肝脏去唾液酸糖蛋白受体的生物学特性研究及其特异性适配子的筛选与鉴定
发布时间：2018-08-29 15:51
【摘要】：目的 1.研究肝脏去唾液酸糖蛋白受体(asialoglycoprotein receptor,ASGPR)大亚基异构体的产生机制、表达水平及其编码蛋白的特性,为进一步阐明ASGPR的生物学功能及以其为靶标的肝脏靶向治疗奠定基础。 2.筛选获得能够特异性高亲和力结合肝脏特异性去唾液酸糖蛋白受体的RNA适配子,为开发诊断和治疗肝脏疾病的靶向性试剂和药物奠定基础。 方法 1.从正常人肝组织和HepG2细胞中克隆ASGPR大亚基H1的两种异构体H1a和H1b的cDNA序列,测序并比对分析H1b的产生机制;实时荧光定量PCR检测正常人肝组织和HepG2细胞中H1a和H1b的表达比例,以及在HBV、HCV感染情况下和肝癌组织中H1b表达水平的变化. 2.合成H1b特异性多肽并与钥孔戚血蓝素(KLH)偶联,免疫小鼠制备H1b特异性多克隆抗体,鉴定抗体的效价和特异性;亲和柱层析的方法从人血清和HepG2细胞培养上清中分离并纯化可溶性ASGPR,采用特异性抗体通过Western blot鉴定可溶性ASGPR的组成;免疫组织化学检测H1b在肝组织中表达和定位。 3.合成一个长度为115nt含有25个随机序列的单链DNA随机文库,通过体外转录构建出单链RNA适配子随机文库,从肝组织中分离纯化ASGPR大亚基作为靶蛋白,采用SELEX(systematic evolution of ligands by exponential enrichment)技术筛选高亲和力的ASGPR特异性RNA适配子;测序分析筛选适配子的序列,预测并分析其二级结构特点。 4.同位素~(32)p标记适配子,通过膜结合测定实验、凝胶阻滞实验鉴定筛选适配子对靶蛋白的特异性和亲和力;绿色荧光FITC标记适配子,鉴定其与肝细胞系HepG2和Huh7的特异性结合。 结果 1.人肝组织和肝细胞系HepG2中普遍表达ASGPR大亚基异构体H1a和H1b,H1b的cDNA较H1a缺失了一段117 nt的序列,该序列是ASGPR大亚基编码基因的第二个外显子,其两端具有典型的AG/GT序列,提示H1的两种异构体产生于对ASGPR mRNA的选择性剪接。 2.正常肝组织和HepG2细胞中H1a和H1b的表达比例分别为5.2:1和2.6:1,而且H1b的表达水平在HBV和HCV感染细胞中下降约60%,在肝癌组织中下降90%以上。 3.在从正常人血清和HepG2细胞培养上清中纯化的可溶性ASGPR蛋白中,可以检测到H1b蛋白单体以及H1b和H2构成的多聚体;对肝组织进行的免疫组织化学检测显示H1b不能定位至细胞膜,而主要存在于细胞质中。 4.经过12轮SELEX筛选,适配子文库中与靶蛋白结合的适配子得到明显富集;对第12轮文库中随机挑选的48个适配子进行测序并预测其二级结构,发现文库中适配子从结构上主要由两个家族构成,其占总适配子的比例分别为45.8%和33.3%;并找到一个适配子H1-A25与靶蛋白具有很高的亲和力,Kd值为48.79nM。 5、在膜结合测定实验和凝胶阻滞实验中,反应体系中不添加靶蛋白,或用无关蛋白替代靶蛋白,则不能检测到明显的适配子H1-A25和蛋白结合;而在反应体系中加入过量未标记适配子H1-A25,则能明显阻断放射标记适配子H1-A25与H1蛋白的结合。 6、FITC标记的适配子H1-A25能结合至肝癌细胞系HepG2和HuH-7,但不能结合不表达ASGPR的HeLa细胞;加入ASGPR的多克隆抗体可部分阻断荧光标记适配子H1-A25与HepG2细胞或HuH-7细胞结合,而加入过量未标记适配子H1-A25则几乎可以完全阻断荧光标记适配子H1-A25结合HepG2细胞的荧光信号。 结论 1.人肝组织及肝细胞系HepG2和Huh7中普遍表达ASGPR大亚基异构体H1a和H1b。 2.大亚基异构体H1b编码的蛋白为分泌型H1。 3.血清中的可溶性ASGPR是由分泌型的H1和H2构成的功能性复合物。 4.成功地筛选出了具有高亲和力的肝脏ASGPR特异性RNA适配子H1-A25。 5.适配子H1-A25能特异性靶向结合至肝细胞。 本研究的创新点及意义 1.首次发现人类肝脏去唾液酸糖蛋白受体大亚基H1存在剪接异构体H1b,并证明此剪接异构体编码的分泌型蛋白参与构成功能性的可溶性ASGPR,从而完善了对可溶性ASGPR的认识,并为进一步全面系统探讨ASGPR的生物学功能奠定了坚实的基础。 2.本研究首次筛选得到了肝细胞特异性的RNA适配子并鉴定了其功能,而对其进一步的改进和修饰将使其有望成为新的诊断和治疗肝脏疾病的靶向性试剂和药物载体。
[Abstract]:objective
1. To study the production mechanism of large subunit isomers of hepatic asialoglycoprotein receptor (ASGPR), the expression level and the characteristics of its encoded proteins, so as to lay a foundation for further elucidating the biological function of ASGPR and targeting liver therapy.
2. Screening RNA aptamers with high affinity binding to liver-specific desalialoglycoprotein receptors will lay a foundation for the development of targeted reagents and drugs for the diagnosis and treatment of liver diseases.
Method
1. Cloning and sequencing of H1a and H1b isomers of ASGPR large subunit H 1 from normal human liver tissues and HepG2 cells to analyze the mechanism of H1b production; Real-time fluorescence quantitative PCR was used to detect the expression ratio of H1a and H1b in normal human liver tissues and HepG2 cells, and the expression level of H1b in HBV and HCV infection and liver cancer tissues. Change.
2. H1b-specific polypeptides were synthesized and coupled with key hole hemocyanin (KLH) to immunize mice to prepare H1b-specific polyclonal antibodies to identify the titer and specificity of the antibodies; soluble ASGPR was isolated and purified from human serum and culture supernatant of HepG2 cells by affinity column chromatography, and the soluble ASGPR was identified by Western blot with specific antibodies. Immunohistochemical staining was used to detect the expression and localization of H1b in liver tissues.
3. A 115nt single-stranded DNA random library containing 25 random sequences was synthesized. A single-stranded RNA aptamer random library was constructed by transcription in vitro. A large subunit of ASGPR was isolated and purified from liver tissues as a target protein. SELEX (system evolution of ligands by exponential enrichment) technique was used to screen high affinity ASGPR specific library. RNA aptamers were sequenced and analyzed, and the sequences of aptamers were screened, and their secondary structure characteristics were predicted and analyzed.
4. Isotope ~ (32) P labeled aptamers were used to identify the specificity and affinity of aptamers to target proteins by membrane binding assay and gel block assay, and green fluorescent FITC labeled aptamers were used to identify their specific binding to hepatocyte lines HepG2 and Huh7.
Result
1. ASGPR macroisomers H1a and H1b are widely expressed in human liver tissue and hepatocyte line HepG2. H1b cDNA has a 117 NT deletion sequence compared with H1a. The sequence is the second exon of the ASGPR macrosubunit coding gene. Typical AG/GT sequences are found at both ends of the sequence, suggesting that the two H 1 isomers are produced by selective splicing of ASGPR mRNA.
2. The expression ratio of H1a and H1b in normal liver tissues and HepG2 cells was 5.2:1 and 2.6:1, respectively. The expression level of H1b in HBV and HCV infected cells decreased by 60% and in HCC tissues by more than 90%.
3. The soluble ASGPR protein purified from normal human serum and culture supernatant of HepG2 cells could be detected as H1b protein monomer and a polymer composed of H1b and H2. Immunohistochemical detection of liver tissue showed that H1b could not be located on the cell membrane, but mainly in the cytoplasm.
4. After 12 rounds of SELEX screening, the aptamers binding to target proteins in the aptamer library were significantly enriched; 48 aptamers randomly selected from the 12th round library were sequenced and their secondary structures were predicted. It was found that the aptamers in the library were mainly composed of two families, accounting for 45.8% and 33.3% of the total aptamers, respectively. An aptamer H1-A25 has a high affinity for target protein, and the Kd value is 48.79nM..
5. In membrane-binding assay and gel-blocking assay, the binding of adapter H1-A25 to H1 protein could not be detected without adding target protein or substituting unrelated protein for target protein, while the binding of adapter H1-A25 to H1 protein could be blocked by adding excessive unmarked adapter H1-A25 to the reaction system. Close.
6. FITC-labeled adapter H1-A25 could bind to HepG2 and HuH-7 cells, but could not bind to HeLa cells without ASGPR expression; polyclonal antibodies to ASGPR partially blocked the binding of fluorescent labeled adapter H1-A25 to HepG2 cells or HuH-7 cells, while excessive unlabeled adapter H1-A25 almost completely blocked the binding of fluorescent labeling to HepG2 cells or HuH-7 cells. Aptamer H1-A25 combined with fluorescence signals of HepG2 cells.
conclusion
The ASGPR large subunit isoforms H1a and H1b. are commonly expressed in 1. human liver tissues and hepatocyte lines HepG2 and Huh7.
The 2. largest subunit isoform H1b is a secreted H1. protein.
3. soluble ASGPR in serum is a functional compound composed of secreted H1 and H2.
4. the liver specific ASGPR RNA aptamer H1-A25. with high affinity was successfully screened out.
5. aptamer H1-A25 can specifically bind to hepatocytes.
The innovation and significance of this research
1. The splicing isomer H1b was first found in the large subunit H1 of human liver desalinated glycoprotein receptor, and it was proved that the secretory protein encoded by the splicing isomer participated in the formation of functional soluble ASGPR, thus perfecting the understanding of soluble ASGPR and laying a solid foundation for further systematic study of the biological function of ASGPR. Foundation.
2. The hepatocyte-specific RNA aptamers were screened for the first time and their functions were identified. Further improvement and modification of these aptamers will make them promising new targeted reagents and drug carriers for the diagnosis and treatment of liver diseases.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R346
本文编号：2211673
[Abstract]:objective
1. To study the production mechanism of large subunit isomers of hepatic asialoglycoprotein receptor (ASGPR), the expression level and the characteristics of its encoded proteins, so as to lay a foundation for further elucidating the biological function of ASGPR and targeting liver therapy.
2. Screening RNA aptamers with high affinity binding to liver-specific desalialoglycoprotein receptors will lay a foundation for the development of targeted reagents and drugs for the diagnosis and treatment of liver diseases.
Method
1. Cloning and sequencing of H1a and H1b isomers of ASGPR large subunit H 1 from normal human liver tissues and HepG2 cells to analyze the mechanism of H1b production; Real-time fluorescence quantitative PCR was used to detect the expression ratio of H1a and H1b in normal human liver tissues and HepG2 cells, and the expression level of H1b in HBV and HCV infection and liver cancer tissues. Change.
2. H1b-specific polypeptides were synthesized and coupled with key hole hemocyanin (KLH) to immunize mice to prepare H1b-specific polyclonal antibodies to identify the titer and specificity of the antibodies; soluble ASGPR was isolated and purified from human serum and culture supernatant of HepG2 cells by affinity column chromatography, and the soluble ASGPR was identified by Western blot with specific antibodies. Immunohistochemical staining was used to detect the expression and localization of H1b in liver tissues.
3. A 115nt single-stranded DNA random library containing 25 random sequences was synthesized. A single-stranded RNA aptamer random library was constructed by transcription in vitro. A large subunit of ASGPR was isolated and purified from liver tissues as a target protein. SELEX (system evolution of ligands by exponential enrichment) technique was used to screen high affinity ASGPR specific library. RNA aptamers were sequenced and analyzed, and the sequences of aptamers were screened, and their secondary structure characteristics were predicted and analyzed.
4. Isotope ~ (32) P labeled aptamers were used to identify the specificity and affinity of aptamers to target proteins by membrane binding assay and gel block assay, and green fluorescent FITC labeled aptamers were used to identify their specific binding to hepatocyte lines HepG2 and Huh7.
Result
1. ASGPR macroisomers H1a and H1b are widely expressed in human liver tissue and hepatocyte line HepG2. H1b cDNA has a 117 NT deletion sequence compared with H1a. The sequence is the second exon of the ASGPR macrosubunit coding gene. Typical AG/GT sequences are found at both ends of the sequence, suggesting that the two H 1 isomers are produced by selective splicing of ASGPR mRNA.
2. The expression ratio of H1a and H1b in normal liver tissues and HepG2 cells was 5.2:1 and 2.6:1, respectively. The expression level of H1b in HBV and HCV infected cells decreased by 60% and in HCC tissues by more than 90%.
3. The soluble ASGPR protein purified from normal human serum and culture supernatant of HepG2 cells could be detected as H1b protein monomer and a polymer composed of H1b and H2. Immunohistochemical detection of liver tissue showed that H1b could not be located on the cell membrane, but mainly in the cytoplasm.
4. After 12 rounds of SELEX screening, the aptamers binding to target proteins in the aptamer library were significantly enriched; 48 aptamers randomly selected from the 12th round library were sequenced and their secondary structures were predicted. It was found that the aptamers in the library were mainly composed of two families, accounting for 45.8% and 33.3% of the total aptamers, respectively. An aptamer H1-A25 has a high affinity for target protein, and the Kd value is 48.79nM..
5. In membrane-binding assay and gel-blocking assay, the binding of adapter H1-A25 to H1 protein could not be detected without adding target protein or substituting unrelated protein for target protein, while the binding of adapter H1-A25 to H1 protein could be blocked by adding excessive unmarked adapter H1-A25 to the reaction system. Close.
6. FITC-labeled adapter H1-A25 could bind to HepG2 and HuH-7 cells, but could not bind to HeLa cells without ASGPR expression; polyclonal antibodies to ASGPR partially blocked the binding of fluorescent labeled adapter H1-A25 to HepG2 cells or HuH-7 cells, while excessive unlabeled adapter H1-A25 almost completely blocked the binding of fluorescent labeling to HepG2 cells or HuH-7 cells. Aptamer H1-A25 combined with fluorescence signals of HepG2 cells.
conclusion
The ASGPR large subunit isoforms H1a and H1b. are commonly expressed in 1. human liver tissues and hepatocyte lines HepG2 and Huh7.
The 2. largest subunit isoform H1b is a secreted H1. protein.
3. soluble ASGPR in serum is a functional compound composed of secreted H1 and H2.
4. the liver specific ASGPR RNA aptamer H1-A25. with high affinity was successfully screened out.
5. aptamer H1-A25 can specifically bind to hepatocytes.
The innovation and significance of this research
1. The splicing isomer H1b was first found in the large subunit H1 of human liver desalinated glycoprotein receptor, and it was proved that the secretory protein encoded by the splicing isomer participated in the formation of functional soluble ASGPR, thus perfecting the understanding of soluble ASGPR and laying a solid foundation for further systematic study of the biological function of ASGPR. Foundation.
2. The hepatocyte-specific RNA aptamers were screened for the first time and their functions were identified. Further improvement and modification of these aptamers will make them promising new targeted reagents and drug carriers for the diagnosis and treatment of liver diseases.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R346
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