Screening Of The Recombinant Plasmids With Immunostimulatory And Its Biosafety In Swine

The SPF swine peripheral blood mononuclear cells (PBMCs) by Ficoll gradient centrifugation procedure, were prepared and stimulated by recombinant pcDNAKCpG-1, pcDNAKCpG-2, pcDNAKCpG-3 and pcDNAKCpG-4 with four doses (1.25μg, 2.5μg, 5.0μg, 10.0μg) respectively. The effect of the lymphocyte transformation was detected by MTT assay. It is showed that the four plasmids had different extent immunostimulatory effect; the stimulate index of the 10μg of pcDNAKCpG-4 is 6.3. After elaborate analysis, the conclusion was drawn that the pcDNAKCpG-2 had the most immunostimulatory activity on swine because it had a good immunostimulatory effect even at low doses.The swine injected with 500μg of pcDNAKCpG-2 with the most immunstimulatroy activity were slaughtered to extract the gDNA of heart, liver, spleen, lung, kidney, blood and muscle of the injection site at 4h, 4d, 8d, 15d and 30d after injection. Then the distribution and persistence of the plasmid in the tissues were detected by PCR using specific primers. It is showed that pcDNAKCpG-2 was detected in muscle at 4h after injection; Four days later, the fragment of plasmid was amplified from the blood and muscle of one pig by PCR. The plasmid could exist in organs no more than 8 days after injection.On the base of above research, the gDNA of PCR positive and NO.9 pig at 30d after injection was identified by southern blotting with a DIG labeled probe to determine whether the plasmid had integrated into host chromosome or not. Results showed that the plasmid did not integrate into the gDNA of swine.Generally, pcDNAKCpG-2 has the most immunostimulatroy effect on swine; the distribution and persistence of the plasmid could exist in organs no more than 8 days after injection; and it wouldn't integrate into the genomic DNA of swine.