PVAX-ptx3 Plasmid Extraction Optimization And In Clinical Practice

PTX3 was the first confirmed long pentosan protein.In the early 1990s,PTX3 was first discovered as a cytokine-inducible gene for endothelial cells and fibroblasts.PTX3 was found to bind to the surface of microorganisms such as pyrethroid,And some viruses,but it can not bind to Berkeley,enterotoxin A and B,and LPS,through the study of genetically modified mice found that PTX3 protein has resistance in piaying certain microorganisms important role,it has a potential application value.In order to improve the yield of plasmid DNA and reduce the cost,the best medium was screened,the selection of several alternative basal media was selected by single factor.Seven kinds of the initial medium were selected by Plackett-Burman test design.The functional data were analyzed by Minitab software,and the relationship between the three main factors and the yield of plasmid DNA was established and repeated three times.It was concluded that LB medium was selected as the base culture medium.The optimum nitrogen and carbon sources are sucrose and casein peptides,respectively Finally,The optimal medium(Casein peptides 11.65 g/L,Sucrose 2.55 g/L,Yeast extract 5g/L,NH4Cl 3.33 g/L,NaCl 10g/L,Na2HPO4 12 g/L,KH2PO4 2 g/L)was obtained by Plackett-Bunnan test The concentration of plasmid DNA was 350.5?g/mL,and the concentration of plasmid DNA was(350.5 ± 5)?g/mL.In order to explore the clinical application of porcine pVAX-ptx3,the recombinant plasmid pVAX-ptx3 was extracted with Omega kit.Vaccination of fresh piglets at a dose of 500 ?g/dose was performed at the same time,after weaning a week piglets were randomly selected three piglets,ear vein injection of S.suis 2 HA9801(1 × 108 CFU).By testing the live bacteria load of peripheral blood and various organs,The results showed that the survival rate of peripheral blood and organ viable cells in the immunized group was significantly lower than that in the control group(100%)was significantly higher than that in the control group(33.3%).The results showed that pVAX-ptx3 had a protective effect on piglets,and the expression of PTX3 protein had some antibacterial effect on Streptococcus suis type 2In order to reduce the production cost of recombinant plasmid pVAX-ptx3,the difference of clinical effect of recombinant plasmid pVAX-ptx3 was extracted by comparing Omega kit and non-kit.the recombinant plasmid pVAX-ptx3 was extracted by alkaline lysis method and the best culture medium selected by response surface method.The extracted plasmids were purified by PEG and diatomaceous earth respectively.The recombinant plasmid pVAX-ptx3 was extracted with omega kit and non-kit,Newborn piglets were immunized with a dose of 500 ?g,while each group set the control group.The results showed that the purified plasmids of diatomaceous earth were less,and the quality was higher than that of PEG;and the plasmid extracted from the kit was higher in weight gain than the control group.In the natural state,the plasmid extracted from the Omega kit(5%)was lower than that in the control group(20%),and the noninucleator(0%)and the control group(5%)were not significantly different.The IL-6 And IL-8 were significantly higher than those in the control group.