肝臟去唾液酸糖蛋白受體的生物學特性研究及其特異性適配子的篩選與鑒定
發(fā)布時間：2018-08-29 15:51
【摘要】：目的 1.研究肝臟去唾液酸糖蛋白受體(asialoglycoprotein receptor,ASGPR)大亞基異構體的產生機制、表達水平及其編碼蛋白的特性,為進一步闡明ASGPR的生物學功能及以其為靶標的肝臟靶向治療奠定基礎。 2.篩選獲得能夠特異性高親和力結合肝臟特異性去唾液酸糖蛋白受體的RNA適配子,為開發(fā)診斷和治療肝臟疾病的靶向性試劑和藥物奠定基礎。 方法 1.從正常人肝組織和HepG2細胞中克隆ASGPR大亞基H1的兩種異構體H1a和H1b的cDNA序列,測序并比對分析H1b的產生機制;實時熒光定量PCR檢測正常人肝組織和HepG2細胞中H1a和H1b的表達比例,以及在HBV、HCV感染情況下和肝癌組織中H1b表達水平的變化. 2.合成H1b特異性多肽并與鑰孔戚血藍素(KLH)偶聯(lián),免疫小鼠制備H1b特異性多克隆抗體,鑒定抗體的效價和特異性;親和柱層析的方法從人血清和HepG2細胞培養(yǎng)上清中分離并純化可溶性ASGPR,采用特異性抗體通過Western blot鑒定可溶性ASGPR的組成;免疫組織化學檢測H1b在肝組織中表達和定位。 3.合成一個長度為115nt含有25個隨機序列的單鏈DNA隨機文庫,通過體外轉錄構建出單鏈RNA適配子隨機文庫,從肝組織中分離純化ASGPR大亞基作為靶蛋白,采用SELEX(systematic evolution of ligands by exponential enrichment)技術篩選高親和力的ASGPR特異性RNA適配子;測序分析篩選適配子的序列,預測并分析其二級結構特點。 4.同位素~(32)p標記適配子,通過膜結合測定實驗、凝膠阻滯實驗鑒定篩選適配子對靶蛋白的特異性和親和力;綠色熒光FITC標記適配子,鑒定其與肝細胞系HepG2和Huh7的特異性結合。 結果 1.人肝組織和肝細胞系HepG2中普遍表達ASGPR大亞基異構體H1a和H1b,H1b的cDNA較H1a缺失了一段117 nt的序列,該序列是ASGPR大亞基編碼基因的第二個外顯子,其兩端具有典型的AG/GT序列,提示H1的兩種異構體產生于對ASGPR mRNA的選擇性剪接。 2.正常肝組織和HepG2細胞中H1a和H1b的表達比例分別為5.2:1和2.6:1,而且H1b的表達水平在HBV和HCV感染細胞中下降約60%,在肝癌組織中下降90%以上。 3.在從正常人血清和HepG2細胞培養(yǎng)上清中純化的可溶性ASGPR蛋白中,可以檢測到H1b蛋白單體以及H1b和H2構成的多聚體;對肝組織進行的免疫組織化學檢測顯示H1b不能定位至細胞膜,而主要存在于細胞質中。 4.經過12輪SELEX篩選,適配子文庫中與靶蛋白結合的適配子得到明顯富集;對第12輪文庫中隨機挑選的48個適配子進行測序并預測其二級結構,發(fā)現(xiàn)文庫中適配子從結構上主要由兩個家族構成,其占總適配子的比例分別為45.8%和33.3%;并找到一個適配子H1-A25與靶蛋白具有很高的親和力,Kd值為48.79nM。 5、在膜結合測定實驗和凝膠阻滯實驗中,反應體系中不添加靶蛋白,或用無關蛋白替代靶蛋白,則不能檢測到明顯的適配子H1-A25和蛋白結合;而在反應體系中加入過量未標記適配子H1-A25,則能明顯阻斷放射標記適配子H1-A25與H1蛋白的結合。 6、FITC標記的適配子H1-A25能結合至肝癌細胞系HepG2和HuH-7,但不能結合不表達ASGPR的HeLa細胞;加入ASGPR的多克隆抗體可部分阻斷熒光標記適配子H1-A25與HepG2細胞或HuH-7細胞結合,而加入過量未標記適配子H1-A25則幾乎可以完全阻斷熒光標記適配子H1-A25結合HepG2細胞的熒光信號。 結論 1.人肝組織及肝細胞系HepG2和Huh7中普遍表達ASGPR大亞基異構體H1a和H1b。 2.大亞基異構體H1b編碼的蛋白為分泌型H1。 3.血清中的可溶性ASGPR是由分泌型的H1和H2構成的功能性復合物。 4.成功地篩選出了具有高親和力的肝臟ASGPR特異性RNA適配子H1-A25。 5.適配子H1-A25能特異性靶向結合至肝細胞。 本研究的創(chuàng)新點及意義 1.首次發(fā)現(xiàn)人類肝臟去唾液酸糖蛋白受體大亞基H1存在剪接異構體H1b,并證明此剪接異構體編碼的分泌型蛋白參與構成功能性的可溶性ASGPR,從而完善了對可溶性ASGPR的認識,并為進一步全面系統(tǒng)探討ASGPR的生物學功能奠定了堅實的基礎。 2.本研究首次篩選得到了肝細胞特異性的RNA適配子并鑒定了其功能,而對其進一步的改進和修飾將使其有望成為新的診斷和治療肝臟疾病的靶向性試劑和藥物載體。
[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.
【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2009
【分類號】：R346
本文編號：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.
【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2009
【分類號】：R346
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