Construction Of Neutralizing Epitope Recombinant Proteins Of Porcine Reproductive And Respiratory Syndrome Virus And Study On Their Immunogenicity In Mice

Porcine reproductive and respiratory syndrome（PRRS）,a highly contagious disease,which was caused by porcine reproductive and respiratory syndrome virus（PRRSV）,is one of the most difficult to control in all swine diseases.The neutralizing antibody of PRRSV can resist PRRSV infection or relieve the symptoms after PRRSV infection.The virus neutralizing epitope can induce the body to produce neutralizing antibody.This research mainly studies whether the neutralization linear epitopes of PRRSV structural proteins（M,GP5,GP4,GP3 and GP2）can stimulate the body to produce high titer neutralizing antibodies,so as to preliminarily determine whether this way can provide protection for the body.The neutralized linear epitopes of M,GP5,GP4,GP3 and GP2 proteins of PRRSV are connected directly or with two kinds of linkers（G₄S and FFRK）by artificial synthesis,which are named as GP5-2,GP5-2-G₄S,GP5-2-FFRK（including GP5,GP4,GP3 and GP2）and MGP5-3（including M,GP5,GP4 and GP3）.First,in order to enhance the immune effect,LTB was fused to the neutralizing epitopes of PRRSV,which are named LTB-GP5-2,LTB-GP5-2-G₄S,LTB-GP5-2-FFRK and LTB-MGP5-3.Then,to make LTB fold correctly,we selected molecular chaperone Gro ES/Gro EL-TF co-expression with LTB-PRRSV recombinant protein.Finally,the above-mentioned recombinant proteins were expressed through E.coli expression system.The results were as follows,the MGP5-3 recombinant protein were mainly expressed as inclusion bodies,and GP5-2,GP5-2-G₄S and GP5-2-FFRK recombinant proteins were completely expressed as inclusion bodies.LTB-MGP5-3,LTB-GP5-2,LTB-GP5-2-G₄S and LTB-GP5-2-FFRK recombinant proteins achieve soluble expression by co-expression with Gro ES/Gro EL-TF.The above-mentioned recombinant proteins were purified and made into vaccines.Then the vaccines,mixed with GP96 molecular adjuvant to enhance the immune effect,were emulsified with oil adjuvant to immune mice.Finally,the antibody titer produced by the mice was detected through ELISA,and the antibody titer results was analyzed by statistics.The results were as follows,the mice,immunized with high-dose recombinant proteins composed of PRRSV neutralizing linear epitopes,produced a high level of antibody,and the use of immune enhancers had no effect after second and third immunization.The LTB fused to the recombinant proteins increased the antibody level of the recombinant antigen with a low immune response level in the early stage of immunization（after first immunization）,but it could reduce the immune effect of the recombinant protein with a high immune response level.The GP96 could inhibit immune effect with a large dose of antigen immunization in the early stage of immunization（after first immunization）and reduce the antibody level.Therefore,in the immunization with a large amount of antigen,the use of immune enhancement may suppress the immune response and reduce the antibody titer.