Prokaryotic Expression And Characterization Of Prrsv GP5 Fragments

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) was the pathogeny of Porcine Reproductive and Respiratory Syndrome (PRRS) represented by abortion of sew and respiratory obstacle of piglets. For its infectiousness as well as the immune repression and intercurrent brought to pigs, PRRSV caused huge economic loss to pig's feeding industry. Moreover, the mutants of PRRSV genes increase the difficulty to control the illness. Glycoprotein 5(GP5) is the primary structural protein of PRRSV. It shows the function of combining the virus to the receptor on the host cell as well as inducing the apoptosis of the cell and novel vaccine was designed according to its ability to induce the neutralization antibody. So GP5 provides the virus with important physiological functions and shows great value on the control of PRRS.Before entering host cells, PRRSV (porcine reproductive and respiratory syndrome virus)first binds with membrane receptors of the host cells specificly. And infects permissive cells such as monkey kidney cell (MA-104, Marc-145) and PAM (pig alveolar macrophage) through cell endocytosis. Up to now four independent but functional related PRRSV cell receptors were reported. They are Sialoadhesin, Heparinlike, Vimentin and CD163 receptor.To study the interaction of GP5 and other cell receptors, PRRSV SD-TA strain which is stored and seperated in our laboratory was used. Based on considerable literatures and analysis of DNAStar software to the antigen epitope possiblity of GP5, GP5 gene was divided into four parts: 79-207, 133-330, 229-492 and 382-603, the divided four genes were cloned to pGEX-6p-1 vector, and expressed in E. coli. BL21 competent cells. The four recombinant proteins (GP5-1, GP5-2, GP5-3 and GP5-4) have predicted sizes of 30.9kD, 33.3kD, 35.9kD and 34.5kD, respectively. These four purified proteins were immunized Balb/c mice, and after the third immunization the titer of specific antibody reached to 1:100000, indicating that these four proteins have good immunogenicity. The IFA method to research the interaction of GP5 hole protein and truncated proteins with Marc-145 cells showed that GP5 hole protein, GP5-1 and GP5-2 protein can bind with Marc-145 cell membrane, while GP5-3 and GP5-4 protein can not. GP5-1 protein can bind with CD163 P-1, CD163 P-2, CD163 M-2 and Sn-3 protein by ELISA method.