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研究生:顏宏勳
研究生(外文):Yen, Hong-Hsin
論文名稱:以反轉錄競爭性定量聚合�@連鎖反應探討豬生殖與呼吸綜合症病毒感染對肺臟細胞素基因表現之影響
論文名稱(外文):The impact of porcine reproductive and respiratory syndrome virus infection on lung cytokine gene expression by reverse transcription-quantitative competitive polymerase chain reaction
指導教授:鄭謙仁龐飛龐飛引用關係---
指導教授(外文):Chian-Ren JengVictor Fei Pang
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:獸醫學系研究所
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
論文出版年:1998
畢業學年度:86
語文別:中文
論文頁數:77
中文關鍵詞:反轉錄聚合連鎖反應定量競爭豬生殖與呼吸綜合症病毒細胞素
外文關鍵詞:RT-PCRquantificationcompetitivePRRSVcytokine
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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-1beta、
IL-2、IL-4、IL-8、IFN-gamma、PRRSV、beta-actin及TNF-alpha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-gamma、IL-1beta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-gamma之表現量皆顯著高於對照組;而攻毒組IL-1beta
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-1beta
之表現量無顯著差異;另外,隨病毒量的增加,IFN-gamma與IL-1beta mRNA的表現亦跟著
上升。此結果顯示PRRSV感染會影響IFN-gamma與IL-1beta 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 transcription-quantitative competitive pol
ymerase chain reaction(RT-qcPCR)to assay porcine cytokines and porcine repro
ductive and respiratory syndrome virus(PRRSV)mRNA expression, and investigat
ed the effect of PRRSV infection on cytokine gene expression in the lung tissu
e of nursing pigs. We modified a previously described RT-qcPCR method to const
ruct a DNA fragment to measure interleukin-1beta(IL-1beta), IL-2, IL-4, IL-8
, interferon-gamma(IFN-gamma),tumor necrosis factor-alpha, beta-actin, and P
RRSV mRNA expression in pig. This constructed DNA fragment had identical prime
r sequences to target cDNA except differences in the size of products. After c
o-amplifying of RNA-derived cDNA with serial dilution of the DNA fragment and
calculating the ratios between them, a precise quantification data of sample w
as obtained.By using this DNA fragment, it was possible to analyze IL-1beta, I
FN-gamma and PRRSV mRNA expression in the lung between PRRSV-infected(n=4)an
d sham-inoculated(n=4)piglets. The results revealed that piglets infected by
PRRSV had a higher level of IFN-gamma mRNA expression in their lung than thos
e of sham-inoculation(P<0.05)at 7, 14, and 21 days post-inoculation(PI).
PRRSV-infected piglets also showed higher IL-1beta mRNA expression than sham-i
noculated ones at 21 days PI(P<0.05)although there were no differences betw
een the groups of 7 and 21days PI. Besides, as virus load increased IFN-gamma
and IL-1beta mRNA expression would also elevated. From these results, it is co
ncluded that PRRSV infection would alter IFN-gamma and IL-1beta mRNA levels in
the lung tissue of infected piglets; however thedisturbance of immune system
in piglet by PRRSV infection required further study.
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