鸡Toll样受体15单克隆抗体的制备及其初步应用

Preparation of Chicken Toll-like Receptor 15 Monoclonal Antibody and Its Preliminary Application

旨在制备鸡Toll样受体15(ChTLR15)的特异性单克隆抗体(mAb),并初步应用。利用PCR技术扩增ChTLR15第162—386位氨基酸,克隆于载体pET-30a中进行诱导表达,获得高纯度的重组ChTLR15(162—386 aa)蛋白。然后采用皮下多点注射方式将ChTLR15免疫6周龄的雌性BALB/c小鼠,常规获得针对ChTLR15蛋白的单克隆抗体。运用原核表达系统对ChTLR15基因进行截短表达,对单克隆抗体针对的ChTLR15抗原表位进行鉴定。利用6C7株单克隆抗体以激光共聚焦显微技术对鸡HD11细胞进行定位,同时,利用免疫组化技术确定ChTLR15在鸡部分组织中的分布情况。结果表明:成功构建重组质粒pET-30a-ChTLR15(162—386 aa),经IPTG诱导表达后,获得以包涵体形式存在的约26 ku的重组蛋白ChTLR15(162—386 aa)。方阵试验表明,最适血清稀释度为1∶6400,最适抗原包被浓度为0.35μg·mL^(-1)。采用有限稀释法进行4轮筛选,最终获得4株针对ChTLR15蛋白的单克隆抗体,将其以2G4、6C7、6D10、7C4进行命名。2G4与6C7的亚类为IgG2a,6D10与7C4的亚类为IgG1。Western blot结果显示,4株单抗均能与ChTLR15发生特异性反应,而不与其他受检蛋白反应。运用原核表达系统对ChTLR15(162—386 aa)进行截短表达,对ChTLR15单克隆抗体的抗原表位进行鉴定。结果显示,单抗2G4与7C4识别的抗原表位为^(230)QLGTVLEF^(237),6C7与6D10识别的抗原表位为^(245)EMDLLS^(250)。细胞定位结果显示,ChTLR15蛋白位于细胞表面。免疫组化结果显示,健康对照鸡肝、脾、肺、肾均有微弱阳性染色,禽致病性大肠杆菌(avian pathogenic Escherichia coli,APEC)感染鸡肝、脾、肺、肾阳性染色有所增强。本研究通过淋巴细胞杂交瘤技术成功获得4株抗ChTLR15蛋白的单克隆抗体,通过截短免疫原蛋白法对其识别的线性表位进行鉴定,可用于进一步研究ChTLR15的结构与功能,为后续信号通路、免疫机理、疫苗研发等相关领域的研究提供了有力工具。

The preparation and preliminary application of monoclonal antibodies(mAbs)specific to chicken Toll-like receptor 15(ChTLR15)is described.The gene fragment encoded 162-386 amino acids of ChTLR15 were amplified by PCR and cloned into the vector pET-30a.IPTG was used to induce expression of the recombinant plasmid,the collected target protein was first purified by Ni-NTA affinity chromatography medium method,and the purified protein was renatured by dialysis using urea with concentration gradient.The renatured protein was purified again by gel filtration chromatography to obtain high-purity recombinant ChTLR15(162-386 aa)protein.The 6-week-old female BALB/c mice were immunized with multi-loci subcutaneous injections,and the spleen cells of mice with higher serum antibody levels after immunization were fused with SP2/0 myeloma cells,and the limiting dilution method was used for multiple rounds of screening to obtain mAbs against ChTLR15 protein.The ChTLR15 gene was truncated and expressed by the prokaryotic expression system,and the ChTLR15 epitopes targeted by the mAbs were identified.The mAb 6C7 was used to locate the ChTLR15 in chicken HD11 cells with laser confocal microscopy.At the same time,the distribution of ChTLR15 in some chicken tissues was determined by immunohistochemistry.The recombinant plasmid pET-30a-ChTLR15(162-386 aa)was successfully constructed,and the recombinant protein ChTLR15(162-386 aa)at about 26 ku in the form of inclusion body was obtained after induced expression by IPTG.The optimal antigen coating concentration(0.35μg·mL^(-1))and the optimal serum dilution(1∶6400)were determined by indirect enzyme-linked immunosorbent assay(ELISA).A total of 4 mAb hybridoma cell lines that can stably secrete antibody against ChTLR15 were obtained,and they were named 2G4,6C7,6D10 and 7C4.The subclass of 2G4 and 6C7 was IgG2a,and the subclass of 6D10 and 7C4 was IgG1.Western blot results showed that the 4 mAbs can react specifically with ChTLR15,but not with other tested proteins.The prokaryotic expression system was used to express the truncated ChTLR15(162-386 aa),and the epitopes of ChTLR15 mAbs were identified.The results showed that the epitope recognized by mAbs 2G4 and 7C4 was ^(230)QLGTVLEF^(237),and the epitope recognized by 6C7 and 6D10 was ^(245)EMDLLS^(250).Observation under a laser confocal microscopy suggested that ChTLR15 protein was located on the cell surface.Immunohistochemistry showed that the liver,spleen,lung,and kidney from the health birds had weak positive staining,and the positive staining of liver,spleen,lung,and kidney from the avian pathogenic Escherichia coli(APEC)challenged birds was enhanced.In this study,four mAbs against ChTLR15 protein were successfully developed and the epitopes recognized by them were identified,which is conducive to further research on the structural and functional characteristics of ChTLR15.It provides a robust tool for subsequent research in related fields such as signal pathways,immune mechanisms,and vaccine development.