Construction Of PcDNA3/hIL-22 And PcDNA3/hIL-22-VP1 And Study On Its Immunological Effect On Mice

Objective: Human interlerkin-22 is a novel cytokine discovered by Dumoutier et al. in 2000. Mature hIL-22 is constituted of 179 amino acids, and has 23% homogeneity with interleukin-10; It is produced mainly by T lymphocyte, including CD4(+) T lymphocyte, and NK cells can produce little of it. By binding with its receptor, hIL-22 could activate STAT1,3,5 signaling pathways. JAK1 TYK2 and MEK-ERK-RSK,JNK/SPAK and P38 MAPK Signaling pathways, that medicate the transcription and translation of genes. Its main biological effect including: up-regulating the expression of acute reactive proteins in liver cells; inducing the expression of pancreatitis associated Protein PAP1 in pancreatitis. In addition, it was found to be related to lymphoma inducing. asthma susceptibility , anti-parasite immune response and cell expansion of B1 lymphocyte; Regarding Immunological regulation, hIL-22 inhibits the production of IL-4 by Th2 lymhpocytes but has no influence on the production of IFN-γin these cells. Wolk et al. attributed IL-22 to Th1 type cytokine and believed it could mediate cell immune response. More biological functions of IL-22 remain to be investigated, and the construction ofeukaryotic expression plasmid of hIL-22 will lay a foundation for this. Thus, we cloned the gene of hIL-22, constructed the plasmid of pcDNA3/hIL-22 and observed its protective effect on CVB3 challenged BALB/c mice. Coxsackievirus belong to picornavirus, and coxsackievirus group B (CVB) is the prodominant pathogen of human viral myocarditis. Clinically, at least 50% acute myocarditis are caused by this virus, with CVB3 being the most common. Until now, there is no effective CVB3 vaccine available and DNA vaccine provide an alternative for treatment and prevention of CVB infections. The gene of CVB3 VP1 protein is a conservative gene, and the protein that encoded can stimulate the production of antibody. At present, most investigators have constructed the eukaryotic expressing plasmid of CVB3 VP1 gene to use as a gene vaccine. But the ported observations suggested that although the vaccine constructed with VP1 alone could induce the production of antibody, the titers of antibody produced were usually too low to provide protection. Therefore, enhancing the protective effect of VP1 gene vaccine is so far a problem need to be resolved. Adding genes of cytokine, costimulatory factor, ISS or immunological regulatory molecular as a molecular adjuvant, to the vaccine seems to be a possible way to solve this problem. As a cytokine, whether the IL-22 has or not the immune enhancing effect is worthy to investigate. Thus, we constructed a double expressing plasmid of pcDNA3/hIL-22-VP1 and observed its effect on theproduction of antibody and protective effect against CVB3 infection in CVB3 challenged mice.Methods: ⑴ Lymphocytes were isolated from human peripheral blood and the RNA was extracted after stimulating with ConA; PT-PCR was performed using a pair of primers specific to hIL-22, P57(+)5'-GAAGGATCCGTTATGGCCGCCCTG CAGAA-3',P596(-)5′GGACTCGAGTCAAATGCAGGCATTTCTCAG-3′,to amplifying the gene fragment of hIL-22, ⑵ The amplified product was linked to the plasmid pGEMT and competent DH5αwas transformed. The plasmid DNA was extracted and identified by enzymes digesting and sequencing. ⑶ The pGEM-T/hIL-22 was cut by BamHⅠand XhoⅠ, and linked to pcDNA3 that had be cut using the same enzymes to construct a eukaryotic expressing plasmid pcDNA3/hIL-22. After transformation, the recombined plasmid was identified by enzymes cutting. ⑷ Using the plasmid pcDNA3/VP1 as a plate, PCR was performed to amplifying a VP1 gene fragment that carried the genes fragments (CMVp-VP1-BGHp) of CMV promotor and BGH Ploy A tailing Signal. The primers used were P209(+)5'-GCATAGATCTGCGATGTACGGGCCAGATAT-3'和P1249 (-)5'-ATACAATTGTCCCCAGCATGCCTGCTAT-3'⑸ The amplified PCR product CMV-VP1-BGHp was cut by BglⅡand NruⅠand linked to pcDNA3/IL-22 that had be cut by the same Enzymes, to construct the eukaryotic double expressing plasmid pcDNA3/IL-22-V...