| Feline herpesvirus type 1(FHV-1)can cause feline herpesvirus disease after infection,which mainly manifests symptoms in the upper respiratory tract,thus it is also known as feline infectious rhinotracheitis.FHV-1 mainly infects kittens with the morbidity up to 100%and mortality rate of nearly 50%.In contrast,adult cats do not show symptomes after infection,but cats carrying the virus are highly contagious.In addition,recovered cats still have the possibility of infection,because the latent virus in trigeminal ganglion could be reactivated under stress.As a result,the FHV-1 infection are mainly from acute infected cats and clinically recovered cats.FHV-1 is widely prevelent all over the world,however,there are no diagnostic and detective strips in our country at present.The purpose of this work is to establish a simple ELISA to diagnose FHV-1 with high specificity,sensitivity,and at low cost.In order to obtain FHV-1 specific antibodies for the establishment of sandwich ELISA,the phage display technique was used to screen monoclonal antibodies against FHV-1.Firstly,the FHV-1 virus preserved in our laboratory was inoculated into CRFK cells,which then the inactivated virus was used as immunogen to immunize New Zealand rabbits.After four times immunization,the antibody titer in rabbit serum reached 5.6×10~5,indicating there were a large number of FHV-1-specific plasma cells.At this time,the heavy chain variable region(VH)and light chain variable region(VL)were amplified from the c DNA of immunized rabbit spleen,and then spliced into single chain variable region sc Fv.After sc Fv was cloned into phage vector p Comb3XSS,a rabbit single chain antibody phage library with a capacity of 1×10~9 cfu/m L was obtained.In order to screen the FHV-1 specific sc Fv clones,capsid protein g B protein was used as antigen in two rounds of amplification-enrichment-elution process,and the specificity of selected colonies was further identified by phage ELISA.Five sc Fv clones with the strongest binding ability were sequenced and named as 33-10,43-1,303-8,303-12,405-10,respectively.The VH and VL genes were amplified from the sc Fv genes and ligated to the vector containing constant regions of heavy chain and light chain,respectively.The recombinant expression plasmids of heavy chain and light chain were co-transfected into HEK 293F cells to express the full-length antibodies.The best capture antibody(43-1)and detection antibody(303-8)were selected from five antibodies by chessboard method.Other ELISA reaction conditions were optimized,including the best coating concentration of capture antibody was 4μg/m L,the coating condition was 4℃for 12 hours,the detection antibody was diluted in 3%BSA and incubated at 37℃for 2 h,and TMB solution was incubated at 37℃for 10 min.Under the optimal reaction conditions,the detection sensitivity is 12.5 ng/m L,and the assay do not show cross-reactivity with other viruses,indicating that the sandwich ELISA established in this study has high sensitivity and specificity.To sum up,this project successfully established an FHV-1-specific and highly sensitive ELISA detection method,which provides convenience for clinical diagnosis and contributes to the prevention and treatment of feline herpesvirus. |
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