開發豬環狀病毒二型血清抗體之酵素連結免疫吸附分析法

Abstract

目前，豬環狀病毒二型（porcine circovirus 2; PCV2）在豬場是與離乳豬多系統性消耗症候群有關的一種重要緊急病原。此病常發生於5-12週齡豬隻。 臨床上，發生離乳豬多系統性消耗症候群（post-weaning multisystemic wasting syndrome；PMWS）症狀的豬隻，呈現體重持續下降、呼吸道症狀及偶爾可見到黃疸。 本研究的目的乃是發展一PCV2病毒之重組結構外殼蛋白（recombinant capsid protein; rORF2）， 以為檢測血清PCV2抗體之酵素連結免疫吸附分析法 (enzyme-linked immunosorbent assay；ELISA) 套組之抗原。根據豬環狀病毒二型的ORF2基因，從5端123鹼基對，開始合成專一引子。經由聚合酶鏈反應（polymerase chain reaction ；PCR）放大基因產物，選殖標的基因至pMAL-c2X基因載體，重組蛋白質表現於大腸桿菌。純化的重組蛋白質 (MBP-D41) 當作ELISA檢測套組之抗原，作為檢測豬環狀病毒二型抗體之用。 為了探討豬場內，PCV2抗體的產生與病毒循環情形之關係，我們將每一豬場，分成以下階段：1週齡、3-4週齡、8週齡、肉豬（70-80Kg）及母豬，每場總共收集50個血液樣本。 經由商業套組及自行開發的診斷套組，兩者檢測從野外豬場所採集的血清PCV2抗體。ELISA法的檢測結果，抗體陽性豬隻之比例為：90%（1週齡）、47.4 %（3-4週齡）、20 %（8週齡）、97.5 %（肉豬）、85 %（母豬）。出生後，1-8週抗體的分佈曲線呈下降趨勢。而在8週齡時，幾乎偵測不到PCV2的抗體，此時對病毒感染之敏感性增加。 檢測PCV2在不同豬隻生長階段之抗體結果，發現自行開發的ELISA與商業套組，並無明顯不同。這些結果顯示，重組蛋白MBP-D41是一潛在良好的ELISA檢測抗原。

Currently, porcine circovirus 2 (PCV2) is an important emerging pathogen associated with post-weaning multisystemic wasting syndrome ( PMWS) in pig farm. This syndrome of disease occurs commonly in 5-12-week-old pigs. Clinically, PMWS is characterized by progressive weight loss, respiratory signs, and occasional the jaundice in pigs. The objective of this study was to develop a recombinant protein of the viral capsid protein (ORF2) as antigen of enzyme-linked immunosorbent assay (ELISA) kit for the detection of antibody to porcine circovirus type 2. Specific primers were synthesized according to PCV2 ORF2 DNA sequence starting from the 123 bp at 5’ end. The PCR amplicom were cloned into pMAL-c2X vector and expressed in E. Coli. The purified protein (MBP-D41) was used as ELISA antigen for detecting PCV2 antibody. To investigate the dynamics of PCV2 antibody production in relation to virus protection or circulation within the farm, we collected 50 blood samples from each farm at the following stages: 1 week, 3-4 weeks, 8 weeks, finishing pig (70-80 kg), and sow. Serological tests of PCV2 antibody were performed with commercial and “recombinant antigen (MBP-D41)” diagnostic kits in wild field. The average percentage of pigs with PCV2 antibody response in ELISA test was 90% (1 week), 47.4% (3-4 weeks), 20% (8 weeks), 97.5% (finishing pig) and 85% (sow), respectively. The distribution of the PCV2 antibody decreased from one to eight –week-old pigs. The level of antibody was almost undetectable at eight-week-old pigs. Consequently, the sensitivity to viral infection was found to be increased. There was no significant difference between our developing diagnostic kit and the commercial diagnostic kit for the detection of PCV2 antibody in various growth stages of pig. These results demonstrate that the MBP-D41 protein is a potential candidate as an ELISA antigen.