Secreted Expression Of Japanese Encephalitis Virus PrME In Pichia Pastoris And Immunogenicity Evaluation Of The Virus-like Particles In Mice

Japanese encephalitis virus(JEV)is the major pathogen to cause infection of the central nervous system in humans and swine.And JEV is also can cause reproductive diseases in birth sow,such as stillbirths,abortion,mummified fetuses and orchitis in boar.JEV is transmitted in a circulation primarily between Culex species and pigs.Swine are concerned as the major reservoir and amplifying hosts.JEV is a single positive strand RNA virus,which belongs to the genus Flavivirus.The single ORF is translated into one large putative polyprotein,which contains three structural proteins(capsid C,premembrane PrM/M,envelope)and(NS1,NS2A,NS2B,NS3,NS4A,NS4B,NS5)seven nonstructural proteins.The envelope glycoprotein(E)of JEV is the major structural protein on the surface of the mature virions.The complexes of premembrane(prM)and E proteins play important roles in virus assembly and infection cells modulation and in potential immunity-inducing antibody.As previous study,expression prME proteins of flavivirus which could stimulate mice produce higher level of specific antibody.In this article,we express the target protein through reasonably design and compare signal peptides.It concludes that JEV ? prME was not cleaved between prM and E during secreted expression in Pichia pastoris and both the culture supernatant and the purified protein,examined by electron microscopy,we found to contain JEV virus like particles(VLPs).JEV prME protein stimulated mice produce high level of JEV specific antibody.Meanwhile,the high neutralizing antibody titer was examined in immunized mice serum.In the end,as the methods of expression JEV ? prME protein,JEV ??? prME proteins were expressed and investigated.All in all,this study may provide a new train of thought for the development of effective and economic JEV subunit vaccine.The whole studies contain four parts as below:1.The effects of expression JEV ? prME in Pichia pastoris by using different signal peptidesThe JEV ? prME gene was amplified by RT-PCR with genome RNA of vaccine strain SA14-14-2 and then cloned into Pichia pastoris expression vector pPICZa-A,designated as pPICZa-prME.pPICZa-SprME was the same with pPICZa-prME besided with the additional 19 Aa signal peptides coding gene of the JEV cap protein C terminal.The linearized expression vector was transformed into Pichia pastorisX-33 by electroporation and induced with methanol during fermentation expression.The expression JEV ? prME protein was identified by SDS-PAGE and Western blot,which was found the target protein expression content of pPICZa-SprME/X33 higher than pPICZa-prME/X33.Meanwhile,both the specific mAb of E and prM can react with the expressed protein,it is logical to conclude that the prME was not cleaved between prM and E during secreted expression in Pichia pastoris.Then pPICZa-SprME/X33 expression protein purified by S-400 High Resolution HiPrep 16/60 Sephacry.Both the culture supernatant and the purified protein expressed by pPICZa-SprME/X33,examined by electron microscopy,we found to contain VLPs with diameters of 30?50 nm.2.The immunogenicity evaluation of the JEV?virus-like particles in miceFemale ICR mice aged 4 weeks were used in the immunization experiments.Four groups of the mice were immunized with different doses purified JEV?prME protein with complete Freund's adjuvant(Sigma)at a volumetric ratio of 1:1 and the fifth group injected with sterile PBS as blank control.The immunization was developed according to subcutaneous injection.The level of JEV specific antibody in serum of the immunized mice was detected by ELISA.The JEV specific antibody reached peak during 3 to 4 w post-injection and still maintain in mice at 7 wpi inoculated with 10 ?g and 15 ?g JEV ?VLPs.The weight of immunized mice had no obvious disparity among different groups.3.Neutralization assays of the JEV ? prME immunized mice25 female ICR mice aged 4 weeks were divided 5 groups,four groups were vaccinated with 10 ?g/dose purified JEV ? prME protein mixing different volume nucleic acid adjuvant and the fifth group injected with sterile PBS as blank control.All the immunization was developed according to subcutaneous injection.JEV specific antibody in serum of immunized mice was detected with ELISA and enhanced immune response was observed.The neutralization titers were determined based on a TCID50 reduction assay in BHK cells,which confirmed the neutralizing antibody titer of serum from 4 wpi mice was among 1:80 to 1:160.4.Expression JEV I?V prME in Pichia pastorisThe JEV ? prME gene was amplified by RT-PCR with genome RNA of synthetic yeast addiction by JEV ? strain LMG21421(GenBank:JF702686)and then cloned into Pichia pastoris expression vector pPICZa-A,designated as pPICZa-SprME/JEV ?,which contained the additional 19 Aa signal peptides coding gene of the JEV cap protein C terminal.The linearized expression vector was transformed into Pichia pastoris X-33 by electroporation and induced with methanol during fermentation expression.The JEV ?prME gene was amplified by RT-PCR with genome RNA of JEV ? strain XZ0934(GenBank:JF915894).JEV ? prME protein was expressed in the same way with JEV ?.The expression JEV prME protein was identified by SDS-PAGE and Western blot,both the specific mAb of E and prM can react with the expressed protein,it is logical to conclude that the prME was not cleaved between prM and E during secreted expression in Pichia pastoris.The JEV ??? prME protein expression situation was same with JEV ?,which put foundation for study in the future.