Constraction?Efficient Expression And Preparation Of Nanocellulose Single Chain Viariable Fragment Anti Porcine Epidemic Diarrhea

The frequent outbreak of porcine epidemic diarrhea in recent years and the high lethality of suckling piglets have not only brought huge economic losses to the pig breeding industry,but also demonstrated that traditional vaccines(inactivated vaccines,live vaccines,etc.)that rely on active immunity have no effect on suckling piglets.So the use of genetic engineering methods to develop new vaccines has positive implications for the treatment of swine epidemic diarrhea.The imperfections of the piglets' immune system at the age of 7-14 days and below prove the urgency of passive immunization of piglets.Therefore,this study used genetic engineering methods to prepare and detect antibodies that can be used orally.In this study,based on the anti-PEDV COE single-chain antibody,the cellulose binding domain was inserted into the sc Fv sequence sequence 5' while the E.coli prokaryotic expression was optimized,and the prokaryotic expression vector p ET19B-CBD-sc Fv was constructed..The expression conditions of BL/p ET19B-CBD-sc Fv were optimized,and the effects of temperature,induction dose of IPTG,length of induction and other factors on the growth and protein expression of the bacteria were studied.Preparation of nanocellulose by sulfuric acid hydrolysis,50 g of cellulose paper was added to 500 m L of 64% s?Lfuric acid,45 °C water bath 1h.The reaction product was added 2.5L of deionized water to terminate the reaction,centrifuged after centrifugation,dialysis,ultrasound.The renatured CBD-sc Fv was combined with CNC and the conditions for binding were optimized from the conditions of cellulose concentration,binding time,and bonding temperature.The laboratory-preserved PEDV vaccine virus was inoculated with 10% vero cells to 3 passages.The cells were frozen and thawed 3 times after 72 hours.The supernatant was collected by centrifugation and stored in a-80? refrigerator.The virus and renatured sc Fv samples were used for the virus neutralization test.PEDV and sc Fv at different dilutions were added to a 96-well plate to observe the cell morphology in each well.The final authentic BL/p ET19B-CBD strains were studied.The best condition of sc Fv was at 37°C,the IPTG induction concentration was 0.6m M,the induction time was 7h,and the protein content induced by the optimal induction conditions was up to 0.952mg/m L.The concentration of CNC prepared by sulfuric acid hydrolysis is 4.02 mg/m L measured by thermal drying method.CNC with a hydrodynamic mean radius of 229.8 nm were detected by dynamic light scattering.CNC and refolding sc Fv binding experiment results show that the best combine condition was at room temperature(25°C)and incubation for 4h.The ReedMuench method was used to measure the TCID50 of the cultured PEDV virus by TCID50=10-1.32/100 ?l.The final results show that the cell morphology changes per well did not reach the ideal state,but also further cell optimization and the next step in animal experiments to verify the neutralization of PEDV and single-chain antibody.In summary,codon-optimized CBD-sc Fv can be highly expressed in prokaryotic expression system;meanwhile,CBD-sc Fv can be effectively combined with prepared CNC,and successfully prepared the complexes CBD-sc Fv and CNC that can be stored at room temperature.PEDV successfully cultured in vero cells identified by RT-PCR;neutralization test of virus and CBD-sc Fv had failed,but the next step can continue at viral titers and its concentration with CBD-sc Fv to optimize the ratio.This study laid a solid foundation for the stable preservation of CBD-sc Fv and the efficient expression of CBD-sc Fv,and laid a certain research foundation for the study of passive vaccines immunization against PEDV,providing a new approach for the prevention and treatment of PEDV.