Construction and immunogenicity of recombinant adenovirus tandem expressing M and GP5 of porcine reproductive and respiratory syndrome virus / 生物工程学报

ABSTRACT

The M protein gene of porcine reproductive and respiratory syndrome virus amplified by PCR was tandem linked with its GP5 gene in shuttle vector in correct frame, resulting in shuttle vector pShuttle-CMV-M-GP5. The positive clone was identified by PCR and further confirmed by sequencing. The constructed plasmid was linearized with Pme I and co-transformed BJ5183 host bacteria with pAdEasy-1 to produce recombinant adenovirus DNA by homologous recombination. Then the adenovirus DNA was linearized with Pac I and transfected into HEK-293A cells to obtain recombinant adenovirus. The specific expression of target proteins by the recombinant adenovirus was verified by indirect immuno-fluorescence assay (IFA) with monoclonal antibodies against M and GP5.The results showed that the tandem linked M with GP5 could be co-expressed by adenovirus vector. Mice immunized with the constructed recombinant adenovirus induced strong humoral immunity (ELISA antibody and virus neutralizing antibody) and cellular immunity (lymphocyte proliferation and CTL responses). The results showed that the recombinant adenovirus has strong immunogenicity and provided the basis for the further experiments in pigs.