抗猫细小病毒嵌合抗体基因在昆虫细胞中的表达与鉴定

Expression and identification of felineparvovirus chimeric antibody gene in insect cells

为降低单克隆抗体(MAb)在犬体内的免疫原性,本研究克隆了具有中和猫、犬细小病毒(FPV、CPV)活性的MAb(3EB)的轻、重链可变区基因及犬天然抗体(NAb)轻、重链恒定区基因,通过融合PCR获得鼠-犬嵌合抗体基因。构建能够表达嵌合抗体的重组质粒,经昆虫杆状病毒表达系统对重组嵌合抗体进行表达。SDS-PAGE和western blot试验显示鼠-犬嵌合抗体重链为55ku、轻链为25ku。间接免疫荧光试验显示该嵌合抗体能够与抗犬IgG和FPV特异性结合。上述结果表明,嵌合抗体保留了原MAb3EB对病毒特异结合能力的基础上,将鼠源恒定区替换为犬源NAb的恒定区。病毒中和试验表明嵌合抗体保留了3EB对FPV、CPV的中和活性。本研究首次通过昆虫杆状病毒表达系统表达了抗FPV、CPV的嵌合抗体,降低了原MAb的免疫原性,对FPV、CPV病临床治疗用抗体药物的研制具有重要指导意义。

To reduce the immunogenicity of monoclonal antibodies in dogs, light and heavy chain variable region genes of anti-FPV and CPV monoclonal antibodies 3EB's and light and heavy chain antibody constant region genes of canine antibody were cloned, and successfully composed to chimeric antibody genes by overlapping PCR. Then recombinant plasmid expressing light and heavy chain of chimeric antibody simultaneously was constructed, and the chimeric antibody was expressed by the insect baculoviral expression system. The SDS-PAGE and western blot analysis showed that the murine-canine chimeric antibody has a heavy chain size of 55ku and a light chain size of 25ku. IFA results showed that the chimeric antibody was able to bind to the canine IgG and FPV, sepcifically the virus. Viral neutralization tests showed that the chimeric antibody retained neutralizing activity against FPV and CPV. This was the first study to construct a recombinant plasmid and express a chimeric antibody against parvovirus by an insect baculoviral expression system, the chimeric antibody would reduce murine immunogenicity when injected into a different species, which has great significance for the development of drugs for clinical treatment of parvovirus infection.