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Characterization of the monoclonal antibody against pseudorabies virus glycoprotein E and its application to diagnostic reagent development
swine herpesvirus type 1
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|摘要:||假性狂犬病毒(pseudorabies virus, PRV)引起豬隻重要傳染病-假性狂犬病(pseudorabies, PR)。PRV醣蛋白gE之缺損並不影響病毒的生長但會使其毒力下降，常做為標示疫苗(marker vaccine)使用，而檢測gE抗體成為重要的血清診斷依據。本研究之目的為製備抗PRV gE之單株抗體並進一步開發做為簡單操作且價格低廉的診斷試劑，以利豬場PR之疫情監控。首先利用酵母菌Pichia pastoris表現系統進行gE N端第31-372個胺基酸片段的大量表現，接著以Western blotting進行分析，此表現蛋白(gEN31-372) 約於分子量55-72kDa處有明顯條帶，經鎳離子親和管柱純化後，以此蛋白免疫BALB/c小鼠，在確定抗體揚升之後進行融合瘤實驗，並進而得到六株持續分泌專一性抗gE抗體之單株抗體(monoclonal antibody, MAb)。選取其中表現穩定的三株單株抗體(FY1-4, FY2-10, FY3-5)進行特性分析，以間接免疫螢光分析法(indirect immunofluorescence assay, IFA)分析顯示能特異性辨認PRV感染之PK-15細胞，而中和試驗(neutralization assay)之結果顯示不具中和抗體能力，以blockng ELISA測試之結果也不具有和豬隻血清競爭epitope的能力。MAbs FY1-4及FY3-5之抗體亞型為IgG1，MAb FY2-10亞型則為IgG2a，三者輕鏈皆為kappa chain。抗原決定位分析(epitope mapping)顯示MAb FY1-4可辨認gE胺基酸135-268片段，而其餘兩株則皆無法辨別任一片段。進一步應用MAb FY1-4做為捕捉抗體，並以gEN31-372做為抗原建立間接三明治(indirect sandwich) ELISA抗體檢測方法，分別以商品化blocking ELISA所測定之48支陽性及48支陰性豬隻血清進行分析，當OD405讀值之cut-off值設定為0.5225時，結果顯示敏感性為85.4% (41/48)而特異性可達89.6% (43/48)。|
Pseudorabies is a significant infectious disease in pig, caused by the pseudorabies virus (PRV). Deletion of the PRV glycoprotein E (gE) does not affect the growth of virus, but reduces the viral virulence. PRV gE is also considered to be a candidate for effective marker vaccine, and the antibody against gE in swine becomes an important indicator for serological diagnosis. The purposes of this study are to prepare and characterize the monoclonal antibody (MAb) against PRV gE, and employ gE to develop diagnostic reagents. At first, the PRV gE subunit consisting of the amino acid (a.a.) residues 31-372 was expressed by yeast Pichia pastoris expression system, and the expressed protein (gEN31-372) was about 55-72 kDa in size identified by Western blotting analysis. The gEN31-372 was purified by metal affinity chromatography and then used as antigen to immunize BALB/c mice for hybridoma experiment. The MAbs FY1-4, FY2-10 and FY3-5 were able to specifically recognize the PRV gE in immunofluorescence assay but did not have neutralizing activity in the neutralization assay. In addition, MAbs FY1-4 and FY3-5 were determined to be the type of IgG1 subclass, but MAb FY2-10 belonged to the subclass of IgG2a. The epitope recognized by MAb FY1-4 was mapped to the region of a.a. 135-268 of gE. However, MAbs FY2-10 and FY3-5 were not able to recognize any region of gE. Furthermore, MAb FY1-4 was applied as a capture antibody to develop a gEN31-372-based indirect sandwich ELISA for detecting swine antibody against gE. The assay was demonstrated to be high sensitivity and specificity of 85.4% (41/48) and 89.6% (43/48), respectively, and may provide an alternative method for routine serodiagnosis of PRV infection at herd level.
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