Expression Of Capsid Protein Of Bluetongue Virus 1 In The Sf9 Cells And Establishment Of Competitive ELISA Assay

To explore the subunit vaccine of bluetongue and the serological diagnostic method, the following aspects were stuidied:1. We use sucrose density gradient centrifugation to isolate and purify BTV1 from BHK-21 infected with BTV1. BTV1 was mixed with freund’s complete adjuvant to vaccinate rabbits and sheep.2. The genes of the capsid proteins were amplified from the BHK-21 cells infected with BTV1 and cloned into pMD18-T to construct clone vectors.3. The four genes of the capsid proteins was cloned into pFastBac HTB by digesting with double enzymes, then the recombinant plasmids were tranformed into DH10 Bac competent cells to make white and blue screening. The recombinant bacmid was transfected to Sf9 cells by Cellfection Ⅱ Reagent. The expressed recombinant proteins was confirmed by SDS-PAGE and Western-blot.4. Recombination VP7 protein was expressed and purified to establish the competitive ELISA with BTV polyclonal antibodiesThe result of the study showed:1. BTV1 was purified successfully. Polyclonal antibodies were prepared with the purified BTV1. The titer of the polyclonal antibodies was is greater than 1:1024.2. The genes of the capsid proteins were cloned, the sizes of the capsid were 2 886 bp、2706 bp、 1581 bp、1050 bp, respectively.3. Sf9 cells infected with P3 recombinant baculovirus were analyse by SDS-PAGE and western-blot. The recombinant proteins were expressed and can specifically react with BTV polyclonal antibodies. The sizes of these protein were 115 ku、107 ku、61 ku、38 ku.4.86 samples were tested with competitive ELISA, the result of test is the same as the result of the assay kits of Nation Key Laboratory for Animal Abovirology. But the mumber of the sample of the test is limited, we need a large number of field samples to study the sensitivity and the specificity of the competitive ELISA...