Production And Immunogenicity Of Japanese Encephalitis Virus Gene Engineering Oral Vaccine LTB-NS1_△63

Japanese encephalitis（JE）,which caused by Japanese encephalitis virus（JEV）,and JE is a mosquito-borne zoonotic infectious disease that seriously threatens the health of human and animal.Pigs are the most important storage and proliferation hosts for the virus in nature.China is one of the highest-prevalent area.The predominant genotypes are GI,GIII and GV.Currently,GI has become the main genotype in many regions of China.JEV-infected sows can cause piglet encephalitis and boar breeding obstacles,causing huge economic loss in the swine industry.JEV infection causes about 14%of the deaths each year and about 30%of patients leave different degrees of neurological sequelae.JE vaccines used at home and abroad include inactivated vaccines and live attenuated vaccines.Although these two vaccines can prevent Japanese encephalitis virus infection,they have the drawbacks of high production cost,large side effects,short immune protection time,and potential for virulence recovery.Therefore,the development of safe,efficient,and inexpensive new genetic engineering vaccines has become the development direction of JE vaccine research both at home and abroad.The JEV genome is a single and positive stranded RNA that encodes a protein which includes three structural proteins:a core protein（C）,an anterior membrane protein（pr M）,an envelope protein（E）,and seven non-structural proteins（NS1,NS2A,NS2B）.NS3,NS4A,NS4B,NS5).NS1 is involved in the replication and release the virus genome,and can induce immune protection.The heat-sensitive enterotoxin B unit（LTB）is a good oral adjuvant.In this study,we used E.coli to express LTB-NS1 recombinant fusion proteins including LTB and JEV NS1 and to study their immunological properties.The recombinant LTB gene and JEV NS1 gene were cloned into p ET28a vector using DNA recombination technology,and the recombinant expression vector p ET28a-LTB-NS1expressing the fusion antigen was obtained.By analyzing the hydrophilicity of NS1 protein,it was found that the C-terminal hydrophobicity was stronger.Therefore,the expression vector p ET28a-LTB-NS1△₆₃ was constructed to delete the 63-amino acid mutant protein NS1△₆₃ with high hydrophobicity at the C-terminus of NS1 protein.p ET28a-NS1Δ63 and p ET28a-LTB were also constructed as controls.The constructed recombinant expression vectors p ET28a-LTB-NS1,p ET28a-LTB-NS1_Δ63,p ET28a-NS1_Δ63 and p ET28a-LTB were identified by double enzyme digestion and sequencing.The recombinant expression vector was introduced into the prokaryotic expression strain BL21 and optimized for inducing expression conditions（temperature,time and IPTG concentration）.The results showed that the higher expressed recombinant protein LTB-NS1_△63,NS1_△63was obtained at 1 m M IPTG for 20 hours at 18°C.And LTB.The expression of the recombinant protein was detected by SDS-PAGE and Western blot.The expressed protein was purified by Ni column.The purified expressed proteins(LTB-NS1△₆₃,NS1△₆₃ and LTB)were orally immunized and intraperitoneally injected immunized with 3-5 week-old female BALB/c mice,PBS-immunized mice as negative control,and the SA14-14-2 vaccine-immunized mice as positive controls.Mouse sera were collected and their antibody titers were detected by indirect ELISA.The results showed that the titers of NS1 antibody induced by oral administration of LTB-NS1△₆₃ immunized mice were higher than those induced by oral administration of NS1_Δ63 immunized mice,and similar NS1 antibody titers were observed in peritoneal injection of LTB-NS1_Δ63immunized mice and NS1_Δ63 immunized mice.Negative control LTB and PBS immunization groups did not induce NS1 antibodies.Western blotting results indicated that LTB-NS1△₆₃ immune mouse serum reacted specifically with JEV NS1 protein.The protective ability of the vaccine was analyzed by in vivo challenge,and the survival status of the mice was continuously observed and recorded.The results showed that oral LTB-NS1△₆₃（8/8）was equivalent to the positive control SA14-14-2 vaccine group（8/8）in immunoprotection efficacy.This was significantly higher than that of the other control groups(NS1_Δ63,LTB and PBS),and the side effects after immunization of the mice were obviously reduced as compared with the intraperitoneal injection group.In summary,the novel genetically engineered oral vaccine LTB-NS1△₆₃ prepared in this study has good immunogenicity and immune protective efficacy,and is expected to become a substitute for the existing JEV vaccine.