豬第二型環狀病毒抗原決定位之篩選

Abstract

豬第二型環狀病毒 (PCV2)是一種小且無封套膜的去氧核醣核酸病毒，導致豬淋巴細胞消耗及嚴重影響養豬產業的病原。本研究的目的是評估特異性胜肽的抗原性和免疫原性，並尋找有潛力的PCV2胜肽疫苗候選物。首先用胜肽檢測PCV2感染的豬血清及胜肽免疫的小鼠血清，進行免疫測試。其中分析合成胜肽（C1，C2，C3，N1，N2和N3）與PCV2感染的田間豬群血清的結合反應。本研究按每季逢機選取分娩仔豬，每胎距平均體重最近之仔豬公母各一頭，共從11頭母豬中選取仔豬22頭，試驗中的畜試黑豬一號母豬及豬隻均未施打該病毒的疫苗。豬隻1日齡(吸到初乳)、1月齡、3月齡及6月齡時，抽血並以間接性酵素連結免疫吸附法進行血清的抗胜肽的抗體檢測。我們還探討了特定的胜肽在體液免疫中可能是免疫原的候選物。為了證明這些胜肽可以模擬天然PCV2殼鞘蛋白質(CP)的抗原決定位，我們利用鍵結的胜肽接種小鼠並製造單株抗體。我們使用抗原決定位拼圖法和液相阻斷免疫測定法找到單株抗體在PCV2殼鞘蛋白質的最小結合位置。數據顯示，PCV2感染的豬血清可以與PCV2殼鞘蛋白質的近氨基(N)端區(C1)，中間區(C2)和羧基(C)端區胜肽(C3)、開放閱讀框架三(ORF3)蛋白質(N1)、開放閱讀框架六(ORF6)蛋白質(N2)及開放閱讀框架九(ORF9)蛋白質(N3)等進行反應。本研究顯示可以藉由特異性合成胜肽(C3和N2)來生產製造具有識別PCV2病毒蛋白質能力的抗PCV2小鼠抗血清，亦發現三級結構或線型結構C端區序列的PCV2殼鞘胜肽(C3)僅存在PCV2感染的豬腎(PK)細胞之細胞核中，而PCV2病毒樣顆粒主要分佈位於豬腎細胞之細胞質中，PCV2的ORF 6蛋白質有異樣分佈位於豬腎細胞之細胞質中。此外，在殼鞘蛋白質的3度空間結構中，C1和C3的大多數胺基酸殘基都呈現在PCV2殼鞘蛋白質的表面上。另外，結果證實PCV2感染的豬隻在1日齡具有最高的抗C3特異性免疫球蛋白A(IgA)量(p <0.01，成對t-測試)。且證實PCV2感染的豬隻在1日齡、3月齡及6月齡的抗C3之免疫球蛋白G(IgG)的OD405值比在1月齡的血清為高(p <0.05，成對t-測試)。這暗示哺乳仔豬在出生24小時內，從初乳和母乳中吸收母體移行抗體(抗C3特異性免疫球蛋白A和G)。3月齡和6月齡的抗C3 之免疫球蛋白M(IgM)量高於1日齡(p <0.01，成對t-測試)。這些證據表明仔豬在斷奶後或3個月齡時，發展出適應性免疫反應，靠增加體內免疫球蛋白(抗C3特異性免疫球蛋白A、G和M)的合成。並確認抗C3的特異性抗體是PCV2感染豬隻的血清學標記。此外，利用PCV2b(PCV2b-1A/1B)殼鞘蛋白質的胜肽(C3)模擬羧基端區誘導小鼠體液免疫，並生產融合瘤，透過西方墨漬法分析證實所得單株抗體對PCV2殼鞘蛋白質的陽性反應性，這些單株抗體藉間接免疫螢光染色在PCV2b感染的豬淋巴細胞上顯示陽性訊號。單株抗體1H3分別與PCV2b-1A/1B，PCV2b-1C和PCV2a-2A的殼鞘蛋白質的羧基端上的三個最小線性抗原決定位 (P62, DPPLNP; P67, DPPLNPK; P73, LKDPPLKP)結合。單株抗體3B2僅與一個最小線性抗原決定位 (P59, KDPPLNP)結合。單株抗體6B8與兩個最小線性抗原決定位 (P59和P67)結合。在液相阻斷免疫測定(LPBI)方法中，P59中的核心基序(P62)，以自由游離狀態可以被單株抗體(3B2和6B8)識別，但在間接性酵素連結免疫吸附法(iELISA)檢測該固定形式P62，卻不能被單株抗體(3B2和6B8)所識別。然而，在間接性酵素連結免疫吸附法檢測中，單株抗體1H3可以識別P73，但是在液相阻斷免疫測定方法中，無法抑制C3和單株抗體1H3的相互作用結合。本研究還表明IgM單株抗體和有Ig缺陷的單株抗體具有廣泛的結合能力、中度特異性和低親和力。本研究證實單株抗體具有多能結合，這可能是抗體對PCV2b殼鞘蛋白質的羧基端的反應現象。

Porcine circovirus 2 (PCV2) is a small, non-enveloped DNA virus causing swine lymphocyte depletion and severe impact on the swine industry. The aim of this study was to evaluate the antigenicity and immunogenicity of specific peptides, and to seek the potential candidate of PCV2 peptide-based vaccine. It was initiating from peptides reacting with PCV2-infected pig sera and peptide-immunized mouse sera. The synthetic peptides (C1, C2, C3, N1, N2, and N3) were analyzed for the binding with field sera collected from PCV2-infected herds. This study involved 22 newborn piglets of TLRI Black Pig No.1 (TBP), delivered from 11 sows during 4 seasons of one year. One male and one female piglet were selected from each litter, which body weights were close to the average of their littermate’s. All of pigs had not been immunized with PCV2 vaccine. Blood samples from each pig were collected 4 times during this experiment: on the 1st day (after colostrum uptake), 1st month, 3rd month, and 6th month of life, respectively. Serum samples from these pigs were used to detect anti-PCV2 specific antibodies by an indirect enzyme-linked immunosorbent assay. We also explored specific peptides could be candidates of immunogen involved during humoral immunity. To demonstrate these peptides can mimic the epitopes present on the native PCV2 CP, we utilized the conjugated peptides to inoculate mice and generate mAbs. We generated mAbs and defined their minimal binding region on PCV2 CP using epitope mapping and liquid phase blocking immunoassay. The data showed that the sera from PCV2-infected pigs could react with the N-terminal (C1), middle region (C2), and C-terminal peptide (C3) of PCV2 capsid protein (CP), ORF3 protein (N1), ORF6 protein (N2) and ORF9 protein (N3). This study demonstrated that anti-PCV2 mouse antisera could be generated by specific synthetic peptides (C3 and N2) and recognized PCV2 viral protein. We found that the tertiary or linear form C-terminal sequence (C3) of PCV2 capsid peptide only appeared a local distribution in the nucleus of PCV2-infected PK cells, virus-like particles of PCV2 major appeared a local distribution in the cytoplasm, and ORF 6 protein of PCV2 were shown unusually in cytoplasm. Furthermore, most residues of the C1 and the C3 were presented on the surface of PCV2 CP, in the view of 3-D structure of the CP. The results indicated that these pigs had the highest C3-specific IgA level on Day 1 in 6 months of life (p < 0.01, paired Student’s t-test). These data demonstrated that PCV2-infected pigs had higher OD405 value of anti-C3 IgG on Day 1, Month 3 and Month 6 than that in Month 1 (p < 0.05, paired Student’s t-test). This suggested that suckling newborn piglets absorbed maternal transferring antibodies (C3-specific IgA and IgG) from colostrum and milk in the first 24 h. These pigs had higher anti-C3 IgM level in Month 3 and Month 6 than that on Day 1 (p < 0.01, paired Student’s t-test). This suggested that piglets developed the adaptive immune response by increase synthesis of globulin (C3-specific IgA, IgG, and IgM) at aged 3 months or after weaning. The specific antibody against the C3 were confirmed as the serological marker in PCV2-infected pigs. Further, we utilized the peptide (C3) mimetic carboxyl-terminus (C-terminus) of PCV2b CP (PCV2b-1A/1B) to induce humoral immunity for hybridomas preparation. The positive reactivity of the mAbs to PCV2 CP was demonstrated by western blot assay. Those mAbs also showed positive signals on PCV2b infected swine lymphocytes by indirect immunofluorescence staining. The mAb 1H3 bound to three minimal linear epitopes (P62, DPPLNP; P67, DPPLNPK; P73, LKDPPLKP), which was located at C-terminus of the capsid protein of PCV2b-1A/1B, PCV2b-1C, and PCV2a-2A respectively. The mAbs 3B2 bound to only one minimal linear epitopes (P59, KDPPLNP). The mAbs 6B8 bound to two minimal linear epitopes (P59 and P67). This data demonstrate the core motif (P62) within the P59 could be recognized by mAbs (3B2 and 6B8) in the free status by liquid phase blocking immunoassay (LPBI) but not be recognized in the fixed form on the plate by indirect ELISA (iELISA). However, the P73 could be recognized by mAb 1H3 by iELISA but no inhibition of the interactive binding of C3 and mAb 1H3 by LPBI. This study also indicated that IgM mAbs and defective Ig mAb have broad binding, moderate specificity and low affinity. This study confirm that mAbs have pluripotency of binding. It might be a phenomenon of antibody response to C-terminus of PCV2b CP.