Construction Of Co-expression σB-σC-LTB Of ARV Subunit Vaccine And Immunogenicity Analysis

Museovy duek reovirus is a kind of Avian reovirus, which lies in the second subgroup ofReovirus genus. It shares common features with Avian reovirus family members. It usually invadesMuseovy duck between the the age of4-45days in summer. Its clinical mortality rate showed greatdifferentiation with the lowest at about10%~30%and and the highest around90%whenco-infected with other pathogens. Survival individuals showed stunted growth and thus becamestiff ducks, which seriously hindered the development of Museovy duck industry.S3(1104bp) and S40RF2(810bp) gene fragments encode protein aB and protein aC of thevirus respectively. Immunized with each protein could induce specific immune immunity to itsparental virus and provide protection against. ARV. To test immunity of the viral ptoteins, weselect the σB Gene σC gene, the cluster combination of fragment σB-σC and σB-σC-LTB as thetarget, respectively. We used E.coli expression system to produce the above four proteins, thenanalyzed and compared the immunogenicity of the four proteins by animal immunizationexperiments.We designed several pairs of primers to acquire σB gene (1100bp) and σC gene (810bp) byPCR amplification, according to the Genbank published sequence. The two gene fragments werecloned into plasmid pMD18-T simple followed enzyme digestion, PCR identification and sequenceanalysis. The recombinant plasmid was named as pMD18-T-σB and pMD18-T-σC,respectively.With the same enzyme digestion, σB and σC fragment is ligated by T4DNA ligase and thissequentially clustered fragment σB-σC was cloned and named as pMD18-T-σB-σC. Meanwhile,the fragment σB-σC-LTB were fused by overlap extension PCR using the σB-σC gene fragmentand the LTB gene fragment. The four gene fragments were cloned into the prokaryotic expressionvector pET-30b and expressed in E. coli BL21(DE3) followed enzyme digestion, PCRidentification and sequence analysis. The recombinant plasmid was named pET30b-σB,pET30b-σC, pET30b-σB-σC, pET30b-σB-σC-LTB.The expression of the recombinant four plasmids in E.coli BL21(DE3) was induced anddetected by SDS-PAGE analysis. These proteins showed molecular weight of40kD,29.7kD,70kD,83.93kD, respectively. The four expressed protein can be recognized by the positive serum ofARV by Western blotting analysis. The results showed that the expressed proteins were antigenical.The four fusion proteins existed as inclusion body. Chicken were immunized respectively with thefour fusion proteins after inclusion body extraction and purification and renaturation.Serum of Chicken were collected before immunization and7,14,21,28,35days after immunization. To assess systematic immune responses, specific IgG levels were determined byELISA. Indirect ELSA result showed that a relatively low level serum antibody were detected atthe7th day after the first immunization. Serum antibody gradually increased and attained thehighest lever at the35th day, which showed significant difference (p＜0.01) compared with thePBS group. σB-σC-LTB IgG titers was the highest, while σC was the lowest, with σB-σC, σBranked at the second and third. Taken together, our reults indicated that these recombinant proteinscould stimulate animal body to generate benign humoral responses as immunogens, which provieda good base for further clinical protective trial tests.