Construction And Immunogenicity Of Nucleic Acid Vaccines Containing C3d As Molecular Adjuvant

In recent years, with the development of molecular biology and immunology theory and technology, the research of biological products using to prevent and treat have already become a hot spot in the immunology field by genetic restructuring express technology. Among them the nucleic acid (DNA) vaccine is extremely popular. The nucleic acid vaccine is a new generation of vaccine after the inactivated vaccine, live attenuated vaccine, unit vaccine and genetic engineering vaccine, which has many advantages, such as inducing the humoral immune and cellular immune response, safety in the use, easy to produce, and so on. The gene combination vaccine could be made by integrating some genes together. However, the speed of DNA vaccines'application development is limited because its'low protein expression and weaker immune response. Thus, improving the immunity effect of DNA vaccine is an urgent problem to solve in the immune study. At present, application of molecular adjuvants is an important measure to improve the immunity effect of DNA vaccines. The molecular adjuvants have interleukin, interferon, thymosin and complement molecular adjuvants etc, which complement molecules C3d as a new molecular adjuvants is of growing concern.C3d is the final cleavage product of complement C3 while C3 is activated, which can promote antigen presenting cells (APC) to carry on the uptake of antigens in , to present antigens, and improve the immune response capability. Therefore, C3d has been defined to be a new and effective molecular adjuvant in the nucleic acid vaccine. Whereas C3d as molecular adjuvants has different animal species diversity, the immuno-enhancing differences of C3d among different animals need further study.GP5 protein is a glycosyl capsule membrane protein coded by PRRSV GP5 gene, which has good immunogenicity and could induce the specific humoral immune and cellular immune. InvH gene is the highly conservative gene at salmonella A-E groups, which can code the surface protein to adsorb and invade epithelial cells. The capacity of salmonella bacteria to invading intestinal mucosa cells is decided by this protein.Firstly, C3d genes of mammals (pig and mouse) were cloned and sequence analyses were investigated in this study; Subsequently,the relevance and difference between mammals and poultry animals (pig, mouse, chicken and duck) was investigated in gene level. And then, nucleic acid vaccines containing PRRSV GP5 model gene with the complement p28 polymersomes of mammals (swine or mouse) (complement C3d receptor binding domain), or the complement p29 polymersomes of poultry (chicken or duck) and nucleic acid vaccine containing Salmonella invH model gene with C3d-p28.6 of mouse were constructed; mice were inoculated by the nucleic acid vaccines and the main immune indexes of humoral immune and cellular immune were evaluated to observe the immuno-enhancing effect of the different animals C3d (C3d-p28(29).n(n=2,4,6))in the nucleic acid vaccines. This research mainly including four sections:1. Cloning and Sequence Analysis of Complement Component C3d from the mammals pig and mouse: In order to obtain mammals (pig and mouse) C3d gene cloning and compare the differences with poultry animals (chicken and duck) C3d gene sequences, total cell RNA was extracted from the liver tissue of the mammals (pig and mouse), and the cDNA of C3d was amplified by reverse transcription polymerase chain reaction (RT-PCR). The cDNA fragments were directly cloned into pMD18-T plasmid, and the recombinant plasmids pMD18-T-C3d were identified by restriction endonucleases digestion and sequencing. The cloned C3d genes and CR2 binding region on C3d were compared between mammals and poultry. Electrophoresis showed the C3d gene cloning of pig and mouse were obtained successfully for the bright strips in 936bp and 888bp respectively. The results of sequence analysis indicated that the homology of complement C3d gene between mammals (pig and mouse) and poultry (chicken and duck) is only 64%; the phylogenetic tree showed C3d had varieties in species mammals and poultry, more close relationship, more close evolution. Structural analysis in CR2 binding region indicated that there were 28 amino acids in mammals but 29 amino acids in poultry; the homologous amino acids were only 60% between poultry and mammals, however there were more homologous between mammals pig and mouse 82.8% and between poultry chicken and duck 84%, which indicated that the complement C3d and the CR2 binding region had the genus-specificity. 2. Construction of nucleic acid vaccines containing PRRSV GP5 gene with complement C3d of different animals as molecular adjuvants: In order to find out the immuno-enhancing effect of molecular adjuvant C3d of different animals in the nucleic acid vaccine, after cloning the C3d cDNA, four pairs of primers were designed to subclone the C3d-p28(29) gene to the pUC19 plasmid. Several tandems of C3d-p28(29) were constructed in the pUC19 plasmid used a pair of isoschizomers BamHI and BglII. Digested the pUC19-C3d-p28(29).n to get the gene of C3d-p28(29).n, and then cloned the products to pcDNA3.1 (+) plasmid. After this, the GP5 gene of PRRSV was cloned through RT-PCR and inserted to the upstream of the C3d-p28(29).n which is in the pcDNA3.1-C3d-p28(29).n, and the nucleic acid vaccines containing PRRSV GP5 gene with C3d-p28(29).n as molecular adjuvants were constructed. Electrophoresis showed that the bright strips appear in 807,987,1167bp after digested the reconstructive plasmids (pcDNA3.1-GP5-C3d- p28(29).n) respectively, which indicates that the reconstructive plasmids containing PRRSV GP5 gene with C3d-p28(29).n as molecular adjuvant were constructed successfully.3. Study on immunity enhancement effect of nucleic acid vaccine containing PRRSV GP5 with C3d of different animal as molecules adjuvants: In order to explore the immuno-enhancing effect and difference of the PRRSVGP5 nucleic acid vaccine containing molecules adjuvant C3d of different animals, the reconstructive plasmids were extracted and expressed instantaneously in the Marc145 cells by liposomes carrying after the reconstructive plasmids of four kinds of animals (pcDNA3.1-GP5-C3d-p28(29).n) and the plasmid (pcDNA3.1-GP5) were constructed. Subsequently, the vaccines'abilities to elicit the humoral and cellular immune responses were investigated in BALB/c mice. The result showed that the reconstructive plasmids well expressed as GP5 protein in the Marc145 cells and that significantly enhanced GP5-specific ELISA antibody, GP5-specific neutralizing antibody, IFN-γlevel, and IL-4 level, could be induced in mice immunized with nucleic acid vaccines encoding the pcDNA3.1-C3d-p28(29).n-GP5 than those received nucleic acid vaccine expressing only the pcDNA3.1 vector and pcDNA3.1- GP5 group (P <0.05), although these were not as effective as inactivated oil-emulsion vaccine. The increase in the immune response elicited by six copies of p28(29) was higher than four copies of p28(29) (P <0.05), which is also higher than two copies of p28(29) (P <0.01). Furthermore, there were no differences of the GP5 antibody level, IL-4 and IFN-gamma content in the serums of mice immunized with nucleic acid vaccines containing the same copies of C3d-p28(29).4. Construction and immunogenicity of nucleic acid vaccines containing invH gene of salmonella with murine complement C3d as molecules adjuvants: In order to further explore the immuno-enhancing effect of C3d as molecular adjuv- ants in the bacteria nucleic acid vaccine. Nucleic acid vaccines containing the invH as model gene with six copies of mC3d-p28 as molecules adjuvant were constructed; subsequently, mice were inoculated by the nucleic acid vaccines and the invH antibody level, IL-4 and IFN-gamma content in the serums were detected; and then, tapping poison protection test was performed in mice. Result showed differences of the invH antibody level, IL-4 and IFN-gamma content in the serums were significant compared with those received nucleic acid vaccine expressing only the pcDNA3.1 vector and pcDNA3.1-GP5 group (P<0.05).The results of poisoning experiment showed that the protective rate of the vaccine containing six copies of mC3d-p28 was higher than pcDNA3.1-invH group, significant difference (P<0.05), and was extremely significant difference (P<0.01)compared the blank group and pcDNA3.1 group, although these were not as effective as inactivated oil-emulsion vaccine.The results of this study has proven the immuno-enhancing effect of molecular adjuvants C3d in the viruses and bacteria nucleic acid vaccine, which may provide theoretical basis and technical support for the development of other pathogens'nucleic acid vaccine using C3d of different animals as molecular adjuvants.