Construction And Therapeutic Evaluation Of Chimeric Antibody C10H4 Against Canine Parvovirus

Canine parvovirus disease is an infectious disease caused by canine parvovirus(CPV)that can infect a variety of animals,especially canines.CPV can be transmitted by the fecal-oronasal route,and CPV can even remain infectious in feces for up to 6 months.CPV is one of the pathogens that seriously threatens the health of dogs.Dogs infected with CPV will have symptoms such as depression,hemorrhagic gastroenteritis,vomiting,fever and diarrhea,and may die if not treated in time.There are commercial murine monoclonal antibodies for the prevention and treatment of canine parvovirus disease.Murine monoclonal antibodies have strong specificity,efficiency and stability,but they are also prone to immune rejection in dogs.In order to reduce the heterologous immune rejection or allergic reaction that may be produced by murine monoclonal antibody in the treatment of canine parvovirus-infected dogs.In this study,a murine monoclonal antibody 10H4(deposited by the National Research Center For Veterinary Medicine)with high neutralizing activity against CPV was transformed into a chimeric antibody c10H4.In the first part of this study,the heavy and light chain variable regions of murine monoclonal antibody 10H4 were sequenced to obtain the heavy and light chain variable region sequences.Consult relevant literature and websites,the sequences of the heavy chain and light chain constant regions of the canine monoclonal antibody were determined,and the sequence of the junction between the variable region and the constant region of the chimeric antibody c10H4 was determined.The murine monoclonal antibody 10H4 heavy chain variable region sequence and the canine antibody Ig G-B heavy chain constant region sequence are spliced together,the murine monoclonal antibody 10H4 light chain variable region sequence and the canine antibody Ig G-κ light chain constant region sequence After splicing,the antibody gene sequence suitable for CHO expression system was synthesized.The chimeric antibody c10H4 heavy chain sequence(Ig G-Heavy,hereinafter referred to as H)and light chain sequence(Ig G-Light,hereinafter referred to as L)were inserted into p CHO1.0expression vector in the order of H first and L last,construct p CHO1.0-H-L expression plasmid,then transfer p CHO1.0-H-L into Expi CHO-S cells for transient expression,and identify the chimeric antibody c10H4 can be expressed in the cell supernatant by SDS-PAGE and Western-Blot.The detection titer of Hemagglutination inhibition test is 1:640,and the neutralization titer is 1:2 881.8.In the second part of this study,the chimeric antibody c10H4 expression plasmid p CHO1.0-H-L was stably transfected into CHO-S cells.After pressure screening and two rounds of limiting dilution cloning,27 monoclonal cell lines were obtained,and the 27 cell lines expressed The chimeric antibodies have both HI activity and neutralizing activity.Among them,the HI titer of the chimeric antibody c10H4 expressed by cell line 11D9 was1:2 560,and the neutralization titer was 1:11 494.3.The chimeric antibody expression,HI activity and neutralizing activity are all optimal.Through the cell line stability evaluation,it was determined that the cell line 11D9 capable of stably expressing the chimeric antibody c10H4 was obtained.In the third part of this study,the chimeric antibody c10H4 was used for the therapeutic evaluation of canine parvovirus infection in beagle dogs.The clinical results showed that all dogs in the challenge control group died,and all dogs in the challenge treatment group survived.Combined with the detection of fecal virus content and the results of pathological tests,the chimeric antibody c10H4 can be applied to the treatment of canine parvovirus disease.In conclusion,in this study,the murine monoclonal antibody 10H4 was transformed into chimeric antibody c10H4 by genetic engineering.The chimeric antibody c10H4,which can be used for the treatment of canine parvovirus disease,was obtained,which can provide clinical treatment for canine parvovirus disease.This study provides a novel genetically engineered antibody for the clinical treatment of canine parvovirus disease.