龐飛臺灣大學：獸醫學研究所陳菀蓉Chen, Wan-JungWan-JungChen2007-11-282018-07-092007-11-282018-07-092007http://ntur.lib.ntu.edu.tw//handle/246246/60015為探討是否不同的田間PCV2病毒株間，在誘導巨噬細胞產生干擾素α ( IFN-α ) 的作用上有所不同，而此不同與其基因序列的差異有關，因此進行此實驗。由田間獲得的病弱豬隻之脾臟、淋巴結、扁桃腺等組織塊，先抽取DNA，經PCR確定為PCV2陽性後，實驗分成兩部份進行：第一部份為DNA萃取，進行全長基因定序、分析及比較。第二部分則在基因序列比對後，將呈現顯著不同的病毒株再經病毒分離、增殖、定量﹙in PK–15 cell line﹚後，分別接種於豬肺泡巨噬細胞，收集上清液進行IFN-α檢測，獲得的結果再與基因序列做比較，以了解彼此間的關聯性。本實驗共收集58個經PCR檢測含有PCV2之病材，有45個完全定序，其中有7個PCV2分離株可進行病毒分離並檢測其力價。在完全定序的45株由田間偵測到的PCV2樣本中，分析發現所得的序列全長可分為1767 bp或1768 bp兩組，且其中有五株PCV2含有可抑制豬周邊血液單核球 ( PBMCs ) 產生IFN-α的作用的ODN，其基因序列全長都為1768 bp；經分析已發表於GenBank的PCV2，發現均有類似的情形。自完全定序之病毒株中選出12株進行核酸序列比對，全長核酸序列相似性為94.7 -99.9％，主要差異發生在ORF2的區域 ( 核酸序列相似性為89.5-100.0％ )。在IFN-α檢測方面，不同的PCV2病毒株彼此間誘導IFN-α產生的能力上確實有所不同，而PCV2確實是IFN-α誘導者，但與含或不含有抑制作用的ODN關連性較小，進一步與多段已發表之ODNs進行比對發現，PCV2核酸序列中含有多段具刺激IFN-α產生能力的ODNs；並且發現近年來PCV2核酸序列似有轉變的現象，變為以1767 bp的病毒株為主，而1768 bp的病毒株與含有抑制IFN-α產生的ODN的病毒株皆有減少的趨勢。由本實驗可知，PCV2核酸序列全長、可抑制IFN-α產生的ODN及誘導IFN-α產生能力之間有關連性存在，而此或許與PCV2感染後造成差異甚大的臨床症狀的致病機制有關。口試委員會審定書 致謝 中文摘要 ------------------------------------------------------------------------------------- I Abstract --------------------------------------------------------------------------------------- II 目錄 -------------------------------------------------------------------------------------------- III 表次 -------------------------------------------------------------------------------------------- V 圖次 -------------------------------------------------------------------------------------------- V 第一章 序言 --------------------------------------------------------------------------------- 1 第二章 文獻回顧 --------------------------------------------------------------------------- 3 第一節 環狀病毒 ------------------------------------------------------------------------ 3 1.1環狀病毒的病毒學分類 ------------------------------------------------------- 3 1.2豬環狀病毒特徵------------- ---------------------------------------------------- 3 1.3第二型豬環狀病毒序列特色 ------------------------------------------------- 4 第二節 離乳後多系統消耗性症候群------------------------------------------------- 5 2.1 歷史背景與流行病學 --------------------------------------------------------- 5 2.2 臨床症狀與病理變化 --------------------------------------------------------- 6 2.3 PCV2的細胞組織親和性與主要感染之標的細胞 ----------------------- 7 2.4 PCV2與其他病原的共同感染 ----------------------------------------------- 8 2.5 PCV2與免疫的關係 ----------------------------------------------------------- 9 2.6 其他與PCV2相關之疾病或症候群 ---------------------------------------- 11 第三節 干擾素 --------------------------------------------------------------------------- 12 3.1 IFN-α之介紹 -------------------------------------------------------------------- 12 3.2 IFN-α之功用 -------------------------------------------------------------------- 13 3.3病毒感染與IFN-α之關係 ----------------------------------------------------- 14 第四節 CpG 雙核苷酸序列 ----------------------------------------------------------- 17 4.1 CpG 的介紹 --------------------------------------------------------------------- 17 4.2 CpG 的作用機制 --------------------------------------------------------------- 18 4.3 CpG ODNs於臨床上的應用 -------------------------------------------------- 19 4.4 CpG-S DNA於臨床上的副作用 --------------------------------------------- 19 第三章 實驗材料與方法 ------------------------------------------------------------------ 21 第一節 實驗設計 ------------------------------------------------------------------------ 21 1.1 實驗設計流程圖 --------------------------------------------------------------- 21 第二節 材料與方法 -------------------------------------------------------------------- 22 2.1 第二型環狀病毒 ( PCV2 ) 核酸定序 ------------------------------------- 22 2.2 聚合酶鏈鎖反應產物電泳分析 --------------------------------------------- 23 2.3 核酸序列比對 ------------------------------------------------------------------ 23 3.1 第二型環狀病毒 ( PCV2 ) 分離 ------------------------------------------- 24 3.2 第二型環狀病毒 ( PCV2 ) 力價測定 ------------------------------------- 24 3.3 免疫螢光染色分析法 ( immunofluorescence assay, IFA ) -------------- 25 4.1 豬肺臟巨噬細胞 ( porcine alveolar macrophages, PAMs ) 細胞培養液 ( RPMI-C ) ------------------------------------------------------------------ 25 4.2 豬肺臟巨噬細胞 ( PAMs ) 之採集 ---------------------------------------- 26 4.3 第二型環狀病毒 ( PCV2 ) 的選擇 ---------------------------------------- 26 4.4 第二型環狀病毒 ( PCV2 ) 感作 ------------------------------------------- 26 5.1 豬腎細胞株 ( PK-15 cell line ) ----------------------------------------------- 27 5.2 細胞培養液 ( DMEM-C ) ----------------------------------------------------- 27 5.3 細胞清洗液 ( D-PBS ) --------------------------------------------------------- 27 5.4 D (+) glucosamine, hydrochloride --------------------------------------------- 27 5.5 Hanks’ balanced salt solution ( HBSS ) -------------------------------------- 27 6.1 IFN-α之生物測定 -------------------------------------------------------------- 28 6.2 豬水泡性口炎病毒 ( vesicular stomatitis virus；VSV ) ------------------ 28 7.1 統計分析 ------------------------------------------------------------------------ 29 第四章 實驗結果 --------------------------------------------------------------------------- 30 第一節 PCV2之病材收集、病毒檢測 ---------------------------------------------- 30 第二節 PCV2基因序列定序 --------------------------------------------------------- 30 第三節 PCV2病毒分離與力價測定 ------------------------------------------------- 33 第四節 PCV2基因序列分析 ---------------------------------------------------------- 35 4.1 PCV2田間分離株 -------------------------------------------------------------- 35 4.2 PCV2 基因庫 ( GenBank ) 發表株 ---------------------------------------- 38 4.3 PCV2基因序列全長與有抑制能力ODN之間的相關連性 ------------ 40 第五節 豬肺泡巨噬細胞感染PCV2結果 ----------------------------------------- 48 第六節 IFN-α生物活性測定結果 --------------------------------------------------- 49 第五章 討論 --------------------------------------------------------------------------------- 54 第六章 參考文獻 --------------------------------------------------------------------------- 65 表 次 Table 1 The basic information and genetic analysis result of samples collected from the field ---------------------------------------------------------------------- 36 Table 2 The total number of reported PCV2 and genomic sequence characterization from various countries reported on the GenBank nucleotide database at the National Center for Biotechnology Information ( NCBI ) ------------------------------------------------------------- 39 Table 3 The total member of reported PCV2 and genomic sequence characterization from various countries reported before and after the year of 2005 on the GenBank nucleotide database at the National Center for Biotechnology Information ( NCBI ) ------------------------------------------ 40 Table 4 The frequency of PCV2 from various sources containing the reported inhibitory ODN ------------------------------------------------------------------- 42 圖 次 Figure 3.1 The figure of completed PCV2 DNA genomes, the three pairs of primers, and the inhibitory ODN site --------------------------------------- 23 Figure 4.1 The detection of PCV1 and PCV2 by multiplex PCR -------------------- 30 Figure 4.2 The result of partial amplification of porcine circovirus type 2 ( PCV2 ) genome in the segment of 491-1586 nucleotides from purified PCV2 and PCR PCV2-positive tissue samples ----------------- 31 Figure 4.3 The result of partial amplification of porcine circovirus type 2 ( PCV2 ) genome in the segment of 1323-496 nucleotides from purified PCV2 and PCR PCV2-positive tissue samples ----------------- 32 Figure 4.4 The result of partial amplification of porcine circovirus type 2 ( PCV2 ) genome in the segment of 1-741 nucleotides from purified PCV2 and PCR PCV2-positive tissue samples ---------------------------- 33 Figure 4.5 The PCV2 antigens in PCV2-inoculated PK-15 cells. PCV2-positive signals are noted by IFA under the low power field. ( 40x ) ------------ 34 Figure 4.6 The intracytoplasmic PCV2 antigens in PCV2-inoculated PK-15 cells------------------------------------------------------------------------------ 34 Figure 4.7 The intranuclear PCV2 antigens in PCV2-inoculated PK-15 cells ----- 35 Figure 4.8 Alignment of the full length of the nucleotide sequences of GenBank-reported Taiwan PCV2 stains and field-collected PCV2 in this study ------------------------------------------------------------------------ 43 Figure 4.9 Pair comparison of the complete PCV2 nucleotide sequences of 21 strains selected in this study -------------------------------------------------- 47 Figure 4.10 Phylogenetic analysis of PCV2 complete genomes among 9 GenBank reported PCV2 strains and 12 field-collected PCV2 --------------------- 48 Figure 4.11 The distribution of PCV2 antigens in PCV2-inoculated porcine alveolar macrophages ( PAMs ) --------------------------------------------- 49 Figure 4.12 The morphological characteristics of Madin-Darby bovine kidney ( MDBK ) cells with and without the inoculation of vesicular stomatitis virus ( VSV ) ------------------------------------------------------- 51 Figure 4.13 The correlation between the production level of interferon alpha ( IFN-α ) and the multiplicity of infection ( MOI ) of PCV2 inoculated ----------------------------------------------------------------------- 52 Figure 4.14 The IFN-αexpression levels in the supernatants of porcine alveolar macrophages ( PAMs ) inoculated with various field isolates ---------- 532076901 bytesapplication/pdfen-USPCV2PMWSIFN-αCpG motifsODNsthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/60015/1/ntu-96-R94629001-1.pdf