Detection of Subgroup J Avian Leukosis Virus with Monoclonal Antibodies to the N-terminus of Envelope Protein gp85

應用抗膜蛋白質gp85N端單源抗體偵測家禽白血病J亞型病

王金和(Ching-Ho Wang) ； 陳慧真(Hui-Jen Chen) ； 張清華(Allan Ching-Hwa Chang)

《臺灣獸醫學雜誌》 33卷3&4期 (2007/12) Pp. 144-151

https://doi.org/10.7009/TVJ.200712.0144

摘要

由於檢測群特異抗原的方法無法分別外源性及內源性家禽白血病病毒，因此發展一種具特異性檢測家禽白血病J亞型病毒抗原的方法有其必要性。為了生產對抗病毒膜蛋白質表面部份的單源抗體，選取1株台灣分離的家禽白血病J亞型病毒，將其膜蛋白質表面部份(gP85)N端選殖至pRSET及真核表現載體，進行表現，並在小白鼠製造單源抗體，結果獲得2株單源抗體mAb14及mAb22。以免疫墨點法證明此抗體可以特異與家禽白血病J亞型病毒及其原核表現的gp85蛋白質反應，而無法與原核表現家禽白血病A亞型的gp85蛋白質反應。mAb14與gp85表現蛋白質Jgp85N反應但無法與ALV-J病毒反應，顯示mAb14可能辨識gp85蛋白質的內部序列。反之mAb22可以與gp85表現蛋白質Jgp85N及ALV-J病毒，顯示mAb22可以辨識gp85蛋白質的外部序列；以西方墨點法分析mAb22仍可以與變性的蛋白質結合，顯示其抗原決定位不受蛋白質構型的影響。進一步進行抗原ELISA證明mAb22可以偵測到表現蛋白質Jgp85N，因此mAb22可以做為家禽白血病J亞型病毒及其蛋白質的診斷之用。

關鍵字

gp85蛋白質 ； 單源抗體 ； 家禽白血病J亞型病毒

並列摘要

There is a global need to develop a specific antigen detection method for subgroup J avian leukosis virus (ALV-J) since the group-specific antigen detection is unable to distinguish exogenous from endogenous viruses. In order to obtain monoclonal antibodies specific to ALV-J, the N-terminus of the gp85 unit of the envelope protein from a Taiwanese ALV-J was cloned in pRSET and then in a eukaryotic expression vector for monoclonal antibody production in mice. After selection from hybridoma cells, two monoclonal antibodies, mAbl4 and mAb22, were obtained. Both monoclonal antibodies specifically detected ALV-J and the expressed gp85 protein from ALV-J but not that from subgorup A avian leukosis virus by immunodot. The mAbl4 reacted with expressed Jgp85N protein but not the ALV-J virion, it might detect the internal part of the gp85 protein. On the contrary, mAb22 recognized both expressed protein and the ALV-J virion. Thus, mAb22 reacts with the amino acids at the external of the gp85 protein. The epitope recognized by mAb22 is linear and conformation-independent since it recognized expressed Jgp85N and viral gp85 by Western blotting with denaturing protein. Furthermore, we developed an antigen capture ELISA system to detect expressed gp85 protein with mAb22. Thus, mAb22 might be useful for the diagnosis of ALV-J virus or ALV-J viral proteins.

並列關鍵字

gp85 protein ； monoclonal antibody ； subgroup J avian leukosis virus