Cloning, Characterization, and DNA Vaccine Development of Capsid Protein Gene from Classical Swine Fever Virus

• Main Authors: Wei-Nong Chang, 張緯農
• Other Authors: Wen-Hsing Chang
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/35965979243215727073

碩士 === 國立嘉義大學 === 農業生物技術研究所碩士班 === 93 === Classical swine fever (CSF), also known as hog cholera (HC), is a highly contagious and deadly disease in swine. The virus that can cause CSF has been identified and characterized as classical swine fever virus (CSFV). To prevent the outbreak of CSF in domestic swine caused by CSFV derived from wild fields, vaccination is a needed mean. The commonly used vaccine in Taiwan against CSF is an attenuated LPC strain of CSFV. However, applying this vaccine on domestic swine will increase the difficulty of detecting and distinguishing CSFV derived from the wild. To circumvent this dilemma, developing subunit vaccine or DNA vaccine other than utilizing the attenuated LPC strain of CSFV seems to be a better alternative. In this study, open reading frame (ORF) of capsid gene from the LPC strain of CSFV was replicated by reverse transcriptase (RT) and subsequently amplified by polymerase chain reaction (PCR). The PCR product was cloned and sequenced. By comparing at least five separately cloned sequences with the previously published one, two amino acids were found to mutate from Arginine (R) to Lysine (K) at residues 18 and 35 in the capsid protein. The cloned capsid gene was further constructed with a mammalian expression vector and used as a DNA vaccine to immunize chicken. By dot-blotting and Western blotting assays, the immunized chicken was shown to produce specific IgG in its serum that can recognize the protein extract derived from the LPC strain of CSFV. Moreover, the technology of RT-PCR that can amplify the CSFV’s capsid gene was also proven to be capable of detecting virus in the swine’s serum after being immunized by the attenuated CSFV of LPC strain. These observations establish the foundation of developing better vaccines against CSFV in the future.