Studies on the Expression of Recombinant ORF3 Fusion Proteins of Porcine Circovirus Type 2 in Transgenic Tobacco

碩士 === 中國文化大學 === 生物科技研究所 === 102 === Porcine circovirus type 2 (PCV2) is the causative agent of postweaning multisystemic wasting syndrome in pigs and has impact on swine-producing industry worldwide. The open reading frame 3 (ORF3) of PCV2 which encodes an 11.9 kDa ORF3 protein has been found to b...

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Main Authors: Tran Quang Dan, 陳光瑩
Other Authors: Huang, Pung-Ling
Format: Others
Language:en_US
Published: 2014
Online Access:http://ndltd.ncl.edu.tw/handle/xwceag
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spelling ndltd-TW-102PCCU01110262019-05-15T21:23:12Z http://ndltd.ncl.edu.tw/handle/xwceag Studies on the Expression of Recombinant ORF3 Fusion Proteins of Porcine Circovirus Type 2 in Transgenic Tobacco 利用菸草表達豬第二型環狀病毒融合蛋白之研究 Tran Quang Dan 陳光瑩 碩士 中國文化大學 生物科技研究所 102 Porcine circovirus type 2 (PCV2) is the causative agent of postweaning multisystemic wasting syndrome in pigs and has impact on swine-producing industry worldwide. The open reading frame 3 (ORF3) of PCV2 which encodes an 11.9 kDa ORF3 protein has been found to be involved in the pathogenesis of PCV2 infection by its apoptotic activity. The ORF3–based antigens thus represent a traditional approach to develop efficacious oral vaccines for immunization against PCV2. In this study, various ORF3-containing expression vectors, namely pGKF/U-ORF3, pGKF/U-L-ORF3, pGKF/U-ORF2-3b, and pGKF/U-L-ORF2-3b, were transformed to tobacco plants in order to produce the respective subunit recombinant vaccines. Following the Agrobacterium tumefaciens-mediated transformation of 200 leaf samples, 20 ORF3, 15 L-ORF3, 14 ORF2-3b, and 14 L-ORF2-3b putative transgenic tobacco plants were regenerated on the selective medium supplied with 200 mg/L kanamycin and showed GUS expression. The expected sequences of respective ORF3-containing trangenes, 359 bp, 369 bp, 1056 bp, and 1439 bp were amplified from genomic DNA of 19 ORF3 (9.5%), 14 L-ORF3 (7.0%), 12 ORF2-3b (6.0%), and 12 L-ORF2-3b (6.0%) GUS-positive transgenic plants, using specific pair of primers in PCR analysis. On the other hand, integration of 1-2 copies of independent ORF3-containing transgenes into tobacco genome was determined by means of Southern hybridization. The mRNA transcripts of particular transgenes were also found in the PCR-positive transgenic plants. Particularly, expression of respective ORF3 fusion proteins was qualitatively examined via immunobloting (Western blot) assay. However, the results have not clearly shown presence of target proteins, due to either low level of expression or interfering background. Huang, Pung-Ling 黃鵬林 2014 學位論文 ; thesis 93 en_US
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description 碩士 === 中國文化大學 === 生物科技研究所 === 102 === Porcine circovirus type 2 (PCV2) is the causative agent of postweaning multisystemic wasting syndrome in pigs and has impact on swine-producing industry worldwide. The open reading frame 3 (ORF3) of PCV2 which encodes an 11.9 kDa ORF3 protein has been found to be involved in the pathogenesis of PCV2 infection by its apoptotic activity. The ORF3–based antigens thus represent a traditional approach to develop efficacious oral vaccines for immunization against PCV2. In this study, various ORF3-containing expression vectors, namely pGKF/U-ORF3, pGKF/U-L-ORF3, pGKF/U-ORF2-3b, and pGKF/U-L-ORF2-3b, were transformed to tobacco plants in order to produce the respective subunit recombinant vaccines. Following the Agrobacterium tumefaciens-mediated transformation of 200 leaf samples, 20 ORF3, 15 L-ORF3, 14 ORF2-3b, and 14 L-ORF2-3b putative transgenic tobacco plants were regenerated on the selective medium supplied with 200 mg/L kanamycin and showed GUS expression. The expected sequences of respective ORF3-containing trangenes, 359 bp, 369 bp, 1056 bp, and 1439 bp were amplified from genomic DNA of 19 ORF3 (9.5%), 14 L-ORF3 (7.0%), 12 ORF2-3b (6.0%), and 12 L-ORF2-3b (6.0%) GUS-positive transgenic plants, using specific pair of primers in PCR analysis. On the other hand, integration of 1-2 copies of independent ORF3-containing transgenes into tobacco genome was determined by means of Southern hybridization. The mRNA transcripts of particular transgenes were also found in the PCR-positive transgenic plants. Particularly, expression of respective ORF3 fusion proteins was qualitatively examined via immunobloting (Western blot) assay. However, the results have not clearly shown presence of target proteins, due to either low level of expression or interfering background.
author2 Huang, Pung-Ling
author_facet Huang, Pung-Ling
Tran Quang Dan
陳光瑩
author Tran Quang Dan
陳光瑩
spellingShingle Tran Quang Dan
陳光瑩
Studies on the Expression of Recombinant ORF3 Fusion Proteins of Porcine Circovirus Type 2 in Transgenic Tobacco
author_sort Tran Quang Dan
title Studies on the Expression of Recombinant ORF3 Fusion Proteins of Porcine Circovirus Type 2 in Transgenic Tobacco
title_short Studies on the Expression of Recombinant ORF3 Fusion Proteins of Porcine Circovirus Type 2 in Transgenic Tobacco
title_full Studies on the Expression of Recombinant ORF3 Fusion Proteins of Porcine Circovirus Type 2 in Transgenic Tobacco
title_fullStr Studies on the Expression of Recombinant ORF3 Fusion Proteins of Porcine Circovirus Type 2 in Transgenic Tobacco
title_full_unstemmed Studies on the Expression of Recombinant ORF3 Fusion Proteins of Porcine Circovirus Type 2 in Transgenic Tobacco
title_sort studies on the expression of recombinant orf3 fusion proteins of porcine circovirus type 2 in transgenic tobacco
publishDate 2014
url http://ndltd.ncl.edu.tw/handle/xwceag
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