【摘要】 清道夫受体A6(MARCO)是清道夫受体A类家族成员，主要表达于巨噬细胞表面，在机体抵御病原体入侵的免疫防御机制中具有重要作用。为了制备针对猪源MARCO蛋白的单克隆抗体，本研究以NCBI数据库中猪MARCO蛋白基因序列(GenBank:NM＿001256366.1)为参考，构建原核表达质粒pET-28a-MARCOΔTM。通过Rosetta表达系统表达重组蛋白MARCOΔTM,纯化后免疫BALB/c小鼠。利用细胞融合技术制备杂交瘤细胞，间接ELISA筛选阳性克隆。结果显示，本研究成功表达了可溶性MARCOΔTM蛋白，并获得了4株能稳定分泌抗MARCO单克隆抗体的杂交瘤细胞。其中，1H7、2H11和9A12的重链为IgG1,8E8的重链为IgG3,4株抗体的轻链均为Kappa。Western blot结果表明，所获得的单克隆抗体能够特异性识别外源性和内源性表达的MARCO蛋白。间接免疫荧光(IFA)检测结果显示，4株单克隆抗体均能与过表达的MARCO蛋白发生特异性结合，产生绿色荧光信号。免疫共沉淀(IP)试验进一步证实，单克隆抗体8E8和1H7能够有效富集过表达的MARCO蛋白。本研究制备的抗体为深入探究猪MARCO蛋白的生物学功能及其在免疫防御中的作用机制提供了重要的生物材料和研究工具。更多还原

【Abstract】 The scavenger receptor A6(MARCO) is a member of the scavenger receptor class A family.It is primarily expressed on the surface of macrophages and plays a significant role in the immune defense mechanisms against pathogen invasion.In order to prepare monoclonal antibodies against porcine MARCO protein,this study used the porcine MARCO protein gene sequence(GenBank:NM_ 001256366.1) from the NCBI database as a reference,the prokaryotic expression plasmid pET-28a-MARCOΔTM was constructed.The recombinant protein MARCO ΔTM was expressed by Rosetta(DE3) expression system,and BALB/c mice were immunized with the purified protein.Hybridoma cells were prepared by cell fusion technology,and positive clones were screened by indirect ELISA.The results showed that the soluble MARCOΔTM protein was successfully expressed in this study,and four hybridoma cells capable of stably secreting anti-MARCO monoclonal antibodies were obtained.Among them,the heavy chains of 1H7,2H11 and 9A12 are IgG1,the heavy chain of 8E8 is IgG3,and the light chains of the four antibodies are all Kappa.Western blot analysis demonstrated that the obtained monoclonal antibodies could specifically recognize both exogenously and endogenously expressed MARCO proteins.Indirect immunofluorescence(IFA) assays revealed that all four monoclonal antibodies could specifically bind to overexpressed MARCO protein and produce green fluorescence signal.Immunoprecipitation(IP) experiments further confirmed that monoclonal antibodies 8E8 and 1H7 could effectively enrich overexpressed MARCO protein.The antibodies prepared in this study provided essential biological materials and research tools for further exploring the biological functions of porcine MARCO protein and its mechanism of action in immune defense.更多还原