The Preparation And Identification Of Monoclonal Antibodies With Cross-Neutralizing Duck Hepatitis A Virus Type A And C

Duck hepatitis A?DHA?is one of the major diseases harming the duck industry caused by the duck hepatitis A virus,which can cause acute,highly lethal infections in ducklings.According to the difference of gene sequence,duck hepatitis A virus was divided into three genotypes?DHAV-A,DHAV-B and DHAV-C?.DHAV-A and DHAV-C prevailing mainly in China lack effective cross-neutralization,which has seriously affected the prevention and control of this disease.Many studies had shown that the preparation of broad-spectrum neutralizing antibodies againsting conserved neutralizing epitopes was a new strategy to deal with the above problems.In this research,we aimed to develop monoclonal antibodies?McAb?againsting the conserved sequences of DHAV-A and DHAV-C.The broad-spectrum neutralizing antibodies againsting these two genotypes of DHAV were further screened.The main achievements were as followed:1 Development of a monoclonal antibody that is cross-reactive with both DHAV-A and DHAV-CAccording to the amino acid sequence shared by VP1 proteins of DHAV-A and DHAV-C,14-amino acid-length 12 biotin epitopes with the highest score were screened by bioinformatics software,and each peptide was recombined with KLH carrier protein as immune antigens.The McAb were obtained from immunizing BALB/c mice,and 12hybridoma cell lines that stably secreting McAb were obtained.Indirect ELISA test results showed that 6 McAbs?C1,C4,C7,C12,C13,C16?cross-reacted with DHAV-A and DHAV-C simultaneously,but wihtout duck plague virus?DPV?or Newcastle disease virus?NDV?and avian influenza virus?AIV?,Laid the foundation for screening broad-spectrum neutralizing of McAb.2 Screening for monoclonal antibodies with cross-neutralizing activity2.1 Cross-neutralization activity of McAb in duck embryosTo evaluate the neutralizing activity of McAb,the cross-reactive McAb was mixed with200ELD50DHAV-A and DHAV-C in equal volumes,and inoculated with 12-day-old SPF duck chorioallantoic cavity,0.2 mL/embryo,for neutralization test.Processing group of5 duck embryos.The viral load was determined by Real-time PCR with duck-derived?-actin gene as the internal reference gene.The relative quantification was performed by 2^-??CT method to evaluate the neutralizing activity on duck embryos of 6 McAbs that with cross-reactivity to DHAV-A and DHAV-C.The results showed that the inhibition rates of 6 McAbs on DHAV-A and DHAV-C proliferation were:C1?84.4%&82.69%?,C4?90.79%&75.34%?,C7?86.23%&99.83%?,C12?14.74%&25.26%?,C13?38.87%&2.06%?and C16?99.61%&100%?.Acording to he duck embryo neutralization test results,the neutralization titers of 6 McAbs to DHAV-A and DHAV-C were:C1?1:3&1:5?,C4?1:3&1:3?,C7?1:10&1:11?,C12?0&0?,C1:3?0&0?,and C16?1:9&1:9?,According to this screening,four strains?C1,C4,C7,C16?having McAb with cross-neutralizing activity were selected.2.2 Preventive effect of McAb on cross protection of ducklingsIn order to evaluate the effect of McAbs with cross-neutralizing titer activity on DHAV-A and DHAV-C on the cross-prevention rate of ducklings,the ducklings were intramuscular injected with McAb at 1 day,1 mL/only,24 h.After the intramuscular injection of DHAV-A,DHAV-C,100ELD₅₀/only,each treatment of 5 ducklings.and protection test was carried out.The results showed that the protection rates of 4 McAbs against DHAV-A and DHAV-C were:C1?30%&40%?,C4?40%&40%?,C7?70%&80%?,C16?100%&60%?.The results of this study showed that 4 McAbs had different degrees of cross-prevention effect on two different genotype strains,and this experiment identified two strains of C7 and C16 against DHAV-A and DHAV-C with good cross-prevention.,and the protection rate reached 60.%the above.In summary,this study developed McAb with broad-spectrum neutralizing activity against DHAV-A and DHAV-C,which provided new idea for effective prevention and control of animal infectious diseases such as duck hepatitis.