猫细小病毒嵌合抗体基因在CHO-K1细胞中的稳定表达及其特性分析  

Stable expression and identification of feline parvovirus chimeric antibody gene in mammalian cells

作　　者：王文静 薛雨佳 陈慧宇 姚楠 刘明[1] WANG Wen-jing;XUE Yu-jia;CHEN Hui-yu;YAO Nan;LIU Ming(State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,the Chinese Academy of Agricultural Sciences,Harbin 150069,China)

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2021年第12期1318-1323,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家重点研发计划(2016YFD0501001)。

摘　　要：为降低鼠源单克隆抗体(MAb)在猫体内引起的免疫原性,本研究针对猫细小病毒(FPV)制备具有猫源抗体恒定区基因的鼠-猫嵌合抗体。本研究首先克隆具有FPV中和活性的MAb(4F6)的重、轻链可变区基因及猫天然抗体(NAb)重、轻链恒定区基因,通过融合PCR获得鼠-猫重组重、轻链基因片段(C_(H)/C_(L)),构建嵌合抗体表达载体(pMC-C_(H)、pMC-C_(L)),并经菌落PCR鉴定正确后转染CHO-K1细胞,并使用100μg/mL博来霉素(Zeocin)筛选,经有限稀释法将其克隆纯化后经双抗体夹心ELISA与western blot筛选出同时表达鼠-猫重组重、轻链基因的细胞株并命名为CHO-MC,将CHO-MC上清中表达的嵌合抗体纯化后调整浓度为1 mg/mL,间接ELISA检测嵌合抗体的效价,并分析其对病毒感染细胞的中和活性。结果表明,经SDS-PAGE检测鼠-猫嵌合抗体重链和轻链大小分别为55 ku和25 ku,western blot显示鼠源恒定区替换为猫源NAb的恒定区,双抗夹心ELISA显示CHO-MC细胞系在传代至15代仍能够稳定表达嵌合抗体,间接免疫荧光试验显示该嵌合抗体能够与抗猫IgG和FPV特异性结合,ELISA及中和效价分别为1:1280(0.78μg/mL)和1:16(62.5μg/mL)。本研究首次通过哺乳动物细胞表达系统表达了FPV的嵌合抗体,对治疗FPV抗体类药物的研究具有重要指导意义。To reduce the heterogenous immunogenicity of murine monoclonal antibodies in cats,the murine-feline chimeric antibody against feline parvovirus was constructed containing the constant region genes of canine antibody.In this study,the sequences of light and heavy chains were cloned for both the variable region of anti-FPV monoclonal antibodies 4F6 and constant region genes of canine antibody.And the murine-feline recombinant heavy and light chain gene fragments(C_(H)/C_(L))were obtained by overlapping PCR.The chimeric antibody expression carriers(pMC-C_(H),pMC-C_(L))was constructed.The obtained recombinant plasmid identified by colony PCR was used to transfect CHO-K1 cells screening with 100μg/mL of zeocin.The CHO-K1 cells were monoclonally purified by the limiting dilution method,and the cell line simultaneously expressing the murine-feline recombinant heavy and light chain genes was screened by double-antibody ELISA and western blot,and it was named CHO-MC.The concentration of the resulting CHO-MC chimeric antibodies was adjusted to 1mg/mL.The antibody titers in the supernatant of cell culture of different generations were determined by indirect ELISA.And the neutralizing activity was analyzed on virusinfected cells.The results showed that the sizes of the murine-feline chimeric antibody separated by SDS-PAGE were 55ku and 25ku for heavy and light chain,respectively.Western blot showed that the murine constant region was replaced with the feline constant region.The double antibodies sandwich ELISA showed that chimeric antibodies could be expressed stably in CHO-MC cell lines and passaged up to 15 generations.The indirect immunofluorescence test showed that the chimeric antibody could bind specifically with both the anti-feline IgG and FPV,and the neutralizing titers of the ELISA were 1:1280(0.78μg/mL)and 1:16(62.5μg/mL)respectively.This was the first study on the construction of chimeric antibodies against FPV with mammalian cell expression system,which had important guiding significance for the research of a

关 键 词：基因工程嵌合抗体 猫细小病毒 哺乳动物表达系统 

分 类 号：S852.65[农业科学—基础兽医学]