| Giant Panda is considered a "national treasure" in China, and it is also a kind of rare wild animal all over the world. However, it is close to extinction. Except for man’s encroachment on the territories of giant panda, diseases are probably the greatest threats. Among40kinds of disease of giant panda, canine distemper become the most serious disease that threat the population and life safety of giant panda. Vaccination is now the most effective method for the animal. But current routine vaccines are shown not good enough to defense pathogen infection because of the shortages on efficiency and safety. Hence, how to improve the efficiency of vaccine under a safe dosage, become one of the hottest topics in vaccine research area. Cytokine, Produced by immune cells and other correlative cells, is a kind of pleiotropic protein which Plays important roles during induction and modulation various immune responses. Interleukin2and interleukin6are important members of IL family, it is often reported that they have been studied extensively as potential vaccine adjuvants to enhance the efficiency of routine vaccine.In view of this, we make a research about the adjuant effects of giant panda recombinant IL-2、IL-6and their fusion protein on canine distemper vaccine in a murine model. The main research included as below:1. In this report, the cDNA for giant panda’s IL-2and IL-6gene was cloned from the ConA-stimulated giant panda peripheral blood mononuclear cells (PBMCs) by RT-PCR. The amplified products of PCR were cloned into PMD18-T vector. After identification by restriction enzyme and sequencing, Bioinformatics analysis was performed to predict the characteristics of this gene. Sequencing revealed that IL-2gene was composed of468nucleotides, encoding a polypeptide with164amino acids, the signal peptide contained20amino acids and the mature peptide was147amino acids, exhibiting a molecular mass of17.92kDa. Compared with other mammals in Genbank, including human, dog, cat, rat mustela putorius and So on, the homology of amino acid sequence Was between44%and 90.4%. IL-6gene was composed of636nucleotides, encoding a polypeptide with211amino acids, the signal peptide contained25amino acids and the mature peptide was composed of186amino acids, exhibiting a molecular mass of23.67kDa. Compared with other mammals in Genbank, including human, dog, cat, rat, mustela putorius and So on, the homology of amino acid sequence was among34.9%～77.9%. Phylogenetic tree based on amino acid both showed that giant panda has the most closed relationgship genetic distance with mustela putorius.2.By the technology of DNA recombination, the IL-2、IL-6and IL-6/2was cloned into Prokaryotic Expression plasmids PET-32(a)+vector, and was transformed into Ecoli Rosetta. The target protein was detected by SDS-PAGE analysis after induction by IPTG, Then the special band of fusion expressed protein was roughly obtained, and it was approximately35kD,41kD and57kD, respectively. Polyclonal antibody was prepared by using the purification recombinant protein. The antibodies titer of IL-2and IL-6was1:4and1:8. And the analysis by western blot proved that the antibodies could react with the native protein.After purification and renaturation of the fusion protein, its biological activity were detected by MTT. The results indicated that the recombinant proteins both have the high ability to supply the proliferation of peripheral blood cells of mouse, and had higher biological activity.After injection the mice with mixture of CDV vaccines and adjuvants factor, blood were collection from tail vein of mice on7、14、21、28、35、42d. Anti CDV antibody levels, IL-4, IFN gamma levels of serum were detected by method of ELISA. The results showed that among three recombinant cytokines, IL-2could furthest enhance CDV-specific humoral and cellular immune responses. Unfortunately, we did not observe obvious positive effect of IL-6and IL6/2administration on CDV vaccination. |
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