發展與分析具檢測雞貧血病毒潛力之單株抗体

雞貧血病毒 (Chicken anemia virus, CAV) 是環狀病毒科唯一的Gyrovirus屬成員。CAV會造成雞隻貧血、淋巴組織萎縮和免疫缺陷，為一免疫抑制病毒。CAV的genomic DNA為一單股環狀DNA，會轉錄出一段帶有部份重疊閱讀框架的mRNA，並且轉譯出三種蛋白分別為VP1、VP2及VP3。VP1蛋白是雞傳染性貧血病病毒的唯一外殼蛋白。由於VP1蛋白對於CAV的生活史扮演相當重要的角色，因此若能表現VP1蛋白並產製具專一性抗体，相信對於CAV的防治與診斷將有極大的助益。為了取得大量VP1蛋白做為研究材料，我們將VP1基因構築於大腸桿菌中表現重組蛋白之表現載體，並經過親和性管柱層析純化後取得VP1蛋白。並將純化後蛋白以皮下免疫與脾臟免疫BalB/c小鼠，並每週檢測小鼠血清之抗體效價。於血清效價達到高值後，取脾臟細胞與骨髓瘤細胞進行融合以產生融合瘤細胞，並以酵素連結反應篩選出對VP1具有高度專一性之單株抗体E-3。E-3單株抗体經由腹水生產後進行A蛋白親和性純化已取得大量單株抗体，並對E-3抗体進行親和力分析與免疫球蛋白亞型鑑別，最後我們將E-3單株抗体用於分離感染雞貧血病毒之雞隻組織中的病毒顆粒，證實E-3抗体對於VP1蛋白具有高度專一性。未來E-3單株抗体將可以用於開發雞貧血病毒之免疫檢測套組或純化感染雞隻組織中病毒顆粒，並於開發雞貧血病毒之次單位疫苗時，作為免疫效價評估依據。

關鍵字

免疫親和性管柱；單株抗体；雞貧血病毒； VP1蛋白

並列摘要

Chicken anemia virus (CAV) is an important viral pathogen and causes anemia and severe immunodeficiency syndrome in chickens worldwide. In this study, a potential diagnostic monoclonal antibody against CAV VP1 protein was developed that can recognize the CAV antigen precisely for diagnostic and virus recovery purposes. The VP1 gene of CAV encoding the N-terminus deleted VP1 protein, VP1Nd129, was cloned into an E. coli expression vector. After IPTG induction, VP1Nd129 protein was shown to be successfully expressed in E. coli. By ELISA screening using two coating antigens, purified VP1Nd129 and CAV-infected liver tissue lysate, E3 MAb was identified to have higher reactivity against VP1 protein than the other positive clones by the limiting dilution method from 64 clones. Using immunohistochemistry, the presence of the VP1-specific MAb, E3, was confirmed using CAV-infected liver and thymus tissues as positive-infected clinical samples. Additionally, CAV particle purification was also established using an immunoaffinity column set up with E3 MAb. The developed monoclonal antibody, E3 MAb, it will not only be very useful for detecting CAV infection and for histopathology studies of infected chickens but may also be employed to purify CAV particles in the future.

並列關鍵字

immunoaffinity column ； monoclonal antibody ； chicken anemia virus

參考文獻

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發展與分析具檢測雞貧血病毒潛力之單株抗体

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