Establishment And Preliminary Application Of ELISA For Detection Of BoHV-1 Antibody

Infectious bovine rhinotra-cheitis(IBR),also known as "necrotizing rhinitis" or "red nose disease",is a bovine respiratory tract infection cause of bovine herpesvirus 1(bohv-1)and is an important pathogen of bovine respiratory disease.Clinical symptoms such as encephalitis,conjunctivitis,respiratory tract infection,decreased milk production and bovine pathogenic bacterial pneumonia were often observed.The infection rate of BoHV-1 is high due to environmental factors,and calves have a higher mortality rate than adult cattle.With intensive farming,BoHV-1 spreads rapidly.Because BoHV-1 is prevalent in most areas of China,the Ministry of Agriculture of China has classified BoHV-1 as the second-class animal disease,and which is also identified as a mandatory animal disease in the Law on Quarantine Inspection of Animals and Plants Entering and Leaving the Country.Therefore,the study of BoHV-1 is of great significance for the prevention and control.In order to meet the needs of diagnosing BoHV-1 in China,an indirect ELISA method was established,through the expression of gL,gG gennes and the construction of gL,gG prokaryotic expression vectors.This study is divided into the following three parts:1.The gL,gG genes of BoHV-1 were cloned and expressed in E.coliBovine kidney cells(MDBK)were inoculated with BoHV-1,then extracts virus DNA.By designing specific primers,the gL,gG genes of BoHV-1 were amplified by PCR.The PCR products were digested by Bam H ? and eco R ?,which were directionally cloned into the prokaryotic expression vector pET-32 a to obtain recombinant expression plasmids pET32a-gL and pET32a-gG.After shaking culture,IPTG induction and SDS-PAGE,the gL and gG proteins were successfully expressed in pET-32a/BL21(DE3),and their molecular weights were about 37 kD and 67 kD,which were consistent with the expected results.The expressed recombinant proteins were respectively for the expression of inclusion bodies and supernatant,Ni-Agarose Resin method was used to purify the fusion recombinant protein,which achieved better results.Western blot analysis showed that the purified pET-gL and pET-G recombinant fusion proteins showed good reactivity with the BoHV-1 standard positive serum.2.Establishment of gL-iELISA methodUsing purified pET-32a-gL as a diagnostic antigen to coat a polystyrene microreaction plate,an indirect ELISA method for detecting BHV-1 antibodies was initially established.The optimized reaction conditions are: the optimal coating concentration of antigen is 0.773 ug/mL,the dilution of serum is 1:20,the coating is overnight at 4°C,the blocking solution is 3% BSA for 1 hour,the serum is incubated at room temperature for half an hour,the dilution of HRP labeled Rabbit anti bovine IgG is 1:3000,incubated at 37°C for half an hour,and the best substrate reaction condition is 37°C for 5 minutes.An indirect ELISA method for the detection of bovine infectious rhinotracheitis was established and the negative and positive cutoff value was determined.The established gL-iELISA method was used to detect bovine infectious rhinotracheitis positive serum,bovine foot-and-mouth disease O-positive serum and Brucella bovis positive serum,and the specificity was good;the IBRV positive serum was still positive when diluted 1:640.171 clinical sera were tested and the results showed that 67 were positive,with a positive rate of 39%.3.Establishment of gG-iELISA methodThe purified pET-32a-gG protein was used as the coating agent to establish an indirect ELISA method for the detection of BoHV-1 and the critical value was determined.The optimized reaction conditions are: optimal coating concentration 0.1563ug/mL,coating at room temperature for 1h + 4°C overnight,blocking with 1% BSA blocking solution for 1h,serum 1:20 dilution,incubation at 37°C for 30 min,The dilution of HRP labeled rabbit anti-bovine IgG 1:15000,incubation at 37? for 30 min,and the best substrate coloring condition is 37? for 5min.The established gG-iELISA method is used to detect bovine infectious rhinotracheitis positive serum,bovine foot-and-mouth disease type O positive serum and brucella bovis positive serum with good specificity;IBRV positive serum is still positive after 1:640 dilution test.The method of gG-iELISA was used to detect the positive sera of infectious rhinotracheitis,foot-and-mouth disease and Brucella;the test results of IBRV positive serum were still positive at 1:640 dilution.171 samples of sera were tested for clinical samples using established the indirect ELISA method,results show that 69 sera were positive,positive rate as 40.35%.This study can be used for diagnosis of BoHV-1 infection and antibody monitoring after vaccination.It is the foundation of ELISA kit for recombinant antigen.