The Production Precess And Effectiveness In Dtap Study Of Animal-free Tetanus Toxoid

In the appropriate medium,Tetanus bacteria produce tetanus toxin which can obtain high purity toxoid by purification,detoxification and column chromatography.Tetanus vaccine absorbed by adjuvant has great protection effect in immunization.Now,main nutriment of Tetanus medium was beef heart infusion or casein digest,thus can't obviating the possibility of prion disease.Also,beef heart infusion be bad for production in industry because of complicated manufacturing precess and low stability in all batch.In this study,we develop a animal-component-free medium by single factor test,orthogonal test and response surface optimization.Then,through centrifuge,filter,column chromatography,detoxification and refinement to obtain high purity tetanus toxiod.At last,DTaP titer was compared with different adjuvants.Through a series experiments,the following conclusions we made:1.In this study,carbon source was glucose and the level of tetanus toxin was compared by peptone and yeast extract act as medium carbon source respectively.Result of those experiments show that yeast extract was better nitrogen source.Through response surface analysis,the 60 Lf/mL toxin was obtained by using animal-free-medium containing 9.2g/L yeast extract and 6.66g/L glucose.2.The fermentation liquor supernatant from centrifugal action are filtered and then purified by column chromatography.Those steps can recovery 80%tetanus toxin at 80 mmol/L salt concentration.In this study,four conditions detoxification experiments show that obtaining tetanus toxiod the hightest limit of formaldehyde and lysine are 27nmol/L and 20 mmol/L.We obtain 2500 Lf/mgPN tetanus toxiod and 85%recovery by using hydrophobic colume chromatography.3.Adult vaccines contain DT,TT,PT,PRN,FHA and FIM 2 and FIM 3 antigens absorbed by aluminum phosphate adjuvant or aluminum hydroxide adjuvant respectively.Immunnogenicity results of NIH mice showed that antibody titers above national standards.Aluminium phosphate is the better adjuvant in DTaP than aluminum hydroxide,ELISA results show that,because higher antibody titer of antigens absorbed by aluminium phosphate.Outside the PRN antigen,we found no statistically significant diffrents between antibody titer that contain FIM or don't.