Expression Of The Epitope-G₁ Of Bovine Ephemeral Fever Virus Glycoprotein And Establishing Of Indirect ELISA Using Recombinant G₁ Protein As Antigen

Bovine ephemeral ferver (BEF) caused by bovine ephemeral fever virus (BEFV), is an acute epidemic infection of cattle and water buffalo. The disease was first found in East Africa and spreaded rapidly in many countries of Africa, Asia and Oceania. The infection causes considerable economic impact in cattle industry including the drops of output and quality of the milk, lameness and paralysis or abortion.BEFV contains five structural proteins and G protein is the primary antigen. Antigenic site G₁ is specific among the five sites on G protein, which only reacts with anti-BEFV sera, however other antigenic sites have cross-reactions with the sera against the correlative viruses besides BEFV, so the site G₁ is cloned, expressed and purified. The indirect ELISA for detecting anti-BEFV sera using recombinant G₁ protein as antigen and correlative Kit are developed in order to diagnose BEF and monitor the antibody level after immunization.The total RNAs of BEFV (JB76H strain) were prepared, then the Epitope-G₁ gene was amplified by RT-PCR. By comparison of the epitope-G₁ gene with the data from NCBI/GenBank, the homologies of the nucleotide acid sequence and speculated amino acid sequence of the epitope-G₁ gene with Taiwan's BEFV are 96.4% and 96.5%, respectively, and the homologies of the epitope-G₁ gene with Australia's BEFV are 89.1% and 90.8% respectively.A soluble target protein (approximately 26 kDa) was obtained from Pichia pastoris GS115. The recombinant G₁ protein (approximately 41.54 kDa) expressed in E.coli BL21(DE3) was inclusion body most. The two expressed products above were analyzed by SDS-PAGE, Western blot, ELISA, immunizing rabbits and specificity experiments. The results indicated that the target proteins had higher reaction activity, immunocompetene and specificity, which could be used as coating antigen to develop ELISA.The indirect ELISA detecting anti-BEFV sera was established using the recombinant G₁ protein expressed in BL21(DE3) as antigen by optimizing reaction conditions. The results indicated that the antigen concentration was 0.574μg/well, and the dilutions of the sera and HRP-IgG were 1:20 and 1:200 ratio, respectively. ELISA plates coated with the antigen were conserved overnight at 4℃after 1h at 37℃. The optimal ELISA reaction conditions were obtained, which included blocking lh at 37℃, sera reaction lh at 37℃and HRP-IgG reaction 1h at 37℃. 2.1 times the average of OD₄₉₀ value obtained from reference negative serum was as criterion. A sample was positive if its OD₄₉₀ value was over or equal to the criterion, or else was negative.The indirect ELISA detecting anti-BEFV sera was also established using the recombinant G₁ protein expressed in GS115 as antigen. This method had higher sensitivity and repetition, however the specificity and coincidence were lower because the antigen purity was not high enough. So the G₁ protein can not be used as antigen until a good method is developed for its purification.The ELISA kit was developed and its composing was confirmed in this test. The data were credible detecting the kit's conservation condition, conservation time, specificity, sensitivity, coincidence, and stabilization between the same or different batchs. Therefore, the kit will be developed to BEF diagnosis widely in near future.