貂產(chǎn)仔數(shù)減少,仔貂大量死亡,給養(yǎng)貂業(yè)造成了極大的危害。由于AMDV基因組的3’-端和5’-端分別存在Y-型和U-型結(jié)構(gòu),給全基因組序列測(cè)定造成一定的困難。目前,僅AMDV弱毒株ADV-G完成了全基因組測(cè)序,而野病毒株序列均為基因組中間部分。另外,弱毒株ADV-G能夠在克蘭德?tīng)栘埬I細(xì)胞系(Crandell feline kidney cells, CRFK)細(xì)胞系中連續(xù)培養(yǎng),而野病毒株均無(wú)法在體外持續(xù)傳代。上述特點(diǎn)使闡明ADV-G與野毒株基因組間的差異無(wú)法進(jìn)行,更無(wú)法闡明其分子致病機(jī)理,給該病的病原學(xué)、免疫學(xué)研究造成了嚴(yán)重的阻礙。本研究首先設(shè)計(jì)AMDV基因組末端特殊克隆方法,成功完成了一野毒株BJ的全基因測(cè)序(GenBank KT329428)。通過(guò)BJ與ADV-G的各部分基因的同源比對(duì),預(yù)測(cè)可能影響病毒體外復(fù)制和致病力的功能基序。以成功構(gòu)建的ADV-G感染性克隆為基礎(chǔ),在其主要結(jié)構(gòu)蛋白VP2中引入系列突變,通過(guò)檢測(cè)突變體克隆在CRFK細(xì)胞中生長(zhǎng)水平的差異,定位影響ADV-G體外增殖的功能殘基。進(jìn)而將ADV-G中的個(gè)別氨基酸殘基或部分片段替換成BJ中的對(duì)應(yīng)氨基酸殘基或部分片段,構(gòu)建出嵌合體感染性克隆。成功實(shí)現(xiàn)體外拯救的感染性克隆對(duì)水貂的感染,為AMDV致病機(jī)理的研究奠定了堅(jiān)實(shí)基礎(chǔ)。主要研究結(jié)果和結(jié)論如下：(1)通過(guò)設(shè)計(jì)特殊末端重復(fù)序列(Inverted terminal repeat, ITR)克隆方法,完成了一野毒株BJ的全基因組測(cè)序。經(jīng)序列比對(duì)分析發(fā)現(xiàn),BJ的結(jié)構(gòu)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殘基,證實(shí)BJ為高致病毒株。通過(guò)與ADV-G進(jìn)行同源比較,發(fā)現(xiàn)兩毒株5'U型ITR結(jié)構(gòu)存在明顯差異。并證實(shí)AMDV毒株非結(jié)構(gòu)蛋白3 (Non-structural protein 3,NS3)開(kāi)放閱讀框終止密碼子的位置并不固定,這個(gè)現(xiàn)象在其它細(xì)小病毒中十分罕見(jiàn)。(2)參照BJ和ADV-G的同源比對(duì)結(jié)果,選擇人工合成非結(jié)構(gòu)蛋白1 (Non-structural protein 1, NS1)a.a.36-52和VP2 a.a.428-446兩個(gè)保守的親水肽段,免疫小鼠制備單克隆抗體,成功篩選數(shù)個(gè)雜交瘤細(xì)胞株,產(chǎn)生的細(xì)胞培養(yǎng)上清液經(jīng)檢測(cè)與拯救毒株rADV-G反應(yīng)靈敏且特異,可用于拯救病毒的檢測(cè)。(3)參照NCBI上公布的ADV-G全序列,人工合成基因組3’和5’端難以克隆的區(qū)域,再利用PCR克隆中間非發(fā)夾序列,利用In-Fusion技術(shù)將各片段連接起來(lái),構(gòu)建成ADV-G全長(zhǎng)基因組克隆質(zhì)粒pADV-G,并對(duì)基因組進(jìn)行無(wú)義突變(A3875C)引入Sac I酶切位點(diǎn)作為遺傳標(biāo)記。將pADV-G轉(zhuǎn)入CRFK細(xì)胞,連續(xù)傳代,從基因組復(fù)制、轉(zhuǎn)錄和翻譯水平證明成功拯救病毒rADV-G,對(duì)盲傳13代rADV-G的cDNA進(jìn)行遺傳標(biāo)記檢測(cè),證實(shí)rADV-G能夠在CRFK細(xì)胞中穩(wěn)定遺傳。(4)利用從國(guó)內(nèi)不同地區(qū)分離鑒定的AMDV毒株(BJ, SD, HLJ和HB)基因序列與其他國(guó)內(nèi)外報(bào)道的17株AMDV序列進(jìn)行同源比對(duì),發(fā)現(xiàn)野毒株與ADV-G在VP2上存在4個(gè)保守的氨基酸位點(diǎn)差異,即92、94、115和116位。在pADV-G引入上述突變,通過(guò)突變體克隆在細(xì)胞中轉(zhuǎn)錄和翻譯水平的檢測(cè),證實(shí)位于VP2衣殼表面三重對(duì)稱軸附近的92H和94Q氨基酸殘基對(duì)于ADV-G在CRFK細(xì)胞中的持續(xù)增殖起著決定性作用。(5)將BJ高致病毒株編碼VP2 455-590位氨基酸區(qū)段的核苷酸序列替換pADV-G感染性克隆中的對(duì)應(yīng)序列,再引入VP2 I352V氨基酸突變,構(gòu)建出兩個(gè)嵌合體全基因克隆,并成功拯救出病毒。水貂接種實(shí)驗(yàn)證明,嵌合體病毒能夠感染水貂,在接種10天后產(chǎn)生短暫的病毒血癥,接種30天仍能檢測(cè)到抗AMDV的抗體,但組織切片顯示未產(chǎn)生典型的AD。以上結(jié)果從全病毒的角度定位了AMDV衣殼蛋白VP2上存在的決定ADV-G體外復(fù)制能力的關(guān)鍵功能殘基,并且通過(guò)構(gòu)建嵌合體病毒的方式證明了ADV-G VP2部分基序的改變可以使病毒獲得感染水貂的能力。同時(shí)在全基因水平比較野毒株BJ同ADV-G間的差異,為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.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S852.65

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