豬環狀病毒第二型ORF1、ORF2、ORF3基因重組蛋白的表現及純化

Expression and Purification the Recombinant Proteins of Porcine Circovirus Type2 ORFl, ORF2 and ORF3

郭村勇(Tsun-Yung Kuo) ； 陳裕森(Yu-San Chen) ； 高志賢(Zu-Shan Cau) ； 吳家霖(Ga-Lin Wu) ； 蘇于婷(Yu-Ting Su) ； 賴秀穗(Shiow-Suey Lai)

《宜蘭技術學報》 9期生物資源專輯 (2002/12) Pp. 117-130

https://doi.org/10.6179/nit.job.2002.09.22

摘要

豬環狀病毒第二型（Porcine circovirus type2, PCV2）是造成仔豬離乳後多系統耗弱症（Post-weaning multisystemic wasting syndrome, PMWS）之致病因子。該病毒可侵害離乳仔豬之呼吸系統、淋巴系統及腎、肝臟等器官並造成致害，近年來已在美加及歐洲地區造成養豬業的重大損害。本報告取1998年疑似受PCV2感染的台灣南部某豬場病材，進行核酸萃取後，以聚合酶鏈鎖反應法（Polymerase chain reaction, PCR）進行該病毒三個基因的增幅。結果ORFl（與病毒複製有關的基因）、ORF2（病毒的結構蛋白基因）、ORF3（其功能則尚未証明），都可順利被增幅。PCR產物經過純化、連同載體一起切割後，再以連接酶連接，送入原核表現載體pET24a中。在定序確認後，進行養菌及IPTG誘導，再以SDS-PAGE進行蛋白質電泳分析，結果發現ORF1、ORF3皆可以大量的表現重組蛋白，而ORF2的表現並不明顯。該三個重組蛋白皆可以西方墨漬法獲得確認。本報告所大量表現的基因重組蛋白，經過純化後，已具有開發成PCV2抗體檢驗試劑的雛型。

關鍵字

豬環狀病毒第二型 ； 仔豬離乳後多系統耗弱症

並列摘要

Porcine circovirus type2 (PCV2), a pathogen that infects the pig's respiration system, lymphoid tissues, kidney and liver, was the cause of porcine post-weaning multisystemic wasting syndrome. Recently, PCV2 caused the economic lose in America, Canada and Europe. Herein we extracted the nucleic acid of the suspect infected pigs from south of Taiwan in 1998 and the three genes (ORF1, viral replication related, ORF2, viral structure protein related and ORF3, function not clear) were all amplified by polymerase chain reaction. After the purification process, the PCR products were cloned into a E. coli expression vector, pET24a. After Sequences confirmation, the log phase cultures were induced by IPTG and the recombinant proteins were analyzed by SDS-PAGE. The results show that ORF1 and ORF3 genes could be highly expressed, besides ORF2. The recombinant proteins were confirmed by western blots. The purified recombination proteins should be the base for development of PCV2 antibody detection kit.

並列關鍵字

Porcine circovirus type2 ； post-weaning multisystemic wasting syndrome