A Study And Establishment Of Indirect ELISA For Detecting Antibody Of Canine Parainfluenza Virus(CPIV) NP Protein

Canine parainfluenza virus(CPIV)is an important pathogen in canine infectious respiratory disease(CIRD).CIRD is commonly known as kennel cough.Kennel cough can cause a series of clinical symptoms such as runny nose,cough,fever on dogs,even leading to death of puppies in severe cases.CPIV is widely prevalent in the environment and often coinfected with canine distemper or other pathogens.Since CPIV has similar clinical signs with other infectious diseases,which cause severe damages to the respiratory system of dogs,the differential diagnosis is quite difficult.In this study,the prokaryotic expression system was used to express CPIV NP protein in vitro.By optimized the expression conditions,the optimal expression condition was ultimately determined:induce expression of NP-GST fusion proteins by adding isopropyl-?D-thiogalactoside(IPTG)to 1 m M final concentration and allow the cells to grow for an additional 10 hours at 30°C.NP-GST fusion protein was finally obtained for 2 m L at a concentration of 1.52 mg/m L and shows a high purity and a good reactogenicity,which was identified with SDS-PAGE and western blot.The purified NP-GST fusion protein was used as the coating antigen to establish CPIV NP antibody indirect ELISA.By optimized each reaction condition,the best conditions for the proposed method were ultimately determined.NP-GST fusion protein was coated to microtiter plates at 0.95?g/m L with Carbonate buffered saline(CBS)overnight at 4?,then blocked for 45 min with 2%skim milk at37?.The testing serums need to incubate for 60 min at 37?,and then incubated with the HRP enzyme-labeled secondary antibody(5000-folds diluted)for 30 min at 37?.Color development was performed for 15 min avoiding light.Finally,the assay was read out with spectrophotometry at OD₆₃₀.The threshold value of determination is 1.29.There is no cross-reactivity with canine parvovirus antibody positive serum,canine respiratory coronavirus antibody positive serum,and rabies virus antibody positive serum,which indicated that the method has high specificity.NP antibodies still can be detected when CPIV antiserum 16000-folds diluted which indicated this method is very sensitive to test NP antibodies.The established method also has a good repeatability and a good reproducibility with IFA of CPIV antibody detection method.The established indirect ELISA method was applied for a serological survey of CPIV in some local dogs of Wuhan regions.The prevalence and vaccination conditions of CPIV in Wuhan regions were preliminary investigated,and the serological datas was obtained.Meanwhile,another experimental trial was conducted to express CPIV HN protein ectodomain with the baculovirus expression system.But it is found that HN protein ectodomain is difficult to express in sf9 insect cells.Further study on trial designing and the optimization of expression conditions is still ongoing.