Using Ferritin Gene Fusion To Express E2 Envelope Protein Of Classical Swine Fever Virus.

Classical swine fever(CSF),also known as "Swine fever"(Swine fever),is a highly contagious infectious disease among pigs.It is a very big problem for the pig industry in China and the world.In recent years,not only the incidence of the disease has increased significantly,but also its epidemic trends and features have become more complicated.The situation of persistent infections is widespread throughout the world.The prevention and control of swine fever has encountered a new bottleneck.At the same time,the preventive effect of the existing vaccines has a clear downward trend.The Chinese strain(strain C)developed by Chinese scholars has been widely applied in the prevention of swine fever for a long time throughout.In addition,there are other types of swine fever vaccines such as inactivated swine fever vaccine,swine fever genetically engineered subunit vaccine,swine fever live carrier vaccine,and swine fever vaccine.Traditional inactivated classical swine fever vaccine and avirulent(attenuated)classical swine fever vaccine have shortcomings such as short immune protection period and strong virulence.Protein-derived subunit vaccine can solve the above problems.Classical swine fever envelope protein E2 is the main antigen that enables pigs to produce protective antibodies against classical swine fever virus,which can be used to diagnose the infection of classical swine fever;E2 protein can induce neutralizing antibodies in the body to prevent classical swine fever virus.The attack,which plays a role in protecting the body,is the main protection antigen.Therefore,this study selected the highly immunogenic envelope protein E2 of classical swine fever virus as the research material for the new swine fever vaccine.With the development of interdisciplinary science,the cross penetration of biology and nanomaterial technology is rapidly developing.Self-assembling proteins are ubiquitous in eukaryotic and prokaryotic cells.The protein subunits assemble spontaneously to form a highly ordered structure,which is the guarantee for maintaining the normal operation of the body and the driving force for the evolution of the body.Nanomaterials formed by self-assembling proteins not only have good biocompatibility and uniform and stable characteristics,but also have great potential application prospects in cell imaging,lesion detection,drug sustained release and vaccine development.Among them,ferritin Self-assembled nanoparticle technology has attracted people's attention.Ferritin can assemble spontaneously to form uniform and stable protein nanoparticles with good biocompatibility.It not only has a unique spatial structure of 24 subunits,but its spherical hollow structure has three interfaces : Inner surface,outer surface and contact surface between subunits.Therefore,ferritin can become an ideal multimeric nanoparticle carrier for antigen presentation.The p ET expression system of E.coli(Escherichia coli,E.coli)is the most commonly used to express foreign proteins.It has a clear genetic background,low production cost,fast reproduction speed,and easy purification and high efficiency of the expressed product.It has the characteristics of expressing foreign proteins and wide application range,but because it is a prokaryotic expression system,it will lack complex phosphorylation,glycosylation and the formation of complex disulfide bonds and other post-translational modification processes.There are certain effects on folding and biological activity.In this study,the gene sequences of classical swine fever envelope protein E2 and ferritin were optimized,and the target gene sequence was obtained by chemical synthesis,and E2-Fe was obtained by fusion PCR..coli BL21(DE3)was used to explore the induction conditions and high-efficiency expression.The expressed fusion protein was subjected to Western-blotting verification,nickel column purification,mass spectrometry and electron microscopy.The results show that the fusion gene E2-Fe has been successfully cloned into the p ET-28 a vector,and the optimal condition for fusion protein expression is induced by 0.15% lactose for 6 hours.The values are consistent,and the fusion protein has been successfully expressed in large quantities in E.coli.The diameter of the nanoparticles observed by electron microscopy is about 20 nm,which is also consistent with the theoretical value.That is,the ferritin-fused classical swine fever virus envelope protein E2-Fe in E.coli Get efficient expression.The fusion protein E2-Fe expressed in Escherichia coli was purified by a nickel column and injected into the abdominal cavity of mice.After two immunizations,it can induce the body to produce specific antibodies with a titer of up to 1:6400,indicating that E2-Fe The fusion protein has good immunogenicity.The baculovirus-insect expression system is a popular choice for recombinant protein production.Protein expression in insect cells involves a post-translational modification process.The protein complexes produced by this system and high protein yield are advantageous features of this eukaryotic expression system.Later,with the deepening of research and the continuous development of biotechnology,baculovirus began to be used in surface display systems,gene therapy,vaccine development and other aspects.When exogenous proteins are expressed in insect cells and mammalian cells,the glycosylation modification sites are the same,but the types of oligosaccharides used in the modification are not the same.Compared with mammalian cells,the expression levels of related sugar chain processing enzymes in insect cells are insufficient or lacking,and the complex sialic acid structure cannot be synthesized after the glycosyl modification processing is completed,but ends in the oligomannose structure.This difference in oligosaccharide modification results in the subunit vaccine expressed in insect cells being theoretically unfavorable for production,but incompletely processed N-glycan modification will instead enhance the avian influenza virus hemagglutinin(HA)virus The affinity of the particles and immune cells.Therefore,this study also chose the baculovirus-insect expression system to fuse and express the protein of interest in order to develop a new swine fever vaccine.In this study,the gene sequences of hog cholera envelope protein E2 and ferritin were optimized,and the target gene sequence was obtained by chemical synthesis,and E2-Fe was obtained by fusion PCR.The fusion gene was cloned into the baculovirus transfer vector p VL1393 polyclonal At the site,the recombinant vector p VL1393-E2-Fe was obtained,and the recombinant transfer vector p VL1393-E2-Fe was cotransfected with the inactivated rescue silkworm baculovirus(re Bm Bac)to obtain silkworm Bm5 cells to obtain the recombinant virus,and the virus solution was collected and infected The silkworm was raised at the fifth instar,and after the silkworm showed obvious signs of baculovirus,hemolymph was collected,followed by Westernblotting and electron microscopy.The results showed that the sequencing results of the recombinant transfer vector p VL1393-E2-Fe were correct,the fusion gene E2-Fe has been successfully cloned into p VL1393,and after retransfection with silkworm Bm5 cells with re Bm Bac,the recombinant baculovirus re Bm Bac-E2-Fe with infectious activity was obtained The Western-blotting diagram of silkworm blood shows that the size of the fusion protein is about 46 KD,which is consistent with the theoretical value.The fusion protein has been successfully expressed in silkworm.The theory is consistent,that is,the ferritin-fused classical swine fever virus envelope protein E2-Fe is highly expressed in silkworm.The silkworm hemolymph with E2-Fe fusion protein was prepared and injected intraperitoneally into mice.After two immunizations,the body can induce the body to produce high-titer specific antibodies,and the titer can be as high as 1:25600,which the original E2-Fe fusion protein has good immunogenicity.The above research results indicate that the fusion protein can be efficiently expressed in E.coli and silkworm,and obtain a considerable amount of nanoparticle antigens,which broadens the thinking for the research of new swine fever vaccine.