重组人肝脏胶凝素1在CHO细胞内定位

发布时间：2018-05-07 09:58

本文选题：肝脏胶凝素1 + 抗体制备　；参考：《郑州大学》2010年硕士论文

【摘要】：背景与目的人肝脏胶凝素1(collectin liver 1, CL-L1)基因首先由Ohtani等克隆和鉴定出的一个胶凝素家族的新成员。推测该序列具有典型的胶凝素特点,由N端富含半胱氨酸区、胶原样区、颈区和糖识别区(carbohydrate recognition domain,CRD)构成。已发现的胶凝素多数为分泌蛋白,是固有免疫的重要分子,它们通过凝集病原微生物、激活补体、调理作用、激活吞噬作用和抑制病原生长等参与机体的免疫防御反应。但对CL-L1在人体组织分布和细胞内定位及其功能了解较少。为此,本研究制备人重组CL-L1的多克隆抗体,尝试利用中国仓鼠卵巢(CHO)细胞真核表达系统表达重组人CL-L1,并检测该蛋白在细胞内的定位。为进一步研究CL-L1在人体组织表达谱及其结构和功能奠定基础。方法将人CL-L1颈区-糖识别区目的基因片段定向插入pRSET-C中,构建原核表达载体,在E.coli BL21中进行原核表达,镍亲和层析柱纯化融合蛋白,免疫家兔,制备多克隆抗体。采用ELISA法检测多抗的效价和抗体的特异性。用构建好的真核表达载体pEGFP-CLL1(含人全长CL-L1 cDNA序列)转染CHO-K1细胞,使用免疫荧光标记技术和Western blot,检测EGFP-CLL1融合蛋白在细胞内的表达部位。结果成功构建了人CL-L1颈区-糖识别区原核表达载体pRSET-C/NECK-CRD△K,在E. coli BL21中表达,镍亲和层析柱纯化,得到相对分子质量19200的融合蛋白,免疫家兔后收获抗血清,ELISA显示抗体效价为1/20000,具有高度特异性；通过免疫荧光标记技术和Western blot分析发现,EGFP-CLL1融合蛋白主要存在于细胞质基质中,不存在于细胞培养上清液、内质网、高尔基体和细胞核等部位。结论本研究成功制备了人CL-L1颈区-糖识别区特异性高的多克隆抗体；CL-L1为已发现惟一存在于细胞质基质中的胶凝素成员。
[Abstract]:Background and purpose Human liver 1(collectin liver 1 (CL-L1) gene was first cloned and identified by Ohtani as a new member of the gelatin family. It is inferred that this sequence has typical characteristics of gelatin, and is composed of N-terminal carbohydrate recognition domain, collagen-like region, carbohydrate recognition domain and carbohydrate recognition region, which are rich in cysteine, collagen-like region, neck region and sugar recognition region. Most of them are secretory proteins, which are important molecules of innate immunity. They take part in immune defense by agglutinating pathogenic microorganisms, activating complement, conditioning, activating phagocytosis and inhibiting pathogenic growth. However, little is known about the distribution, localization and function of CL-L1 in human tissues. In this study, the polyclonal antibody of human recombinant CL-L1 was prepared, and the eukaryotic expression system of Chinese hamster ovary Cho cells was used to express recombinant human CL-L1, and the localization of the recombinant human CL-L1 protein was detected. To lay a foundation for the further study of CL-L1 expression profile, structure and function in human tissues. Method The target gene fragment of human CL-L1 cervical region and glucose recognition region was inserted into pRSET-C, and the prokaryotic expression vector was constructed. The fusion protein was purified by nickel affinity chromatography column and the polyclonal antibody was prepared by immunizing rabbits. The titers of polyantibodies and the specificity of antibodies were detected by ELISA method. The recombinant eukaryotic expression vector pEGFP-CLL1 (including human full-length CL-L1 cDNA sequence) was used to transfect CHO-K1 cells. The expression site of EGFP-CLL1 fusion protein in CHO-K1 cells was detected by immunofluorescence labeling technique and Western blot. Result A prokaryotic expression vector pRSET-C/NECK-CRD K was successfully constructed for human CL-L1 neck region and glucose recognition region. It was expressed in E. coli BL21 and purified by nickel affinity chromatography. A fusion protein with relative molecular weight of 19200 was obtained. After immunizing rabbits, the antibody titer was 1 / 200000.The antibody titer was highly specific, and the fusion protein of EGFP-CLL1 was found mainly in the cytoplasmic matrix, not in the supernatant of cell culture and endoplasmic reticulum by immunofluorescence labeling and Western blot analysis. Golgi body and nucleus, etc. Conclusion In this study, a polyclonal antibody (CL-L1) with high specificity in the cervical and glucose recognition regions of human CL-L1 was successfully prepared, which was the only member of gelatin found to exist in the cytoplasmic matrix.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R346

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