Construction Of Anti-CPV T7 Phage ScFv Immune Library From Chicken-derived And Screening Of CPV-VLPs Specific Antibodies

Canine parvovirus causes the canine infections by vomiting,diarrhea and fever caused by Canine parvovirus 2（CPV-2）infection.The infection rate of morbidity and mortality rate are high,and the death occurs mainly in puppies.At present,there is no specific drug for the treatment.CPV-2 infection is one of the most serious infectious diseases in China,especially in dog industries.The traditional attenuated CPV vaccine has far less immune effect,and a series of problems occur during the immunization process.In order to obtain the best immune effect,in addition to considering the best immunization procedures,combination of vaccines,addition of immune adjuvants,etc.,it is also necessary to consider the animal body’s ability to respond,vaccine selection,immune pathways,breed differences.This raises concerns about the immune process,making CPV infections incompletely preventable and there are still many reports of serious economic losses in the dog industry caused by CPV infection.Therefore,accurate and rapid diagnosis of canine parvovirus is the key to controlling this disease.Traditional diagnostic methods require multiple pairs of primers to amplify the fragment of interest,and the process is complicated and cumbersome.Since the avian antibody light chain has only one lambda type and only one pair of primers is needed to amplify the target fragment,thereby simplifying and accurately preparing the engineered antibody.The purpose of this study was to immunize chickens with canine parvovirus-like particles（CPV-VLPs）,obtain single-chain antibody genes,and establish avian single-chain antibody library by T7 phage display system to screen for antibodies with accurate specificity and affinity and expressed in the prokaryotic system.The research content are as follows:（1）Construction and panning of phage displayed antibody libraries:Using Freund’s adjuvant to emulsify CPV-VLPs（2 mg/mL）immunize the chicken intramuscularly in four different sites of pectoral muscles.On the seventh day after the final immunization,spleens were harvested.Total RNA was extracted,and reverse-transcribed into first-strand cDNA,which was used as a template to amplify VH and VL using the designed HF-EcoR I,HR-Linker/LF-Linker and LR-Hind Ⅲ,and the anti-CPV-VLPs-ScFv was assembled by overlay PCR.The target gene ScFv was digested and ligated to T7Select 10-3b arms,and in vitro packaging with packaging extracts.The recombinant plasmids with ScFv gene were sent to sequence.After four rounds of solid phase panning,the specific antibody was enriched,and the final library was 1.52×10⁸pfu.One hundred clones were selected from the fourth round of elution pools and subjected to Phage-ELISA to screen positive monoclonal.（2）Binding activity of ScFv antibodies:A single-chain antibody with the highest OD_450nmvalue in the Phage-ELISA assay was selected for cloning,digested and ligated into pET-30a vector,transformed into BL21（DE3）strain,optimized expression conditions,and final concentration of IPTG was 0.2 mM at 37℃.After induction for 7 h,the expressed protein existed in the form of inclusion bodies.After denaturation and renaturation,SDS-PAGE identification and Western blot verification showed that the recombinant protein was expressed near 35 kDa,and ScFv could recognize CPV-VLPs.（3）Cross-Reactivity Analysis:Collecting fecal material from dogs infected with CPV-2,Canine distemper virus（CDV）and canine coronavirus（CCV）,respectively,total30 cases.The presence of CPV virus in the disease material was identified by PCR,ELISA and immunochromatographic assay（ICA）（Commercial test strip）,and it was determined whether the prepared ScFv was cross-reactive with other viruses.The results showed that there were 28 cases,28 cases and 24 cases of canine parvovirus in ICA,ELISA and PCR.Furthermore,it was found by ELISA that the anti-CPV-VLPs-ScFv antibody had no cross-reactivity with CCV and CDV.