Study On Optimization Of Suspension Culture Inactivation Technology Of Foot-and-Mouth Disease Virus

The recovery of different process of BHK suspension culture foot-and-mouth disease from harvest to inactivation is different.1.Harvest of foot-and-mouth disease virus is subpackaged to 10 L bottles,and storaged in a cold room.Then they are melted,and suspension is filtered by filter mash,then filtered by 0.22 ?m,0.45 lun and 0.45?m three grades dead filters.Follow the regulations to inactivate the foot-and-mouth disease.The recovery is 60%.2.Harvest of foot-and-mouth disease virus is clarifying by continuous flow centrifugation and horizon depth filtration.Follow the regulations to inactivate the foot-and-mouth disease.The recovery is 90%.3.Mix the chloroform and harvest virus by bottom agitation.Then the virus suspension is centrifugated and go through the horizon depth filtration.Follow the regulations to inactivate the foot-and-mouth disease.The recovery is 70%.4.Mix the chloroform and harvest virus by top agitation.Then the virus suspension is centrifugated and go through the horizon depth filtration.Follow the regulations to inactivate the foot-and-mouth disease.The recovery is 92%.We have been monitoring the process of foot-and-mouth disease inactivation,and drawing the trend graphs.The results show that 1.5%volume BEI(0.1 mol/L)is added in virus suspension,and adjust the suspension temperature to 30?,agitation 10 minutes every 50 minutes for 24 hours.Transfer the virus suspension to another tank.Add 1.5%volume BEI(0.1 mol/L),and keep the temperature in 30? for 24 hours.Add the 2%volume sodium bisulphite(50%)to neutralize the excess of BEI.We make a kinetic from first four hours samples.Horizontal coordinates is inactivation hours,and vertical coordinates is TCID50.The cross of coordinate curves and horizontal coordinate is the zero point theoretically.Zero point must be bigger than a half inactivation time,and smaller than a quarter of inactivation time.The cross of coordinate curves and vertical coordinate is the endpoint.The endpoint must be lower-6.0.1 mol/L BEI is prepared from 0.1 N 2-bromoethylamine HBr and 0.175 N NaOH.?-naphthol violet(BVN)is a indicator for BEI prepared.The color of solution will change from violet to orange,as the PH of the solution will change from 12.5 to 8.5.Safety test of inactivated foot-and-mouth disease virus is passed.0.2 mL inactivated foot-and-mouth disease virus is hypodermic injected in suckling mouse two generations of blind transmission.No symptoms is found.PEG is used for foot-and-mouth disease virus purification and concentration 10 folds with diatomaceous earth.After two times PEG diafiltration,FMDV antigens is concentrated 100 folds.3KD Millipore ultrafiltration centrifuge tube is used for further concentrated 3-5 folds.300-500 folds FMDV antigen is tesed for purity.Liquid chromatographs show that there is only a little impurity in FMDV antigen.Electron microscope photos show that there is little impurity around 146S,and particle size is not bigger than FMDV.Suckling mouse safety test shows there is little allergenic protein in a PEG antigen.0.2 mL concentrated antigen is hypodermic injected in suckling mouse two generations of blind transmission.No foot-and-mouth disease symptoms is found.BHK monolayer is infected by mouse body grinding liquid.No cytopathic effect is found.We find that the mixture of chloroform and BHK suspension culture harvest of foot-and-mouth disease virus is centrifugated and go through the horizon depth filtration by top agitation for 1-2 hours.Follow rules to inactivate the virus.The recovery rate of 146S is the best.