猪环状病毒抗体检测方法之研发.doc

豬環狀病毒抗體檢測方法之研發 豬瘟研究組 潘居祥 副研究員 摘要豬第2型環狀病毒（porcine circovirus type 2; PCV2）包含2種主要開放讀碼區 open reading frames; ORFs ，ORF1 編碼病毒複製相關蛋白，ORF2 則編碼病毒殼體蛋白 capsid protein ，本試驗以原核系統表現 PCV2 之 ORF2 基因片段，研發 Indirect ELISA iELISA 法檢測 PCV2 抗體。以 RT-PCR 方式增幅 ORF2 基因，將 RT-PCR 產物以 TA TA cloning 選殖方式殖入 TOPO 載體，並以定序方式確認序列。將 ORF2 基因分別次選殖入 pET32a 及 pCold 兩種表現載體，經定序確認後質體分別轉形至 BL-21 DE3 菌株，以 IPTG 誘導表現重組蛋白並測試是否為水溶性，結果顯示，pCold 質體表現之重組蛋白為可溶性，pET32a 質體表現之重組蛋白呈現不可溶性包涵體。ORF2 重組蛋白以 Western blot 法測試顯示，可辨識PCV2單株及多株抗體。將純化之 ORF2 重組蛋白以 2 μg/ml 濃度披覆於 96 孔微量盤，以 iELISA 方式測試 2 倍連續稀釋之 PCV2 陽性豬血清，可呈線性曲線。自製套組與進口商品化 iELISA 套組同時檢測田間豬血清 PCV2 抗體價，測得 OD 值之相關系數為 0.9，顯示兩種套組對 PCV2 抗體之檢測結果呈現高度一致性。 Development of an indirect ELISA for the detection of antibodies to porcine circovirus. Chu-Hsiang Pan Abstract Porcine circovirus type 2 PCV2 genome contains two major open reading frames ORFs , ORF1 encoding the viral replication-associated protein and ORF2 encoding the viral capsid protein. This study was to express the ORF2 capsid protein and Indirect ELISA iELISA method was developed for detection of antibody to PCV2. Partial ORF2 gene of PCV2 was amplified by RT-PCR. The RT-PCR amplicons were cloned into a TOPO vector using TA cloning kit; correct clones were confirmed by sequencing. The target genes were subcloned into the pET32 and pCold expression vectors. The expression plasmids were transformed into host cell of BL-21 DE3 , then IPTG was used to induce the expression of cloned genes. The solubility of recombinant proteins was tested after induction. Results showed that pCold system expressed a soluble protein and pET32 system showed insoluble inclusion bodies. His tag protein purification kit was used to purify recombinant proteins. The ELISA plates were coated with 2 μg/ml of purified recombinant ORF2 protein and iELISA method was used to test serial dilutions of pig sera which were obtained from the experimental CSFV infected pigs. Results revealed that linear curve could be observed in the iELISA assay. Com