【摘要】 Toll样受体7(TLR7)可介导机体抗病毒等免疫应答，是重要的胞内模式识别受体之一。为获取双峰驼TLR7多克隆抗体，从双峰驼TLR7序列(GenBank Accession:XM＿010966214.1)选取编码区，经过跨膜结构与信号肽分析，取膜外序列构建重组质粒pET-28a-TLR7,并转化进感受态细胞BL21(DE3)中，对IPTG诱导表达的蛋白进行纯化及SDS-PAGE鉴定，将纯化蛋白免疫家兔，制备兔抗双峰驼重组TLR7多克隆抗体，并进行间接ELISA和Western blot检测分析。结果：诱导表达的重组双峰驼TLR7约为47 kDa,与预期大小一致，IPTG最佳诱导浓度为0.3 mol/L,最佳诱导时间为6 h,以包涵体形式表达；间接ELISA检测显示，制备的兔抗重组双峰驼TLR7多克隆抗体效价为1∶32 000,经Western blot鉴定可知抗体特异性良好；在成年双峰驼的空肠淋巴结中检测验证TLR7,根据SABC免疫组化试验得到该抗体的最佳工作浓度为1∶800,并通过光学显微镜观察到TLR7在淋巴结的副皮质区的树突状细胞中表达，证明本试验已制备出高效价和特异性较好的兔抗重组双峰驼TLR7的多克隆抗体。重组双峰驼TLR7的成功表达及其多克隆抗体的制备，为进一步研究双峰驼体液免疫及抗感染治疗奠定了基础。更多还原

【Abstract】 Toll like receptor 7(TLR7) can mediate immune responses such as being anti-virus. It is one of the important intracellular pattern recognition receptors. In order to obtain the Bactrian camel TLR7 polyclonal antibody, the coding region was selected from the Bactrian camel TLR7 sequence(GenBank Access: xm＿010966214.1). The transmembrane structure and signal peptide of the sequence were analyzed, and the extracellular sequence was taken to construct the recombinant plasmid pET-28 a-TLR7, which was transformed into the competent cell BL21(DE3). The protein induced by IPTG was purified and identified by SDS-PAGE. The purified protein was immunized to rabbits to prepare a rabbit anti-Bactrian camel recombinant TLR7 polyclonal antibody. Then, indirect ELISA and Western blot were performed. The results showed that the induced expression of recombinant Bactrian camel TLR7 was about 47 kDa, which was consistent with the expected size. The optimal induction concentration of IPTG was 0.3 mol/L and the optimal induction time was 6 h. The results of the indirect ELISA showed that the titer of the rabbit anti-recombinant Bactrian camel TLR7 polyclonal antibody was 1∶32 000. Western blot showed that the antibody had good specificity. In addition, TLR7 was also detected and verified in the jejunal lymph nodes of the adult Bactrian camels. According to the SABC experiment, the optimal working concentration of the antibody was 1∶800, and its expression was observed in the dendritic cells in the subcortical area of the lymph nodes under optical microscope. It was proved here that a rabbit polyclonal antibody against recombinant Bactrian camel TLR7 with high titer and good specificity was successfully prepared in this experiment. The successful expression of recombinant Bactrian camel TLR7 and the preparation of the polyclonal antibody laid a foundation for further research on humoral immunity and anti-infection treatment of Bactrian camels.更多还原