Modification And Immunogenicity Research Of GP5 Of Porcine Reproductive And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome (PRRS) , caused by porcine reproductive and respiratory syndrome virus (PRRSV), which is characterized by severe reproductive failure in sows, and respiratory distress in piglets and growing pigs, is really a new viral disease of swine. Since it was first described in 1987 in the United States, and in Europe in 1990, PRRS has been one of the most economically important diseases of the global swine industry.The ORF5-encoded major envelope glycoprotein (GP5) is one of the key immunogenic proteins of the porcine reproductive and respiratory syndrome virus (PRRSV) and is the leading target for the development of the new generation of vaccines against PRRS. However, weak and tardy neutralizing antibodies have been elicited in several developed experimental vaccines expressing PRRSV GP5. More recent evidence has demonstrated a non-neutralizing decoy epitope A ((A/V) ²⁷L²⁸V²⁹N³⁰) upstream of the neutralizing epitope B(S³⁷H³⁸(L/I)³⁹Q⁴⁰(L/S)⁴¹I⁴²Y⁴³N⁴⁴L⁴⁵)of GP5, which might prevent the development of a strong neutralizing antibody response against PRRSV. In the present study, We constructed several different ORF5 gene mutations, and a Pan DR T-helper cell epitope (PADRE) as exogenous helper factor to reconstruct this gene, hope to minimize or eliminate the decoy effect of the non-neutralizing epitope, then to enhanced GP5 protein inducing immunoreaction. The main contents were as follows:1. Expressing the ORF5 gene in Eschrichia coliIn this study, in order to test the immunogenicity of different mutations of GP5 protein and analysis the reason different mutations inducing different immunoreaction, we desiged four mutations including losing N terminal signal peptide, losing N terminal signal peptide and non-neutralizing epitope A. These two fragments were fusion expressed with GST, then named GST-542 and GST-552. Then we got fusion expression protein GST-546 only including neutralizing epitope and non-neutralizing epitope, and GST-556, which only including neutralizing epitope. After induced by IPTG, highly expression fusion proteins were obtained. SDS-PAGE analysis showed that the fusion proteins GST-542, GST-552, GST-546, GST-556 were 42kDa, 41kDa, 30kDa and 29kDa in size respectively, designed as expected. These proteins existed mainly in form of inclusion bodies. These proteins were proved have immunogenicity by western blot.2. Preparation of monoclonal antibodies against GP5 of PRRSVInclusion bodies of GST-552 were as the antigens to immunize mice. One hybridoma clone (52-C) was generated by fusion of SP2/O myeloma cells and the splenocytes obtained from Balb/c mice immunized by pKG-552. Western blot and indirect ELISA...