Cloning And Expression Of The σC Gene Of Avian Reovirus And Primary Study On Its Immunogenicity

Avian Viral arthritis discovered in recent years is a chronic infectious disease caused by ARV.Symptoms characterized by infringing hock joint,articulationes digitorum pedis, tendon and the cardiac muscle.The occurrence is all over the world.It brings huge economic loss in the poultry industry.The ARV proteinσC can stimulate organism to produce the protective neutralizing antibody,the immune effect of this protein is good.The experiment applied gene cloning technique to express theσC protein in vitro and carried out a preliminary study on its immunogenicity as well.TheσC gene of ARV S1133 was designed to amplify by reverse transcription chain reaction(RT-PCR).TheσC gene was cloned to the pMD19-T vector and constructed the cloned vector pMD19T-σC.The cloned gene fragment is 1038bp through PCR, enzyme-cutting,sequencing and identification.Nucleotide sequence homology of the amplified target fragment and the reported S1133σC is 98%.Construct the expression vector pET32a(+) by recombinating pET32a(+) to prokaryotic expression vector.The optimized expression obtained after the recombinant fusion being induced in E.coli BL21 by 1.0mM IPTG for 5 hours.Relative molecular mass of the expression of recombination protein is 54KDa in the form of inclusion bodies.Western-blot analysis shows that a specific reaction may occur between the recombinant protein and the ARV positive serum. This indicates its satisfactory reactionogenicity.Histidine labeled column purification carried out with low pressure liquid chromatography to the production of pET32a-σC recombinant bacterium alter being induced and expressed.The eluted protein of 100mM imidazole buffer is fairly pure.The protein content is 150μg/ml after renaturation.The experimental animal was divided into 3 groups.There are S1133 inactivation vaccine group,σC the protein immunity group and the blank control group.Collect the blood serum at 13th,20th,and 35th days of age respectively.Antibody test carried out by establishedσC-ELISA method.Taking the challenge inoculation tests with ARVS1133 at 36th days of age.The result showed that the protective neutralizing antibody produced by applyingσC protein stimulation is roughly identical with the commercialized S1133 inactivation vaccine.