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標題: | 豬隻口服豬生殖與呼吸綜合症病毒GP5-GP6與大腸桿菌熱溶解性腸毒素次單位B(LTB)融合蛋白之基因轉殖菸草癒合組織之免疫效力評估 Evaluation of the immunogenicity and efficacy in pigs fed with a transgenic plant callus expressing the recombinant fusion proteins of porcine reproductive and respiratory syndrome virus GP5-GP6 and Escherichia coli heat-labile enterotoxin B subunit |
作者: | Yenh-An Wu 吳嶽安 |
指導教授: | 鄭謙仁(Chian-Ren Jeng) |
共同指導教授: | 龐飛(Victor-Fei Pang) |
關鍵字: | 豬生殖與呼吸綜合症,基因轉殖菸草口服次單位疫苗,共同表現GP5-GP6及LTB基因轉殖菸草癒合組織,單獨表現GP5的基因轉殖菸草癒合組織,共同表現GP5-GP6及LTB的基因轉殖菸草葉片之冷凍乾燥粉末,無動物蛋白表現的普通菸草, Porcine reproductive and respiratory syndrome,transgenic tobacco subunit vaccine,GP5-GP6-LTB tobacco callus,GP5 tobacco callus,GP5-GP6-LTB lyophilized tobacco leaves,wild-type tobacco fresh leaves, |
出版年 : | 2014 |
學位: | 碩士 |
摘要: | 豬生殖與呼吸綜合症(Porcine reproductive and respiratory syndrome;PRRS)是自1987 年首度在美國及1990年在歐洲被報導的新興豬病,近年來已成為肆虐全球的豬隻病毒性疾病,本省亦於民國八十二年證實有本病的入侵。臨床上,本病主要造成母豬厭食、懷孕後期的流、死產和木乃伊胎、產下虛弱仔豬、懷孕周期延遲和增加幼豬離乳前的死亡率;對各年齡層豬隻可引起呼吸急促、發燒、間質性肺炎等類感冒症狀,罹病豬隻也常因二次性細菌或病毒感染造成病情加劇甚至死亡,豬生殖與呼吸綜合症為目前造成全球養豬產業嚴重損失的重大豬隻病毒疾病之一。PRRSV疫苗早在1990年已開始使用,但至目前為止,仍無法有效預防或控制PRRSV。疫苗控制 PRRSV的困難點為PRRSV是一種 RNA 病毒,其具有高度抗原變異性,使豬隻對於變異的病毒株無法產生足夠的免疫反應,加上PRRSV在宿主所誘發的免疫反應相當複雜,且具有迴避宿主免疫系統的機制,使發展有效疫苗具有相當困難性。之前的研究顯示表現PRRSV GP5-LTB(Escherichia coli heat-labile enterotoxin B subunit;大腸桿菌熱溶解性次單位B融合蛋白)基因轉殖菸草口服次單位疫苗,已能誘發有效的免疫反應和一定程度的保護力,但由於基因轉殖植物疫苗在製程上面臨台灣法規的限制,故本實驗以能夠於培養基上大量生長的菸草癒合組織等不同劑型給予豬隻,嘗試解決疫苗發展困境。本實驗設計為16隻3-4週齡無特定病原(specific pathogen free; SPF)豬隻以口服的方式,分別給予不同劑型之口服疫苗,疫苗劑型分別為:共同表現GP5-GP6及LTB基因轉殖菸草癒合組織(GP5-GP6-LTB tobacco callus);單獨表現GP5的基因轉殖菸草癒合組織(GP5 tobacco callus);共同表現GP5-GP6及LTB的基因轉殖菸草葉片之冷凍乾燥粉末(GP5-GP6-LTB lyophilized tobacco leaves);無動物蛋白表現的普通菸草(wild-type tobacco fresh leaves),每次間隔兩週共三次,於免疫8週後,攻毒3 x 104 TCID50/mL PRRSV田間毒,每隻豬鼻腔及肌肉注射各1 mL,在動物攻毒三週後犧牲採樣,進行基因轉殖植物癒合組織或冷凍乾燥粉末等不同劑型在豬隻的安全性和免疫效力評估。本實驗結果顯示,連續三次服用特定劑量的菸草癒合組織或冷凍乾燥粉末,於豬隻生長表現上並無不良影響。體液性免疫反應結果顯示,於攻毒前anti-PRRSV ORF7含量低於其Cut-off value 0.4,顯示豬隻為陰性未感染PRRSV,anti-PRRSV ORF7 antibody含量於第8週後明顯上升,推測其結果與攻毒有關;anti-PRRSV ORF5 antibody方面,在第3-8週間,A組數值持續略高於其他組別,顯示抗體含量有輕微上升趨勢,但統計上無顯著差異;而第6、8、9週連續稀釋的結果,亦顯示於第6週三組疫苗組皆略高於陰性陰性控制組,但未達顯著差異。在PRRSV含量檢測結果顯示,在血清、組織和周邊血液白血球的PRRSV含量,A、B、C三組普遍略高於控制組D組,統計上四組數值多無顯著差異。總結本動物實驗結果,於攻毒前可觀察到三組疫苗組有anti-PRRSV ORF5 antibody含量微幅上升,但未觀察到有顯著差異的基因轉殖菸草植物相關性抗PRRSV的免疫反應及保護力。 Porcine reproductive and respiratory syndrome (PRRS), a disease initially described in the United States in 1987 and in Europe in 1990, has caused tremendous economic losses in the swine industry worldwide. PRRS was also diagnosed in Taiwan in 1993. The reproductive form of this disease is characterized by anorexia, late term abortions (days 107-112 of gestation), increased numbers of stillborn, mummified, weak-borne pigs, delayed return to estrus, and increased preweaning mortality. Pronounced hyperpnea, fever, interstitial pneumonia, and mild flu-like signs occur in nursing and growing pigs. Infection of PRRS virus (PRRSV) also predisposes pigs to bacterial and viral infection; therefore, PRRS has been one of the most economically significant viral diseases in the swine industry. Although PRRSV modified live-attenuated vaccines (MLV) and inactivated vaccines have been available in 1990s, the efficacy of both vaccines is limited. The control of PRRSV by vaccination is challenging because of the significant antigenic diversity known to exist among all strains of this RNA virus and the resultant insufficient generation of protective immunity toward heterologous PRRSV strains. The complicate evading immune mechanism by PRRSV makes it difficult to develop vaccine. Our previous study has demonstrated that GP5-LTB transgenic tobacco subunit vaccine could induce mucosal and systemic immune responses and offer acceptable protection against PRRSV infection in pigs. Due to the restrictions of Taiwan regulation on transgenic plant vaccines, different forms of transgenic tobacco plant callus provides the alternative way to resolve this problem. The objective of the present study was to investigate the immunogenicity and efficacy of GP5-GP6-LTB tobacco callus, GP5 tobacco callus, GP5-GP6-LTB lyophilized tobacco leaves, and wild-type tobacco fresh leaves after oral administration. Sixteen 4-6 week old pigs, seronegative for PRRSV, were randomly divided into 4 groups. Pigs were given orally three consecutive doses at a 2-week interval, then were intranasally and intramuscularly challenged with 2 ml of 3x104 TCID50/mL PRRSV at 8 weeks post-initial immunization (WPI) and sacrified 3 weeks later. The efficacy of the three different types of transgenic tobacco plant was evaluated based on clinical parameter, immune responses as well as viral load. The result of changes in the body weights and daily weight gain showed no statistical significance among groups, and no clinical signs were observed throughout the study. In humoral immunity, the levels of serum anti-PRRSV ORF7 antibodies and anti-PRRSV ORF5 antibodies in three vaccinated groups elevated gradually after the intranasal and intramuscular challenge with PRRSV inoculation. The levels of serum anti-PRRSV ORF5 antibodies in three vaccinated groups elevated mildly from week 3 to week 8. In serial dilution test, the levels of serum anti-PRRSV ORF5 antibodies in three vaccinated groups did not have statistically significant differences from control group, while the three vaccinated groups generally showed slightly higher than control group at week 6. The levels of PRRSV viral load of serum, tissues and peripheral blood mononuclear cells of the GP5-GP6-LTB tobacco callus, GP5 tobacco callus, and GP5-GP6-LTB lyophilized tobacco leaf groups were not significantly different from the control group (wild-type tobacco fresh leaves). In summary, the present study has demonstrated that various types of transgenic plants could induce slightly low level of anti-PRRSV antibody. However, no detectable statistically significant vaccine-associated anti-PRRSV immunogenicity and protection in the pigs treated with the various types of transgenic plants. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/56654 |
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