| Porcine epidemic diarrhea virus(PEDV),a coronavirus that causes porcine epizootic diarrhea(PED),is a type of positive-sense single-stranded RNA virus with capsule,which belongs to Alphacoronavirus,part of the family Coronavirinae.This decease mainly occurs in cold and wet seasons,and piglets are more vulnerable.Infected piglets will die for water-like diarrhea,emesis,dehydration and electrolyte disturbance.The severity of morbidity and mortality rate are negatively correlated to the day-age of piglets,and especially for those newborn piglets under seven days without effect maternal antibody,the death rate will reach up to 100%.Because of the outbreak of epidemic wild type strains in recent years,traditional commercial PEDV vaccine are not efficacious enough,and high-mortality rate causes enormous economic loss to pig husbandry.The nucleocapsid protein is composed of 441 amino acids in PEDV genome,which encodes a protein around 55 kDa ~ 58 kDa in molecular size.Noteworthy,PEDV nucleocapsid protein,which mainly responsible for the virus duplication,does not have so much homology compared with other members of Alphacoronavirus,while is highly conservative among the same species.Current researches have showed that nucleocapsid protein have quite strong antigenicity and can induce immunoreaction of both B cells and T cells in parasitifers.Parasitifers,moreover,will generate high level PEDV nucleocapsid protein antibody in the early stage of infection,which can be regarded as an important indicator of the infection of PEDV in the early phase for rapid detection.In our research,pET-N overexpression plasmid will be constructed and transformed to Escherichia coli(strain “Transetta”,DE3).We found that Transetta(DE3)expression host cell can reach the highest level and best quantity of protein expression when carried on the following inducing steps: 37?,250 r/min in shake cultivation till OD600 reach 0.6,andthen injected 1.0 mmol/L IPTG and induced for another 5.5h.The result of SDS-PAGE electrophoresis and Western-blotting revealed that the objected protein was 58 kDa in size,thus confirmed the object protein was the designed protein,for it was similar to the result predicted by software.After induction,purification and desalinization,the concentration of object protein was 0.398mg/mL,then identified by MS/MS mass spectrometry.This result laid the foundation for further monoclonal antibody preparation of PEDV nucleocapsid protein.To generate specific monoclonal antibody,purified and desalinized N protein was then emulsified by Freund's Complete Adjuvant(FCA)and Freund's Incomplete Adjuvant(FIA)then carried on mice immunological study.Experimental group was immunized for tree times in a period of 14 days,and 300 ug per mouse each time.After immunization,all the mouse were used for blood sampling,and indirect ELISA was used for titer detection of serum antibody in all these samples.The BALB/c mice with highest serum antibody titer were chosen for enhanced immunization,then the splenocytes were taken and fused with myeloma cell SP2/0 and cultured in HAT medium.Supernatant of each cell were taken for ELISA assay after ten-days culturing,and the positive cells were cloned using limiting dilution assay and repeated for at least three times till screening out the individual cell line which can stably secret specific monoclonal antibodies.The selected hybridism cell strains were cryopreserved immediately and used for preparation of antibody ascites,and the titer of the ascites were then examined as before.Specific test result showed that the monoclonal antibody had satisfying specificity. |
PDF Full Text Request