双峰驼TLR1原核表达体系构建、抗体制备及组织表达分析

Prokaryotic expression system construction,antibody preparation and tissues distribution of bactrian camel TLR1

本研究运用生物信息学软件分析并通过原核表达系统诱导双峰驼TLR1(Toll‑like receptors 1,TLR1)蛋白表达,制备兔抗双峰驼TLR1多克隆抗体,通过HE染色法和SABC免疫组织化学法检测其在双峰驼心脏、肝脏、脾脏、肺脏和肾脏中的表达。结果表明,得到的目的蛋白大小为62 kDa,与预测结果相符。重组蛋白主要以包涵体的形式表达。通过间接ELISA检测抗体效价为1∶64000,Western blot检测原核表达的重组蛋白TLR1能与对应抗体特异性识别。HE和SABC免疫组化染色结果显示TLR1主要在双峰驼心脏的心肌细胞,肝脏的枯否细胞,脾脏的单核/巨噬细胞,肺脏的呼吸性细支气管上皮细胞、Ⅰ型肺泡细胞和Ⅱ型肺泡细胞,肾脏的远曲小管上皮细胞上呈阳性表达。综上表明,本研究成功制备的兔抗双峰驼TLR1多克隆抗体具有良好的特异反应性并能用于TLR1的免疫组化检测,且为进一步探究TLR1在双峰驼不同组织器官中所扮演的角色奠定了基础。

In this study,we used bioinformatics software to analyze the TLR1(Toll-like receptors 1)gene and used a prokaryotic expression system to inducibly express TLR1 recombinant plasmids into TLR1 protein.Rabbit polyclonal anti-bactrian camel TLR1 antibody was generated using the expressed recombinant TLR1 protein,and the expression of TLR1 in organs including bactrian camel heart,liver,spleen,lung,and kidney was detected by HE staining coupled with immunohistochemical staining of SABC using the prepared TLR1 polyclonal antibody.The size of the protein of interest obtained in this study was 62 kDa,which was the same as predicted by bioinformatics software.TLR1 recombinant protein was mainly expressed in the form of inclusion bodies.In this study,the titer of the TLR1 antibody was determined by indirect ELISA,which showed that the antibody titer result was 1∶64000,and the specificity of the TLR1 antibody was confirmed by Western blot analysis.The results of the present trial of HE staining with immunohistochemical staining of SABC showed that TLR1 was upregulated in cardiomyocytes of bactrian camel hearts,Kupffer cells of the liver,monocytes/macrophages of the spleen,the respiratory bronchiolar epithelium,and typeⅠand typeⅡalveolar cells of the lung.A positive reaction appeared on the epithelial cells of the distal convoluted tubule of the kidney.In conclusion,the rabbit anti-bactrian camel TLR1 polyclonal antibody has good specificity and can be used for immunohistochemical detection of TLR1 and provides a basis for further investigation of the function of TLR1 in different tissues and organs of bactrian camels.