Preparation And Immunization In Mice Of Recombinant Plasmid Carrying PCV2 ORF2 Gene

Preparation of plasmid is a fundamental manipulation in molecular biology experiments. With the development of DNA vaccine and genetic recombination medicine, the quantity of plasmid can't meet the need the fundamental rearch and clinical trials. For example, the DNA vaccine can't reach an ideal efficiency for the small immunizing dose, and the application is limited. In this research a new method of plasmid preparation is founded. Schizolysis the bacteria by alkaline, then filter the lysate by a kind of cloth in order to eradicate the proteins and the genomic DNA. The filtrate was precipitated by dimethyl carbinol, then it is filtered by a mixing cellulose ester micropore film of 0.22μm in pore size. Rinsing the micropore film using 70% alcohol, afer air drying, the plasmid DNA are eluted from the micropore film by distilled water. This method suit for 3 mL to 500 mL bacteria, and the average plasmid DNA yield is 15mg/L, with the average OD260/280 value 1.89. This method is simple and no harm to operate; otherwise both the quantity and the quality are good. It can be used in the laboratory and in the production of DNA vaccine.Porcine circovirus type 2 (PCV2) is the primary pathogen of post-weaning multisystemic wastingsyndrome (PMWS) and other associated conditions in pigs. In this study, a set of eukaryotic expression elements encoding PCV2 ORF2 is inserted into the plasmid vector pMD-18T, constructed the recombinant pMD-T-ORF2. pMD-T-ORF2 is transformed into E.coli DH5αto increase its copies. The Balb/C mice were injected intramuscularly with the recombinant plasmid pMD-T-ORF2 every 14 days three times totally. The control group is injected with physiological saline solution instead. The monitoring of antibody indicates that the specific antibody to the protein encoding by PCV2 ORF2 increases within the 14 days after the first injection. But the following immune almost makes no use of the level of antibody. Comparing the control group, the spleen lymphocytes proliferative ability have a little descent but the NK cells activities were significantly elevated (P Value<0.01). The percentage of the Tc lymphocytes in spleen is much higher than the control group, but the percentage of Th and the percentage of B lymphocytes have no difference between the two groups.