Expression And Immunogenicity Of A Multi-Epitope Of Japanese Encephalitis Virus And Bursin Peptides

Japanese encephalitis virus (JEV) belongs to the genus Flavivirus, and the genus Flavivirus includes many clinically important pathogens. Since swine serve as a reservoir and amplifier of the JE virus, thus mass vaccination of swine can prevent the disease in swine and help to prevent JE epidemics in humans. The disease widespread in our country, not only on the pig industry caused huge economic losses, but also a serious threat to human health. Therefore prevention of JE in swine can benefit for swine production and health of human being. Hence, it is essential to prevent swine JE, which is useful for swine production as well as health of human being. There is no effective treatment method for JE, and treatment of this disease is primarily based on the way immunization vaccines for prevention. Therefore, the development of a swine vaccine against JEV is a high priority, as it could help prevent epidemics in humans.The strategy of the epitope vaccine design has made greater progress up to day. The ideal antigens include T-cell epitopes and B-cell epitopes, which can stimulate complete immune responses, and educe its effects of prevention and treatments. So epitope vaccine became the emphasis of noval virus vaccines in recent years.Bursa (bursa of Fabricus, BF) is an important center immune organs in poultry, BF ultrafiltrate (1KD below) contains a number of biologically active substances, in particular, a number of small peptide have immunomodulation function for poultry, which could promote cell differentiation and development of birds and mammals, and immune organs mature. Bursopentin is the new bursal active peptides, which obtains from bursa of Fabricus. According to the report, the bursopentin has immunomodulation function for poultry.In this study, we constructed three recombinant genes using gene recombination technology, which comprising of a multiple-epitope gene from E protein of Japanese encephalitis virus、bursin-like peptide gene and bursopentin gene. Then three recombinant genes were ligated into prokaryotic expression vector, and expressed in E.coli BL21. The Immunogenicity and protective efficacy of three recombinant proteins were observed in BALB/c mice. The results would be helpful to developed attractive Japanese encephalitis virus multiple-epitope vaccines with high immunoprotection. The contents of the paper contain two parts as follows:1Expression of Multi-Epitope of Japanese Encephalitis Virus Envelope Protein%Bursin-Like Peptide and BursopentineThe artificial multiple-epitope gene (designated MEP), containing six B cell epitopes (amino acid residues75-92aa,149-163aa,258-285aa,356-362aa,373-399aa and397-403aa) and two T cell epitopes from E protein of Japanese encephalitis virus (amino acid residues60-68aa and436-445aa), was amplified by PCR. In order to construct the recombinant expression plasmid pET-MEP, the MEP gene was double-digested with EcdRI and Xhol, and followed by ligation into the corresponding restriction sites of pET-28a(+) vector. Bursin-like peptide as an adjuvant is a potent enhancer of immune response in mice immunized with the JEV subunit vaccine, and we constructed a recombinant expression vector, it’s called pET-MEP-BSA. In addition, the new peptide termed Bursopentin (BP5) was obtained from the bursa of Fabricius (BF) in our laboratory. In order to understand the immune effect, and we constructed another recombinant expression vector, it’s called pET-MEP-BSA-BP5. Then three plasmids were transformed into host bacterium BL21(DE3). After IPTG inducing and spallation with ultrasonic waves, the soluble analysis of expression products were done with SDS-PAGE, and three fusion proteins were purified on the HisBind chelation affinity column, then three purified fusion proteins (rMEP、rMEP-BSA、rMEP-BSA-BP5) were analysis by Western Blotting. The result shows that three recombinant fusion proteins were expressed by means of inclusion. Western Blotting indicated that three recombinant fusion proteins had specific antigenicity of JEV.2The influence of rMEP、rMEP-BSA and rMEP-BSA-BP5on Specific Immunization in Mice4-6weeks old female BALB/c mice were randomly separated into5grouos,10mice per group, mice were intramuscularly immunized with50μg of rMEP、rMEP-BSA、 rMEP-BSA-BP5protein (diluted by100μl PBS) three times at2weeks intervals, and with PBS and JEV inactivated vaccine as control. The effect was determined in the form of protective anti-JEV antibody titers, antibodies (IgGl and IgG2a), spleen cell lymphocyte proliferation, levels of interferon-y and interleukin-4cytokines, the T-lymphocyte sub-type composition, and the mice protection against JEV challenge. The IgG1titer and interleukin-4level suggested that the rMEP protein potentiates a Th2immune response, and the response changed when the immunogen was replaced by rMEP-BSA and rMEP-BSA-BP5protein. However, the IgG2a titer and interferon-y level significantly lower than the attenuated vaccine, but higher than that of rMep protein. The immune response level of rMEP-BSA and rMEP-BSA-BP5are very similar. Challenge experiments showed that the mice which received rMEP、rMEP-BSA and rMEP-BSA-BP5or the inactivated vaccine were completely protected against JEV challenge. The results showed that rMEP、rMEP-BSA and rMEP-BSA-BP5have strong immunogenicity and provide the basis for the further research in pigs. From the above results indicated that bursin-like peptide combined with multiple-epitope gene can enhance the immune response in mice, while bursopentin not obvious enhancing immune response. Therefore, our findings indicate that rMEP and rMEP-BSA protein may be an attractive candidate as a vaccine for the prevention of JEV infection.