Cloning And Prokaryotic Expression Of GP5 Gene Of Of Porcine Reproductive And Respiratory Syndrome Virus

The Porcine reproductive and respiratory syndrome virus(PRRSV) were cultured in the MARC-145 cells. According to nucleotide sequence of PRRSV which were available in GenBank, a pair of specific primers(P1/P2) were designed . The PRRSV-FZ′s gene of glycosylation membrane protein (ORF5) was amplified by RT-PCR. The amplified productions were connected into pMD-18 simple T Vector , and transferred them into E. coli DH5α. The T-vector cloning strategy was employed to generate the recombinant plasmid for sequence. An alignment analysis of the GP5 gene of PRRSV-FZ showed that the homology was 100% between PRRSV FZ and AY885248.1; but which was 99.05% between PRRSV FZ and BJ-4, and 99.3% between PRRSV FZ and America type strain VR-2332, and 27.1% between PRRSV FZ and Europe type strain. Therefore, PRRSV FZ was grouped into Northern American genotypesThe recombinant positive plasmid was cloned into the multiple cloning site of pGEX 6p-1(+) vector. Positive clones were screened. The results of the enzyme digestion and gene sequencing showed that the recombinant expression vector was successfully constructed. The recombinant expression plasmid was identified and transformed into E. coli BL21 and induced with IPTG. The products of the prokaryotic expression were tested by SDS-PAGE , and Western blotting was used to test their immunoreactivity character. The SDS-PAGE showed that the recombinant GP5 fusion protein was 48.5k Da,which was in the forms of inclusion bodies.The results of the western blotting showed that the recombinant protein had a good immunoreactivity with the PRRSV monoclonal antibody.