| Feline parvo virus,also known as feline pan-leukopenia virus,whose clinical manifestations are characterized by high fever,vomiting,diarrhea,dehydration,and reduction of white blood cells in the circulating blood stream,has always been paid attention by people because of the characteristics of strong infectivity,high fatality rate,wide host spectrum and high harmfulness.Hosts for FPV include domestic and wild catsand some wild canids,such as raccoons and foxes.At present,the antibody cure against FPV mainly relies on antiserum.Most of the serum on the market is murine monoclonal antibody.Although it has a certain curative effect,the curative effect is not good because continuous injection is impossible because of the fact that it is prone to allergic reactions due to the application of heterologous protein in the body.In recent years,because the genetically engineered chimeric antibody can reduce the immunogenicity of murine monoclonal antibodies while retaining the affinity and specificity of the original murine antibodies and has the advantages like that it can change the effect function of antibodies through linking different constant region genes,the genetically engineered chimeric antibody has developed into one of the most studied genetically engineered antibodies at present.This experiment aims to reduce the immune side effects caused by murine monoclonal antibodies in felines by expressing the murine-feline chimeric antibodies with FPV neutralizing activity in mammalian cells.Through cloning the MAb(4F6)heavy and light chain variable region genes with FPV neutralizing activity and the feline natural antibody(NAb)heavy and light chain constant region genes,the murine-feline recombinant heavy and light chain gene fragments(C_H/C_L)can be obtained by PCR overlap and extension.The murine-feline recombinant heavy and light chain gene fragment(C_H/C_L)are respectively cloned into p FUSE-m Ig G1-Fc carrier so the chimeric antibody expression carrier(p MC-C_H,p MC-C_L)can be constructed.After colony PCR identification is correct,transfect CHO-K1 cells;use 100μg/m L zeocin to screen;The CHO-K1 cells are monoclonally purified by the limiting dilution method,and the cell lines expressing the murine-feline recombinant heavy and light chain genes are screened by double-antibody ELISA and Western-blot was named CHO-MC.CHO-MC cell lines were constructed and passed to 15 generations.The antibody titers in the supernatant of cell culture of different generations were determined by double antibody sandwich ELISA.Purify the correctly identified murine-feline chimeric antibodies,and adjusted to a concentration of 1 mg/m L,and the titer of the chimeric antibodies is detected by ELISA indirectly,and analyze the neutralizing activity on virus-infected cells.The results shows that the sizes of the murine-feline chimeric antibody heavy and light chain separated by SDS-PAGE are 55 ku and 25 ku respectively.Western blot shows that the murine constant region was replaced with the feline constant region.The double antibody sandwich ELISA showed that CHO-MC cell lines could still stably express chimeric antibodies after passage to 15.The indirect immunofluorescence test shows that the chimeric antibody can specifically bind to anti-feline Ig G and FPV,and the ELISA and neutralizing titer are 1:1280(0.78μg/m L)and 1:16(62.5μg/m L)respectively.The above results indicate that this study expresses the chimeric antibody of FPV through the mammalian cell expression system on the basis of retaining the neutralizing activity of the original feline parvo virus MAb 4F6,and the purpose of reducing antibodies for the immunogenicity of heterogeneous animals is achieved. |
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