Establishment Of Positive Hybridoma Cell Lines Secreting MAbs Against FCV

Feline calicivirus(FCV)is one common pathogen of feline upper respiratory tract disease(URTD).It accounts for 20% to 53% to URTD.When feline is infected by FCV,they always manifest fever,rhinitis,conjunctivitis,oral ulcerations,or chronic stomatitis.Sometimes,it also shows lameness,skin ulcerations,and pneumonia.FCV had high morbidity before,but low mortality.In the past decade,however,it was reported that there were FCVs with high mortality which severely endangered healthy of feline.Infected feline usually infect multiple other pathogens at same time.This condition makes diagnose so difficult.So,it is necessary that we need to develop an assay which can quickly and accurately detect serum.In this research,two FCV strains isolated by our lab were compared with other FCV strains.Comparing with vaccine strains,the nucleotide identity of FCV-SX2 strain is lower than FCV-SX1.We chose FCV-SX2 strain as immunogen.The FCV-SX2 which was cultivated in F81 cell was purified by gel filtration chromatography.The viral titer of antigen which was determined by TCID50 was 10-9.3.The concentration of antigen was 1.3 μg/μL.VP1 gene of FCV-SX2 and nano-luciferase gene were linked to eukaryotic expression vector PVAX I.With nano-luciferase fused antigen expressed in COS 1 cells,LIPS method was established by using positive and negative serum of mice,which could be used to detect the antibody of FCV.The purified virus mixed with Freund’s adjuvant was used to immunize Balb/c mice.By the cell fusion technology,SP2/0 cells and the splenocyte of immune mice whose neutralizing titer was more than 1:3200 were fused.The m Ab-secreting hybaidoma strains were screened by LIPS method.After three subcloning,5 hybridoma cells which could secret antibodies against FCV were screened out and named as 1E2,4C3,5F11,6G9 and 7D2.Two of hybridoma cells had neutralizing activity.By using the LIPS method,the results suggest that 1E2,4C3 and 6G9 were Ig G1 subtybe with κ chain and 5F11 and 7D2 were Ig M subtybe with κ chain.By Western Bloting,all m Abs were reactive with FCV JL-2 and had great specificity.The hybridoma cells were inserted into the enterocoelia of mice.The titers of 1E2 and 6G9 were1:12800.The titers of 4C3,5F11 and 7D2 were 1:25600.This research is very significant to establish the method that can detect FCV quickly and precisely.