Immunogenicity Of Nonstructural Protein NS1 Of Japanese Encephalitis

Japanese encephalitis virus is a virus transmitted by insects,which can cause zoonosis.Human infection with epidemic encephalitis B virus can lead to death.Those who was alive will also have permanent sequela.Since the discovery of Japanese encephalitis,it has been widely used in various countries and regions of the world.The main popular areas include Southeast Asia,Oceania and Africa.Around 50000 people worldwide are ill each year,about 15000 of them die.At present,specific drugs for the treatment of encephalitis B have not been developed,mainly through immunization to reduce the incidence of encephalitis B infection.Japanese encephalitis virus has serious harm to aquaculture,and it also causes significant economic losses in aquaculture.The encephalitis virus is mainly proliferating in pigs.After sows were infected with virus,they mainly showed reproductive dysfunction,while boars infected with virus were mostly unilateral orchitis,which affected the ability of mating.In the five nonstructural proteins of JEV,NS1 is considered to be an important protein related to viral genome replication.In this study,we plan to construct NS1 protein prokaryotic expression plasmid,simultaneously express M and E,compare the immune effect of three proteins,and lay the foundation for exploring the immunogenicity of JEV non structural protein NS1 and developing the vaccine related to JEV NS1 protein.In this study,NS1 protein was expressed by prokaryotic expression vector pET-28 a,and the best conditions for expression of NS1 protein in JE were explored.The gene sequence of NS1 protein expressed on Genbank HW1 was used as a template,and specific primers were designed.The upstream primers were inserted into the BamH I restriction enzyme site,and the downstream primers were inserted into the Hind III restriction enzyme site.The NS1 nucleotide sequence was cloned by PCR method,and the prokaryotic expression vector pET-28 a and NS1 target fragment were ligated by T4 ligase.After Primer5.0 analysis,the sequencing results were compared with NCBI,and the target gene reading frame was completely correct.The constructed prokaryotic expression plasmid was transformed into Escherichia coli Rosetta(DE3)and induced by IPTG for 4 h.The expression of NS1 protein was successfully expressed by SDS-PAGE analysis.At the same time,the original recombinant plasmid pET-28a-E and pET-28a-M were transformed into Escherichia coli Rosetta(DE3),IPTG induced,SDS-PAGE electrophoresis,M protein and E protein express the same success.The relative molecular weights of NS1 protein,E protein and M protein were 69 kD,63 kD and 30 kD respectively.The three expression bacteria of NS1,M and E were broken down,and the protein expression patterns were analyzed.It was found that three proteins were expressed by inclusion bodies.Inclusion bodies were denatured,renatured,dialysate and concentrated,and the target protein containing very little protein was obtained.The BALB/c female mice were immunized with the complete adjuvant and Freund’s incomplete adjuvant,and the serum was collected from the orbit after immunization(5 mice).The other 4 were immunized with third times,and after one week of immunization,each intraperitoneal injection of 0.3mL(≧500 LD50)was injected to observe the condition of the mice.After one week,the blood was collected in the orbit,the brain tissue,the liver and the spleen were aseptic,and the fluorescence quantitative PCR was used to detect the proliferation of the virus;the serum collected at 28 d was used for antibody neutralization experiment.The serum was 1:2-1:256 times diluted,and the encephalitis B cytotoxicity known as TCID50 was used as a positive control.The results showed that the prokaryotic expression of NS1 protein was immunogenic and similar to the protein released by the virus itself.At the same time,NS1 protein could induce neutralizing antibody in mice,and the antibody titer was higher.Comparing the immune effects of the three proteins,the NS1 protein has the best immunization effect,which can effectively control the proliferation of the virus,and the antibody level can be maintained for a long time.Through the prokaryotic expression of NS1 protein,M protein and E protein,this study laid the foundation for the research and development of NS1 related vaccines,and contributed to the exploration of the immunogenicity of NS1.