鄭謙仁2006-07-262018-07-092006-07-262018-07-092000http://ntur.lib.ntu.edu.tw//handle/246246/28685本實驗的目的為建立反轉錄競爭性定量聚合 連鎖反應於豬細胞素及豬生殖與呼吸綜合 症病毒（PRRSV）基因表現的測量，同時運 用此方法觀察PRRSV感染對豬肺臟細胞素表 現的影響。為了克服聚合連鎖反應在定量方 面的困難，我們架構了一段包含了IL-1b、 IL-2、IL-4、IL-8、IFN-g、PRRSV、b-actin 及 TNF-a八對引子序列之DNA 片段，此DNA 片段包含之引子序列與測試樣本用之引子序 列完全相同，唯其PCR 產物大小不一；以此 DNA 片段當模板，利用其與樣本模板共同競 爭PCR 反應物質之原理，將此DNA 片段連續 稀釋，經PCR 反應後，由比對電泳圖上兩種 產物之密度，可推算出樣本之濃度，解決PCR 在定量方面之不足。我們以此DNA 片段，分 別定量測量PRRSV 攻毒組（n=4）與對照組 （n=4）保育豬肺臟IFN-g、IL-1b及PRRSV mRNA 的表現。結果發現，在三個不同時間 點（攻毒後7、14 和21 日），攻毒組IFN-g 之表現量皆顯著高於對照組；而攻毒組IL-1b 之表現量於攻毒後21 日顯著高於對照組，但 攻毒後第7 和14 日，攻毒組與對照組IL-1b 之表現量無顯著差異；另外，隨病毒量的增 加，IFN-g與IL-1b mRNA的表現亦跟著上升。 此結果顯示PRRSV感染會影響IFN-g與IL-1b mRNA 之表現，唯其對免疫系統干擾與否， 則仍需進一步的研究。In this study, we used reverse transcriptionquantitative competitive polymerase chain reaction（RT-qcPCR）to assay porcine cytokines and porcine reproductive and respiratory syndrome virus（PRRSV）mRNA expression, and investigated the effect of PRRSV infection on cytokine gene expression in the lung tissue of nursing pigs. We modified a previously described RT-qcPCR method to construct a DNA fragment to measure interleukin-1b（IL-1b）, IL-2, IL-4, IL-8, interferon-g（IFN-g）, tumor necrosis factor-a, b-actin, and PRRSV mRNA expression in pig. This constructed DNA fragment had identical primer sequences to target cDNA except differences in the size of products. After co-amplifying of RNA-derived cDNA with serial dilution of the DNA fragment and calculating the ratios between them, a precise quantification data of sample was obtained. By using this DNA fragment, it was possible to analyze IL-1b, IFN-g and PRRSV mRNA expression in the lung between PRRSV-infected （n=4）and sham-inoculated（n=4）piglets. The results revealed that piglets infected by PRRSV had a higher level of IFN-g mRNA expression in their lung than those of sham-inoculation（ P＜ 0.05）at 7, 14, and 21 days post-inoculation（PI）. PRRSV- infected piglets also showed higher IL- 1b mRNA expression than sham-inoculated ones at 21 days PI（ P＜0.05）although there were no differences between the groups of 7 and 21 days PI. Besides, as virus load increased IFN-g and IL-1b mRNA expression would also elevated. From these results, it is concluded that PRRSV infection would alter IFN-g and IL-1b mRNA levels in the lung tissue of infected piglets; however the disturbance of immune system in piglet by PRRSV infection required further study.application/pdf46782 bytesapplication/pdfzh-TW國立臺灣大學獸醫學系暨研究所豬生殖與呼吸綜合症反轉錄競爭性定量聚合連鎖反應細胞素Porcine reproductive and respiratory syndrome (PRRS)reverse transcriptionquantitative polymerase chain reaction (RTqcPCR)cytokinesreporthttp://ntur.lib.ntu.edu.tw/bitstream/246246/28685/1/892313B002077.pdf