The Study On Transgenic Lettuce (Lactuca Sativa L.) Expressing The Surface Structural Protein Of Classical Swine Fever Virus

• Main Authors: Ruei-Yuan Su, 蘇瑞媛
• Other Authors: Jinn-Chin Yiu
• Format: Others
• Language: zh-TW
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/10049626801986615131

碩士 === 國立宜蘭大學 === 園藝學系碩士班 === 100 === Swine fever, also called “classical swine fever” (CSF) or “hog cholera” (HC) is caused by classical swine fever virus (CSFV). It is a highly infectious viral disease in pigs that is typified by severe systemic hemorrhage and high mortality. Currently, the most widely used vaccines for controlling CSF are attenuated vaccines, which have the risk of virulence regression through individual subculture infections. Vaccines genetically engineered using plant bioreactors can overcome the disadvantages of attenuated vaccines. Therefore, we transferred the E2 glycoprotein containing epitopes on CSFV into lettuce to develop subunit vaccines for controlling CSF. We constructed the E2 gene, which is a surface structural protein of CSFV, in the transformation vector pBI121, and used the agrobacterium-mediated transformation method to transfer the E2 gene into lettuce. We used cotyledon explants to induce coleoptile regeneration, and then employed kanamycin for selection. The regeneration rates for “Fukuyama,” “Cui-Hua,” and “Romaine” lettuce plants were 6.7%, 2.5%, and 5.6%. We used polymerase chain reactions (PCR) to analyze the DNA of the intended transformed leaf genome and verified that the E2 gene could be inserted into the lettuce genome. The results of the reverse transcription (RT) PCR analysis showed that the E2 gene can perform normal transcriptions of mRNA. Western blot analysis verified that the transformed plants can accurately translate and synthesize the target protein. The molecular analysis results showed that the success rate of the T0 generation-transformed lettuce plants was 0.38%, 0.16%, and 2.27%. The T1 generation plants all showed E2 gene expression and accumulation of the target proteins, indicating that the target genes were steadily inherited in later generations. Enzyme-linked immunosorbent assay (ELISA) analysis of the progeny plants showed that the E2 protein expression in the Fukuyama lettuce accounted for 0.0031% to 0.0093% of the total amount of soluble protein, whereas that in the Cui-Hua lettuce accounted for 0.0012% to 0.0031%, and that in the Romaine lettuce accounted for 0.0020% to 0.0047%. These results demonstrated that using lettuce as a bioreactor to express E2 protein is feasible. We plan to conduct animal experiments in the future to verify its protective efficacy.