鸭TLR5分子的原核表达及其单克隆抗体的制备

发布时间：2018-04-11 18:34

本文选题：Toll样受体 + 鸭Toll样受体5　；参考：《扬州大学》2015年硕士论文

【摘要】：Toll样受体(Toll-like receptors, TLRs)是一类跨膜蛋白家族,通过识别病原相关分子模式(Pathogen-associated molecular patterns, PAMPs)诱导先天性免疫应答。TLRs被认为是最有特点的先天性受体,在宿主抗感染免疫中发挥重要作用。不同的TLRs识别特定的分子标志并且启动先天性免疫应答,如TLR2和TLR3复合物或TLR2和TLR6复合物识别脂肽或脂蛋白,TLR3识别病毒的双链RNA,TLR4识别脂多糖,TLR5识别鞭毛蛋白,TLR7和TLR8识别单链RNA,以及TLR9识别细菌的CpG-DNA。TLR5是一类限制性表达受体,主要表达在上皮细胞、单核／巨噬细胞、树突状细胞(DC)等细胞的表面。鞭毛蛋白是TLR5的特异性配体,TLR5识别鞭毛蛋白后,启动MyD88依赖的信号通路,激活转录因子(NF-κB)诱导前炎性细胞因子的产生。目前,针对先天性免疫应答的研究主要以人或小鼠为模型,而对于家禽的研究则较为少见,主要是由于缺乏与家禽相关的生物试剂材料。因此,本研究构建表达了鸭TLR5分子的胞外区(ECD)片段,并制备了针对TLR5(ECD)的单克隆抗体,以期为家禽先天性免疫应答研究提供材料。本研究以实验室前期制备的鸭TLR5 cDNA为模板,设计合成引物,利用高保真DNA聚合酶扩增鸭TLR5 (ECD)基因片段,片段大小为1608 bp。将扩增的目的基因片段连接于pMD20-T克隆载体,经筛选鉴定正确后,将目的基因分别连接于原核表达载体pCold-1和pGEX-6p-l,经筛选鉴定正确后,分别将构建的原核重组质粒pCold-1-TLR5和pGEX-6p-1-TLR5转化宿主大肠杆菌BL21,从而构建出重组菌pCold-1-TLR5/BL21和pGEX-6p-1-TLR5/BL21.将重组菌pCold-1-TLR5/BL21和pGEX-6p-1-TLR5/BL21分别接种于含有Amp的LB液体培养基中,加入IPTG诱导重组融合蛋白的表达,收集菌体,超声波裂解菌体,离心后分别收集上清和沉淀进行SDS-PAGE凝胶电泳,根据电泳结果判定,重组融合蛋白rHis-TLR5和rGst-TLR5均表达在包涵体中。以重组融合蛋白rHis-TLR5为免疫原免疫6-8周龄的雌性BALB/c小鼠,应用B淋巴细胞杂交瘤技术,通过间接ELISA方法和2次亚克隆筛选,获得6株能够稳定的分泌抗TLR5单克隆抗体的杂交瘤细胞株,分别命名为1C10、485、589、5811、5F9和6D7。对获得的6株细胞株分别进行培养上清效价、腹水效价和抗体亚类测定。结果显示,1C10、485、589和6D7的培养上清效价均在104以上；5811株腹水效价低于106,其余均在107。抗体亚类鉴定显示,5F9株为IgG1,其余5株均为IgM。对收集的腹水单克隆抗体进行透析处理,然后用rProtein A Sepharose 4B亲和层析柱纯化,收集纯化后抗体进行SDS-PAGE凝胶电泳,电泳显示6株抗体的纯化效果整体较好。Western blotting结果显示,每株抗体均与经诱导的重组菌pCold-1-TLR5/BL21和pGEX-6p-1-TLR5/BL21发生特异性反应,出现与目的蛋白一致的特异性条带,不与空载体重组菌pCold-1/BL21和pGEX-6p-1/BL21发生特异性反应。将构建的重组真核质粒pCDNA3.1-TLR5瞬时转染HEK293T细胞,分别以制备的6株单克隆抗体为一抗,以FITC标记的羊抗鼠IgG为二抗,进行间接免疫荧光试验,结果均可以观察到明显的荧光,表明所制备的单克隆抗体与真核表达的TLR5分子可发生特异性结合。将制备鸭脾脏淋巴细胞,以单克隆抗体5F9为一抗,以FITC标记的羊抗鼠IgG为二抗,进行流式细胞术分析,结果表明单克隆抗体5F9可以特异性的识别鸭脾脏淋巴细胞。本研究成功表达了鸭TLR5胞外区的蛋白片段TLR5(ECD),所获得的单克隆抗体可与真核表达的鸭TLR5分子发生特异性的结合,并且单克隆抗体5F9可以用于流式细胞术分析,预示着抗体具有良好的应用前景。
[Abstract]:Toll like receptors (Toll-like receptors TLRs) is a kind of transmembrane protein family, through the recognition of pathogen associated molecular patterns (Pathogen-associated molecular, patterns, PAMPs) induced innate immune response.TLRs is considered to be the most characteristic of the congenital receptors play an important role in the host anti infection immunity. Different TLRs specific molecular marker identification and initiate innate immune response, such as TLR2 and TLR3 complex or TLR2 and identification of TLR6 complexes of lipopeptide or lipoprotein, double stranded RNA virus TLR3 recognition, TLR4 recognition of LPS, TLR5 protein and TLR7 flagellin recognition, TLR8 recognition and identification of single stranded RNA, TLR9 the CpG-DNA.TLR5 of bacteria is a restricted expression receptor, mainly expressed in epithelial cells, monocytes / macrophages, dendritic cells (DC) surface cells. Flagellin is a specific ligand of TLR5, TLR5 to identify flagellar protein, start MyD88 Dependent signal pathway, activation of transcription factors (NF- K B) induced proinflammatory cytokine production. At present, the research on the innate immune response to human or mouse model for poultry research is relatively rare, is mainly due to the lack of raw materials and reagent of poultry related. Therefore, this study on construction of expression of duck TLR5 molecules of the extracellular domain (ECD) fragment, and prepared for TLR5 (ECD) monoclonal antibody, in order to provide materials for the response of poultry innate immunity. In this study, laboratory preparation of duck TLR5 cDNA as template, primers were designed and synthesized, using high fidelity DNA polymerase duck TLR5 (ECD) gene fragments was 1608 bp. amplified fragment of target gene connected to the cloning vector pMD20-T by screening after correct identification, the target genes connected to the prokaryotic expression vector pCold-1 and pGEX-6p-l, were screened by Kam Set right after, respectively construct the Prokaryotic Recombinant Plasmid pCold-1-TLR5 and pGEX-6p-1-TLR5 transformed into Escherichia coli BL21, to construct recombinant pCold-1-TLR5/BL21 and pGEX-6p-1-TLR5/BL21. recombinant strains pCold-1-TLR5/BL21 and pGEX-6p-1-TLR5/BL21 were inoculated in LB liquid medium containing Amp, adding expression of recombinant fusion protein induced by IPTG cells were collected, ultrasonic lysis bacteria, centrifugal after collecting the supernatant and precipitation respectively by SDS-PAGE gel electrophoresis according to the electrophoresis result, the recombinant fusion protein rHis-TLR5 and rGst-TLR5 were expressed in inclusion bodies. The recombinant fusion protein rHis-TLR5 were immunized 6-8 week old female BALB/c mice, lymphocyte hybridoma technique using B by indirect ELISA method and 2 sub clones obtained, 6 strains can stably secrete anti TLR5 monoclonal antibody hybridoma cell lines, named respectively For 1C104855895811,5F9 and 6D7. on the 6 cell lines were cultured supernatant titer, ascites titer and antibody subgroup determination. The results showed that the titer of supernatant of 1C10485589 and 6D7 were more than 104; 5811 strains of ascites was lower than 106, the rest are in the 107. antibody subtype identification showed that 5F9 strain was IgG1, the remaining 5 strains were collected on IgM. ascites monoclonal antibody for dialysis treatment, and then use the rProtein A Sepharose 4B affinity chromatography, the purified antibody was collected SDS-PAGE gel electrophoresis, electrophoresis showed that the purification effect of 6 strains of.Western blotting antibody better overall results show that each antibodies are specifically reacted with the recombinant bacteria induced by pCold-1-TLR5/BL21 and pGEX-6p-1-TLR5/BL21, appear consistent with the target protein specific bands, and the empty weight group of strains pCold-1/BL21 and pGEX-6p-1/BL21 specific response. The recombinant plasmid pCDNA3.1-TLR5 was transfected to HEK293T cells, 6 monoclonal antibodies were prepared for anti FITC labeled Goat anti mouse IgG two antibody, indirect immunofluorescence test, the results can be observed fluorescence indicated that the prepared monoclonal antibody and eukaryotic expression the TLR5 molecules can specific binding. The preparation of duck spleen lymphocytes, a resistance to monoclonal antibody 5F9, FITC labeled Goat anti mouse IgG two antibody, were analyzed by flow cytometry. Results showed that 5F9 monoclonal antibody can specifically identify duck spleen lymphocytes. This study successfully expressed protein fragment TLR5 duck TLR5 extracellular domain (ECD), combined with duck TLR5 monoclonal antibody was obtained from eukaryotic expression can be specific, and the monoclonal antibody 5F9 can be used for flow cytometry analysis indicates that the antibody has good Good prospects for application.

【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.2

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