兔抗长爪沙鼠免疫球蛋白G特异性多克隆抗体的制备与应用

Development and application of specific rabbit polyclonal antibodies against immunoglobulin G of Mongolian gerbils

目的制备兔抗长爪沙鼠免疫球蛋白G1(IgG1)和IgG2的特异性多克隆抗体,并用于蛋白质免疫印迹(WB)和ELISA检测。方法利用质粒-大肠埃希菌系统分别高效表达密码子优化的长爪沙鼠免疫球蛋白IgG1和IgG2重链恒定域CH2和CH3重组蛋白,纯化后免疫新西兰大白兔,分离并纯化获得相应的兔多克隆抗体,用辣根过氧化物酶(HRP)标记后分别作为一抗应用于WB和作为二抗应用ELISA。结果IgG1和IgG2重组蛋白在大肠埃希菌中可高效表达并都以包涵体形式存在,I相对分子质量分别为24800和21200,与预期相符。其纯化后作为免疫原可诱导兔产生相应的多克隆抗体,HRP标记后用于WB可以特异性地识别重组蛋白长爪沙鼠IgG1-CH23和IgG2-CH23,用于ELISA能检测柯萨奇病毒A16型感染长爪沙鼠后诱导产生的特异性IgG抗体。结论成功制备了兔抗长爪沙鼠免疫球蛋白IgG1和IgG2的特异性多克隆抗体,为长爪沙鼠特异性抗体血清学检测提供了有效工具。

Objective To develop rabbit polyclonal antibodies(pAbs)against immunoglobulin G1(IgG1)and IgG2 of Mongolian gerbils,and apply them for Western blotting(WB)and enzyme-linked immunosorbent assay(ELISA).Methods Plasmid-E.coli system was utilized to overexpress the codon-optimized sequences of IgG1 and IgG2 constant heavy chain regions 2(CH2)and 3(CH3)of Mongolian gerbils,respectively.The recombinant proteins were purified and used to immunize the rabbits.The specific pAbs were purified from rabbit serum and labelled with horseradish peroxidase(HRP).The HRP-pAbs were further investigated as primary antibody in WB and secondary antibody in ELISA.Results The recombinant proteins were overexpressed as inclusion bodies in E.coli.The molecular weights of IgG1 and IgG2 were 24800 and 21200,respectively as expected.Specific rabbit pAbs could be induced by the purified recombinant proteins.HRP-labeled pAbs could recognize specifically the recombinant gerbils IgG1 CH23 and IgG2 CH23 in WB,and detect anti-coxsackievirus A16(CAV16)IgG from serum of Mongolian gerbils infected by CAV16 in ELISA.Conclusions Specific rabbit pAbs for IgG1 and IgG2 of Mongolian gerbils are generated successfully,and it provides a very useful tool for unique antibody detection in Mongolian gerbils.