Preliminary Comparison Of Humoral Immunity In Mice Induced By NDV F And IBDV VP2 Multi-epitope Vaccine

Newcastle disease (ND) and Infectious bursal disease (IBD) are two diseases endangering raising industry, it can cause great economic loss with high mortality. Vaccination is the effective means to prevent these two kinds of disease. At present, the use of the vaccine has maken these two kinds of disease under control. However, there are still some atypical ND phenomenon after multiple immunizations, IBD also appears infection of virulent strain, all these often leading to immune failure. At present, the research hotspot in the field of gene engineering is multi-epitope vaccine. Multi-epitope vaccine can be presented efficiently by being identified and combined with MHC molecules which have a variety of genetic backgrounds. It is highly desirable and efficient. Compared with the traditional vaccines, the applicability and operability is great. The joint, modification or reconstruction of epitope can be operated timely and conveniently on the plasmid level.Studies have shown that invariant chain (Ii) plays an important role in antigen presenting process. The four Amino acids LRMK links N fringe of CLIP segment in Ii is called Ii-key. Ii-key can be used as immune carrier to connect epitope and it can enhance the immune responses; other parts of Ii can promote the enhancement. This artical used mouse Ii functional fragments as immune carrier to construct four groups of different series multi-epitope vaccines contained NDV and IBDV. And it is intended to get the best immunogenicity through studying the possibility of resistance against different strains by multi-epitope vaccine and the impact of tandem sequence.Firstly, in the light of the construction of mouse Ii, that the cytoplasmic domain, TM domain, Ii-key, we reconstructed chimera (Cyt/TM/Ii-key/F306 Cyt/TM/Ii-key/F306/VP2、Cyt/TM/Ii-key/VP2、Cyt/TM/Ii-key/VP2/F306) connected with NDV epitope F306 and IBDV epitope VP2. We amplified the fragments according to the designed primers and the reserved plasmid template pEGFP-Cl-Ii, pET-32a-F306 and pET-32a-VP2, the fragments were inserted into the prokaryotic expression vector pET-32a. The results of enzyme analysis and DNA sequence showed the recombined plasmids (pET-32a-Cyt/TM/Ii-key/F306、pET-32a-Cyt/ TM/Ii-key/F306/VP2、pET-32a-Cyt/TM/Ii-key/VP2、pET-32a-Cyt/TM/Ii-key/ VP2/F306) were formed successfully. pGEX-4T-1-F306、pGEX-4T-1-VP2 were used as coating antigen for ELISA.Secondly, recombined plasmids were inserted into the Escherichia coli. The overexpression of the protein was made after the determination of the best expression condition, which was determined by optimizing the induction time (4 h) and concentration (0.8mmol/L). Target protein were obtained by improved KCl plastic cutting method and the molecular weight of the purified protein obtained are 41.35kDa、43.22kDa、31.56kDa、43.22kDa、37.22kDa、27.43kDa.Using Folin-phenol method to detect the concentration of each fusion protein. Result of each fusion protein is:His-Cyt/TM/li-key/F306 1.47mg/mL、His-Cyt/TM/Ii-key/F306/VP2 0.91 mg/mL、His-Cyt/TM/Ii-key/VP20.78 mg/mL、His-Cyt/TM/Ii-key/VP2/F306 0.96 mg/mL、GST-F306 0.93 mg/mL、GST-VP2 0.85 mg/mL, and all reached the requirements of the follow-up experiment.Finally, SPF level female Balb/c mice were immunized with the purified target protein, serum were collected from the eyes after immunization was made 3 times later. The concentration of coating antigen was 2.0μg/mL and the best working concentration of antibodies was 1:2000, which were determined by orthogonal matrix. The antibody titers measured by ELISA respectively reached between 2.1 X 104 and 8.6 X 104. The results show that:compared with those immunized with single epitope (His-Cyt/TM/Ii-key/F306、His-Cyt/TM/Ii-key/VP2), the groups immunized with His-Cyt/TM/Ii-key/F306/VP2、His-Cyty/TMIi-key/VP2/F306 the antibody titers did not decrease. Analysis by statistical software shown that the differences in immune effects is not significant (P>0.05) according to different sequences of epitope (that is the interval size between epitope F306, VP2 and immune vector Cyt/TM/Ii-key). According to the analysis of Western Blot, the purified fusion protein has good immunogenicity.To sum up, this experiment obtained purified fusion protein through prokaryotic expression, immunized mice and obtained specific antibody. The antibody titers were determined by indirect ELISA and they showed that the multi-epitope vaccine could induce antibody of different strains and the effects on the immune effect is not significant according to different sequences of epitope. This also provided experimental basis to the new immune vector Ii applying to prevention and treatment of Newcastle disease and infectious bursal disease.