Cloning And Expression Of Rotavirus SA11 VP7 And Preparation Of IgY Antibodies Against Recombinant VP7

Objective To prepare eukaryotic expression of rotavirus(RV), SA11 capsid protein VP7, generate and purify yolk immunoglobulin (IgY) antibodies against the recombinant VP7 from Roman hens and identify its activity. Methods MA104 cells were infected with the standard SA11 strain and the culture fluid was collected. A DNA fragment of 978 bp encoding SA11 VP7 was obtained by RT-PCR amplification from genomic RNA of rotavirus SA11. The PCR products were ligated to pMD18-T vector following the comfirmation by DNA sequencing and subcloned into pPICZαB. The recombinant pPICZαB-SA11 VP7 was transformed into E coli Top10. The plasmids were linearized by digestion of BstXⅠand transformed into Pichia pastoris X-33 through electroporation by DNA sequencing. The transformants were induced with methanol for expression. The cultural supernatant was subjected to SDS-PAGE and Western blotting. Fusion expression was purified through the column of affinity chromatography. IgY was identified and purified by SDS-PAGE and Western blotting from eggs of Roman hens immunized with recombinant SA11 VP7.The role of activity of IgY was identified in vitro experiment. Results The RNA extracted from the rotavirus culture fluid consisted of 11 bands visualized by silver staining. The expression vector pPICZαB-SA11 VP7 was constructed and the fusion protein in Pichia pastoris X-33 was harvested and purified. The recombinant SA11 VP7 with molecular weight of 40 200 was identified by Western-blotting. The IgY antibodies against the recombinant SA11 VP7 were produced with a purity of 95 percent and yield of 10.2 mg per egg. It was proved that IgY after immunization has relation with the reduction of plaque in vitro.Conclusion The preparation of IgY antibodies to recombinant SA11 VP7 may lay a foundation for the development of vaccines and diagnostic techniques.