[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, and to generate and purify yolk immunoglobulin (IgY) antibodies against the recombinant VP7 from Roman hens. 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 RV SA11. The PCR products were ligated to pMD18-T vector following the confirmation by DNA sequencing and sub-cloned into pPICZalphaB. The recombinant pPICZalphaB-SA11 VP7 was transformed into E coli Top10. The plasmids were linearized by digestion of BstXI and 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. RESULTS: The RNA extracted from the RV culture fluid consisted of 11 bands visualized by silver staining. The expression vector pPICZalphaB-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. CONCLUSION: The preparation of IgY antibodies to recombinant SA11 VP7 might lay a foundation for the development of vaccines and diagnostic techniques.