Study On The Freeze-drying、Security And Antiviral Activity In Vitro Of Chicken Genetic Engineering Composite Antiviral Preparation

Animal disease caused by virus account for a large proportion,and the incidenceand mortality show an increasing tendency. It has increasingly become the primaryfactor to harm breeding industry.And There has been lack of specific drug to cure viraldiseases at present. In order to search for the genetic antiviral drug with new, no-residueand no-drug-tolerance and solve the technical difficulty of the lack of such drugclinically, this study invested the freeze-drying process、safety experiment and antiviralactivity test in vitro of Chicken genetic engineering composite antiviral preparation withthe chief constituent of rIFN-α-linker-IL-2recombinant protein.First, screen the cryoprotectant of Chicken genetic engineering composite antiviralpreparation. The Chicken genetic engineering composite antiviral preparation wasfreeze-dryed with different cryoprotectant prescriptions maked up by skim milk、sucrose、sucrose、trehalose、Vc after110℃，15minor115℃，20min autoclavedsterilization.Then select the ideal cryoprotectant by exterior inspection、 phmeasurement、test of recovery properties of water and potency assay. Place theaquative Chicken genetic engineering composite antiviral preparation and lyophilizedChicken genetic engineering composite antiviral preparation under4℃、-20℃、-70℃for the preservation period experiment. Safety experiment contains sterility test、bacterial endotoxin test、toxicity evaluation(guinea test、mouse test、neonate rattest、 chicken test)and biosafety test （determination of Ampicillin residues、determination of E.coli P）. At last，measure the antiviral activity of Chickengenetic engineering composite antiviral preparation against VSV and IBDV on CEF.The result suggested that after freeze-dryed with the prescription（skim milk＋sucrose and skim milk＋sucrose＋trehalose＋Vc）of cryoprotectant after110℃，15min autoclaved sterilization，the Chicken genetic engineering composite antiviralpreparation has fairly good appearance, the value of pH is7.3, the average rehydrationtime is less than5seconds, the titer of antiviral activity is220, so the two prescriptionscan well protect Chicken genetic engineering composite antiviral preparation. The preservation period experiment showed that the aquative Chicken genetic engineeringcomposite antiviral preparation and lyophilized Chicken genetic engineering compositeantiviral preparation under-70℃can preserve one year with no decline of thetiter,while under4℃、-20℃,the titers of the aquative preparation respectively start todecline after90d、180d,the lyophilized preparation has no obvious variation, theresult indicates the screened lyoprotectants remarkablely extend thepreservation period of Chicken genetic engineering composite antiviral preparation. Theresult of the sterility test and bacterial endotoxin test is eligible,the weights of theguineas、 mouse、 neonate rats、 chickens of the security test all increase. Thedetermination of Ampicillin residues is eligible,and the residue of E.coli P isresidue,which is less than that of the sample. Chicken genetic engineering compositeantiviral preparation has good biological antiviral activity against VSV and IBDV inCEF, while the activity is respectively5.243×10~6IU/mL and6.554×10~5IU/mL.This research selected the cryoprotectant of Chicken genetic engineeringcomposite antiviral preparation, produced lyophilized Chicken genetic engineeringcomposite antiviral preparation with good exterior and steady activity and solved theproblems of poor thermal stability, difficulty in long-term preservation and long-distance transportation,and proved that Chicken genetic engineering composite antiviralpreparation has good safety and good antiviral activity in vitro. It also lay thefoundation for the large-scale production of Chicken genetic engineering compositeantiviral preparation and the completion of application for registration of new veterinarymedicine, and make some contribution to clinic promotion and application.