Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/66314
標題: 大腸桿菌表現雞傳染性喉頭氣管炎病毒醣蛋白在酵素連結免疫吸附分析之應用
Application of Escherichia coli expressed glycoproteins of infectious laryngotracheitis virus in ELISA assay
作者: 陳冠廷
Chen, Kuan-Ting
關鍵字: infectious laryngotracheitis;雞傳染性喉頭氣管炎;ELISA;酵素連結免疫吸附分析
出版社: 獸醫微生物學研究所
摘要: 
雞傳染性喉頭氣管炎(Infectious laryngotracheitis, ILT)是一種嚴重的禽類上呼吸道疾病,此病是由雞傳染性喉頭氣管炎病毒(Infectious laryngotracheitis virus, ILTV)所引起。由ILTV感染後之致死率相當的高,繼又降低產蛋率,常造成養雞業者的嚴重經濟損失。 現今防治此病的方法,是以減毒後的活毒疫苗來免疫雞隻,然而目前所用的活毒疫苗仍可能回復毒力,進而引起ILT之爆發。因此,在進行ILT診斷或其流行病學調查時,如何區分ILTV疫苗株與野外強毒株,仍為一重要的課題。本實驗即嘗試開發ILT標幟疫苗(marker vaccine),與其配套之ELISA區分診斷系統,以血清學快速區分疫苗株與田間強毒株。在方法上,我們首先以各種ILT疫苗株(沙氏株、宇妙株、C7株等)及田間分離中、強毒株(中興嶺株、台南株)免疫雞隻,製備高免血清。結果發現以沙氏株免疫所得之抗體力價最高,其次為宇妙株與C7株等;田間分離毒株則無明顯抗體反應,其原因仍待探討。另一方面,我們也成功地應用原核(E. coli)系統表現出ILTV之glycoprotein 60(gp60)、glycoprotein E(gE)與glycoprotein C(gC)等醣蛋白,並以Western blot技術證實這些蛋白可被抗ILTV高免血清所辨識,其中gp60與gE蛋白經純化後可作為製備ELISA plate之塗鍍抗原。此自製gp60-ELISA系統製備之最佳條件為每個well塗鍍40 ng gp60蛋白,同時將待測血清以100倍稀釋,並以3﹪E. coli超音波震盪後萃取物作前處理。在此條件下免疫組(沙氏株)之ELISA O.D.值皆在0.5-2.5之間,而未免疫組之O.D.值皆在0.5以下,同時免疫組以gp60-ELISA與商用(KPL)ELISA所測得之O.D.值具高度相關性(R2=0.92)。另一方面我們所選擇自製gE-ELISA之抗原塗鍍量為80 ng/well,其它條件與gp60相同,在此條件下,免疫組(沙氏株)之O.D.值在0.5-1.5之間,而未免疫組之O.D.值皆在0.5以下。但是gE-ELISA與商用(KPL)ELISA所測得之O.D.值不具相關性。基於上述結果,我們將進一步利用此gp60-與gE-ELISA來篩選各種疫苗株或田間分離株之高免血清,以尋找gp60或gE缺損之疫苗株,進而一舉建立ILT之標幟疫苗與ELISA區分診斷技術。

Infectious laryngotracheitis (ILT) is an important upper respiratory disease of poultry. This disease is caused by infectious laryngotracheitis virus (ILTV). Infection of ILTV could cause a high mortality, and reduce the egg production; both might lead to a severe economic loss of farmers. ILT is controlled by live attenuated vaccines; however, these vaccines might revert to virulent strains, and cause an outbreak of ILT. How to distinguish vaccine strains from virulent field isolates remain an important problem for the diagnosis and epidemiological study of ILT. This study is aimed to develop a marker vaccine and an ELISA system to different the marker vaccine from virulent isolates. Towards this aim, we first prepared hyper-immune sera of vaccine strains (strains Solvay, Intervet, C7, etc.) and field isolates (strains TW/85 and TW/90) of ILTV. We found that strain Solvay generated the highest ELISA titer of antibody against ILTV, followed by strain Intervet and strain C7. However, the ELISA titer generated by strains TW/85 and TW/90 is not detectable; the reason for this observation remains elusive. We then over-expressed the glycoproein 60 (gp60), glycoprotein E (gE) and glycoprotein C (gC) of ILTV by the procaryotic (E. coli) system. These recombinant proteins were recognized by the anti-ILTV antibodies in Western blot analysis. Moreover, recombinant gp60 and gE protein could be purified and served as the antigen for coating ELISA plates. The optimum condition for the preparation and application of the gp60-ELISA is to coat each well with 40 ng recombinant gp60 protein, and to pre-treat the 100X diluted sera with 3% E. coli sonication extract. Under this condition, the sera of vaccinated chickens (by strain Solvay) produced gp60-ELISA titers between 0.5-2.5 (O.D. value); whereas the control group produced titers of only less than 0.5 (O.D. value). In addition, titers determined by the gp60-ELISA are highly correlated to those determined by the commercial (KPL) ELISA kit. For the gE-ELISA, the optimum condition found is the same as the gp60- ELISA, except that the amount of recombinant gE protein coated is 80 ng/well. Under this condition, the sera of vaccinated chickens (by strain Solvay) produced gE-ELISA titers between 0.5-1.5. In contrast, the control group produced titers of only less than 0.5. The titers determined by the gE-ELISA are not correlated to those determined by the commercial (KPL) ELISA kit. Experiments are underway to screen the hyper-immune sera of various vaccine strains and field isolates of ILTV by the gp60- and gE-ELISA; these experiments will lead to the concomitant development of a marker vaccine strain and its differential ELISA system for ILT.
URI: http://hdl.handle.net/11455/66314
Appears in Collections:微生物暨公共衛生學研究所

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