Application of Escherichia coli expressed glycoproteins of infectious laryngotracheitis virus in ELISA assay

• Main Authors: Kuan-Ting Chen, 陳冠廷
• Other Authors: Pac-Chun Chang
• Format: Others
• Language: zh-TW
• Published: 2000
• Online Access: http://ndltd.ncl.edu.tw/handle/16632458588742277742

碩士 === 國立中興大學 === 獸醫微生物學研究所 === 88 === 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.