| 摘要: |
| 【目的】制备兔抗双峰驼Toll样受体5（TLR5）的多克隆抗体并鉴定其活性。【方法】选择双峰驼TLR5基因序列，经软件分析选择其编码区的膜外区1~499氨基酸位为主要抗原位，再选取对应的核苷酸序列进行基因合成，并与pET-28a（+）载体连接构建重组质粒pET28a-TLR5，对其进行BamH Ⅰ和Xho Ⅰ双酶切鉴定。将pET28a-TLR5转染大肠杆菌原核表达系统，用IPTG进行诱导表达，对IPTG浓度（0.1，0.2，0.4，0.6，0.8，1.0 mol/L）和诱导时间（2，3，4，5，6，7，8，9 h）进行优化。将表达的蛋白经亲和层析法纯化后免疫新西兰白兔以制备多克隆抗体，采用ELISA法检测多抗效价，用Western blot检测和免疫组化验证评价多克隆抗体的特异性。【结果】成功构建了重组质粒pET28a-TLR5，经IPTG诱导后获得了重组蛋白（大小约为57 ku），且以包涵体的形式表达。IPTG最佳诱导表达条件为：IPTG浓度为1 mol/L，诱导时间为6 h。TLR5多抗血清效价为1∶128 000。Western blot鉴定表明，TLR5多抗血清能特异性识别目的蛋白；免疫组化结果表明，TLR5多抗能很好地识别成年双峰驼十二指肠肠系膜淋巴中的TLR5阳性细胞。【结论】成功制备出特异性良好的兔抗双峰驼TLR5多克隆抗体，能够用于TLR5的检测。 |
| 关键词: 双峰驼 TLR5 原核表达 抗体制备 表达定位 |
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| 基金项目:国家自然科学基金项目（31960693，31760723） |
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| Preparation of antibody against TLR5 of Bactrian camel and its expression in lymph nodes |
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WANG Tingwei,LIU Zhuanxia,LU Jia,et al
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| Abstract: |
| 【Objective】This study aimed to prepare rabbit polyclonal antibody against Toll-like receptor 5 (TLR5) of Bactrian camel and identify its activity.【Method】The TLR5 gene sequence of Bactrian camel was targeted,the coding region was determined,and the amino acid site 1-499 was selected as the main resistance site by software analysis.The corresponding nucleotide sequence was selected for gene synthesis and connected with pET 28a (+) vector to construct the recombinant plasmid pET28a-TLR5. The recombinant plasmid pET28a-TLR5 was identified by BamH Ⅰ and Xho Ⅰ double digestion.PET28a-TLR5 was transfected into E. coli prokaryotic expression system,and the expression was induced by IPTG.The concentration of IPTG (0.1,0.2,0.4,0.6,0.8,1.0 mol/L) and induction time (2,3,4,5,6,7,8,9 h) were then optimized.Expressed protein was purified by affinity chromatography and immunized with New Zealand white rabbits to prepare polyclonal antibodies.The titer of polyclonal antibodies was detected by ELISA,and the specificity of polyclonal antibodies was verified and evaluated by Western blot detection and immunohistochemistry.【Result】Recombinant plasmid pET28a-TLR5 was successfully constructed.The recombinant protein (about 57 ku) was obtained after induction by IPTG and expressed in the form of inclusion bodies.The best induction concentration of IPTG and induction time were 1 mol/L and 6 h,respectively.The titer of TLR5 polyclonal antibody was 1∶128 000.The Western blot showed that TLR5 polyclonal antibody could specifically recognize the target protein,and immunohistochemical results showed that TLR5 polyclonal antibody could well recognize TLR5 positive cells in mesenteric lymph of adult Bactrian camel.【Conclusion】The rabbit anti-Bactrian camel TLR5 polyclonal antibody was successfully prepared,which can be used for the detection of TLR5. |
| Key words: Bactrian camel prokaryotic expression of TLR5 preparation of antibody expression localization |
