摘 要：
[目的]NOD样受体家族成员C5(NLRC5)在哺乳动物中广泛表达,并参与调节免疫应答和抗原递呈过程.试验通过真核表达系统表达猪NLRC5基因重组表达产物,并制备猪NLRC5多克隆抗体,为后续研究猪NLRC5的分子机制提供基础支撑.[方法]通过生物信息学在线网站分析NLRC5蛋白的跨膜结构和信号肽.采用PCR方法扩增猪NLRC5基因,构建真核表达载体pCAGGS-HA-NLRC5,并将其转染至HEK-293F细胞.利用Western blotting检测重组蛋白表达,并免疫新西兰大白兔制备多克隆抗体.使用间接ELISA法测定多克隆抗体效价,并检测抗体特异性.采用间接免疫荧光法(IFA)和Western blotting检测NLRC5多克隆抗体与不同细胞的反应原性.以NLRC5多克隆抗体为一抗,通过Western blotting检测经siRNA处理后ST细胞中NLRC5蛋白的表达情况.[结果]生物信息学分析结果显示,NLRC5蛋白不存在跨膜结构域,含有1个信号肽;试验成功构建真核表达质粒pCAGGS-HA-NLRC5.Western blotting结果显示,成功表达NLRC5蛋白,分子质量约203 ku;制备的NLRC5多克隆抗体效价为1∶25 600,且具有良好的特异性.IFA和Western blotting结果表明该抗体能够与多种细胞系发生特异反应;siRNA技术验证其能用于检测细胞中NLRC5的表达.[结论]本研究利用真核表达系统成功获得猪NLRC5重组蛋白并制备兔源多克隆抗体,为下一步探究NLRC5在先天免疫反应中对外来感染的防御作用提供试验材料.
译 名：
Eukaryotic Expression and Polyclonal Antibody Preparation of Porcine NLRC5
关键词：
NLRC5%pig%eukaryotic expression%polyclonal antibody
摘 要：
[Objective]Nod-like receptor family member C5(NLRC5)is widely expressed in mammals and is involved in the regulation of immune response and antigen presentation processes.The objective of this experiment was to express the recombinant expression products of porcine NLRC5 gene through eukaryotic expression system,and prepare porcine NLRC5 polyclonal antibody,so as to provide basic support for the subsequent study of the molecular mechanism of porcine NLRC5.[Method]The transmembrane structure and signal peptide of NLRC5 protein was analyzed by bioinformatics online.The porcine NLRC5 gene was amplified by PCR,and the eukaryotic expression vector pCAGGS-HA-NLRC5 was constructed and transfected into HEK-293F cells.Western blotting was used to detect the expression of recombinant protein,and polyclonal antibodies were prepared by immunizing New Zealand White rabbits.The titer of polyclonal antibody was determined by indirect ELISA,and the specificity of the antibody was detected.Indirect immunofluorescence(IFA)and Western blotting were used to detect the reactivity of NLRC5 polyclonal antibody with different cells.Using NLRC5 polyclonal antibody as the primary antibody,the expression of NLRC5 protein in ST cells treated with siRNA was detected by Western blotting.[Result]The results of bioinformatics analysis showed that NLRC5 protein had no transmembrane domain and contained 1 signal peptide.The eukaryotic expression plasmid pCAGGS-HA-NLRC5 was successfully constructed.Western blotting results showed that NLRC5 protein was successfully expressed with molecular weight of about 203 ku.The prepared NLRC5 polyclonal antibody had a titer of 1∶25 600 and good specificity.IFA and Western blotting results showed that the antibody could react with a variety of cell lines.siRNA validation showed that it could be used to detect the expression of NLRC5 in cells.[Conclusion]The recombinant porcine NLRC5 protein was successfully obtained by eukaryotic expression system and rabbit derived polyclonal antibody was prepared,which provided experimental materials for further investigation of the defense effect of NLRC5 on foreign infection in innate immune response.
