【作者】 李青；

【导师】 吴雄文； 李富荣；

【作者基本信息】 华中科技大学 ， 免疫学， 2007， 博士

【摘要】 磁性高分子微球是通过适当的方法使高分子与无机物结合起来形成具有一定磁性及特殊结构的微球,其表面可赋予多种反应性功能基团,如-OH,-COOH和-NH2等,具有良好的生物相容性,通过与生物活性物质中的配基偶联,能够识别并结合相应的抗原、抗体或核酸等,然后可在外加磁场中进行分离富集。因此,磁性高分子微球在细胞分离、固定化酶、靶向药物和免疫检测等生物学领域有着广泛的应用前景。本课题旨在制备一种新型的纳米磁性复合微球,此种纳米级别的微球与普通的微球相比磁响应敏感度极高,同时具有超顺磁特性,在液相分散体中不易聚集、稳定性高,其比表面积非常大,负载蛋白质的能力强,将其制备免疫磁性微球可作为公共载体和分离工具。然后同免疫分析技术和化学发光技术相结合,设计开发出新一代磁性富集、检测系统和磁性化学发光免疫分析系统用于肿瘤早期诊断。此种检测方法的建立,将会带来巨大的社会效益和经济效益。1.反相微乳-包埋方法制备磁性海藻酸钠复合微球以正庚烷为油相,AOT为表面活性剂,海藻酸钠水溶液和碳包铁纳米颗粒混合作为水相,在高速搅拌和超声的条件下形成微乳液,加入氯化钙和环氧氯丙烷进行交联固化制备磁性海藻酸钠复合微球。系统地考察了反应过程中海藻酸钠的浓度、表面活性剂的浓度及搅拌速度对所制微球性能的影响,并对微球进行了初步的性能表征。实验证明此方法克服了目前磁性高分子微球单分散性差、磁响应性弱和粒径偏大的缺点,得到外观为球形、分散性好、平均粒径为279nm、具有强的磁响应性的磁性高分子微球。2.在磁性微球表面交联抗体制备免疫磁性海藻酸钠复合微球研究磁性复合微球表面化学基团的植入方法,在微球表面接入6-氨基正己酸“手臂”分子,将功能基团的手臂延伸6个碳原子长度,然后以两步法蛋白缩和的方法将羊抗鼠IgG二抗与微球联接,制备免疫磁性微球,并对抗体交联微球的反应条件进行优化。实验证明用此方法制备的免疫磁性微球抗体吸附量达111.2μg/mg,交联抗体后流式细胞术检测98.3%的磁性微球具有免疫活性。3.建立肿瘤细胞磁性富集与检测系统,对肺癌患者外周血微转移癌细胞进行临床验证制备羊抗鼠IgG二抗包被的免疫磁性微球,将免疫磁性分离技术与免疫细胞化学技术相结合建立肺癌细胞磁性富集与检测系统。鉴定该检测系统的特异性、灵敏性和稳定性,并进行肺癌患者外周血肿瘤细胞的临床验证。与单纯的免疫细胞化学检测技术及RT-PCR检测技术相比较,本论文建立的磁性富集与检测系统具有灵敏度高、特异性强和稳定性好的特点,进行肺癌临床验证结果显示本检测系统检测率与RT-PCR检测技术所检测的结果相当,无显著性差异(p>0.05),同时无假阳性结果出现,比单纯的免疫细胞化学检测技术检出率高,存在显著差异(p<0.05)。4.建立磁性化学发光免疫分析系统,应用于肿瘤标志物AFP的测定。制备羊抗兔IgG二抗包被的免疫磁性微球,将免疫磁性分离技术与化学发光技术相结合,建立磁性化学发光免疫分析系统,优化检测系统的条件,鉴定其特异性、灵敏性和稳定性,并应用于患者外周血AFP的测定。实验证明本论文建立的磁性化学发光免疫分析系统具有灵敏度高、特异性强和稳定性好的特点,进行患者外周血AFP的测定结果有很好的临床相符性。本研究制备的新型纳米磁性复合微球,以碳包铁(Fe@C)纳米颗粒作为磁性内核,解决了氧化铁内核磁性能不够的难题,性能优良;在交联抗体时于微球与抗体之间连接一个手臂分子,避免了由于位阻效应导致的微球表面抗体与相应抗原结合不完全的弊端,更有利于微球功能的发挥。在此基础上,纳米磁性复合微球作为公共载体和分离工具应用于磁性富集与检测系统和磁性化学发光免疫分析系统,实验证明方便易行,具有广泛的应用前景。目的:构建生物素-蛋白连接酶(BirA酶)基因的表达载体,并在大肠杆菌BL-21 (DE3)中表达具有生物学活性的BirA酶。方法:用PCR法扩增BirA酶基因。将PCR产物克隆入pGEX-4T-2中构建BirA酶-GST融合蛋白基因的重组表达载体pGEX-BirA。经测序验证后,在大肠杆菌BL-21(DE3)中诱导表达,表达产物采用谷胱苷肽-琼脂糖层析柱进行纯化。以带有生物素酶底物肽(BirA substrate peptide, BSP)的HLA-A2-肽复合物为底物,用ELISA和Western blot鉴定表达产物的生物素化活性。结果:成功地构建了pGEX-BirA原核表达质粒,并在大肠杆菌BL-21(DE3)中诱导表达Mr为61300的BirA酶-GST融合蛋白,表达产物经谷胱苷肽-琼脂糖层析柱纯化后,得到N端带有GST标签的BirA酶蛋白。ELISA和Western blot的结果显示,表达产物具有酶活性,能使HLA-A2-肽复合物生物素化。结论:成功地制备了具有生物学活性的BirA酶,为研究蛋白质分子的相互作用提供了有效的制剂。更多还原

【Abstract】 Magnetic polymer microspheres (MMS) are those microspheres synthesized by polymer and inorganic material on molecule scale, which have the ability of magnetism and special structure. Since varied functional groups linking on the surface of microspheres, such as -OH,-COOH, -NH2 and so on, MMS are biocompatible and can conjugate with some ligands of bioactive compound,these give them the ability to recognize and bind to corresponding antigen, antibody, nucleic acid etc. Then this compound can be separated and enriched through the magnetic field. So MMS are promising for the new tool on varial areas such as cell separation, enzyme immobilization, drug targeting, immunodetection and so on. In this thesis, a kind of neotype nano- MMS were prepared, these nano-MMS had some characteristics when compared with ordinary MMS, such as strongly magnetic response, superparamagnetism, highly stability, largely specific surface area and so on. MMs can be taked as publicly carrier and separate tool. Then associated with immune analytical technique and chemiluminescence reaction, new system of magnetic enrichment and detedtion and magnetic chemiluminescence immune assay system were designed and exploited, and applied in the early diagnose of tumor. These establishments of such detection can bring out strongly social effects and economic returns.1. Preparation of magnetic Alginate Sodium composite Microsphere by means of reverse phase microemulsion and imbed process Take heptane as organic dispersed medium, AOT as surfactant, sodium alginate solution and Fe@C nanoparticle as aqueous phase, the system of reverse phase microemulsion was formed, and calcium chlorination and epichlorohydrin were been taked as crossing agents. Preparation conditions such as the concentration of sodium alginate, the concentration of AOT and stirring speed were described, and then the microsphere properties also were described in this paper. Results showed that the microspheres had a spherical appearance, with narrow size distribution, and the average diameter is 279nm, while the microspheres also showed strongly magnetic response.2. Immunomagnetic Alginate Sodium composite microspheres (IMM) were prepared in this part.The imbedding method of chemical group on the surface of MMS are discussed, and 6-Aminocaproic Acid with six carbon chain length were attached onto MMS as“spacer arm”, then goat anti-mouse IgG antibodies were binded with“spacer arm”through cross-linking reaction by preparing immunomagnetic alginate microspheres. Preparation conditions and MMS properties were described. Results showed that 98.3% immunomagnetic microsphere showed successfully cross-linked actived antibody and there were 111.2μg antibodies immobilized onto 1mg immunomagnetic microspheres.3. Tumor cell magnetic enrichment and detection method were established and applied in the detection of tumor cells in peripheral blood of lung adenocarcinoma patients. IMM coated with goat anti-mouse IgG antibodies were prepared, system of magnetic enrichment and detedtion were established by combining immunomagnetic separation with immunocytochemistry technique. The specificity, sensitivity and stability of the system were identified. In the detection of tumor cells in peripheral blood of lung adenocarcinoma patients, three kinds of method (enrichment and detection method that established in this thesis, immunocytochemistry and RT-PCR method) were used to compare their variability.The result showed that Tumor cell magnetic enrichment and detection system had the abilities of high sensitivity, specificity and stability. In the detection of clinic patients, the new detection system had no significant difference compared with RT-PCR methods (p>0.05), and had significant difference compared with immunocytochemistry methods (p<0.05).4. Magnetic chemiluminescence immune assay system were established and applied in the detection of tumor marker AFP in peripheral blood.IMM coated with goat anti-rabbit IgG antibodies were prepared, magnetic chemiluminescence immune assay system was established by combined immunomagnetic separation with chemiluminescence reaction. The specificity, sensitivity and stability of the system were identified. In the detection of tumor marker AFP in peripheral blood, the result showed that system of Magnetic chemiluminescence immune assay had the abilities of high sensitivity, specificity and stability, and the result of AFP in the blood have matched with clinic diagnosis.In this thesis, neotype Immunomagnetic composite microspheres were prepared taking Fe@C nanoparticle as magnetic materials. This resolved the problem of poor magnetism when taking ferric oxide as magnetic materials. Considering stereo conformation between antibody and antigen reaction, antibodies were binding on microspheres through“arm”molecule. In this basis, nano- MMS are promising for the new tool in more areas besides of system of magnetic enrichment and detedtion and magnetic chemiluminescence immune assay system. Objective: To construct the expression vector of biotin-protein ligase (BirA enzyme) gene and express the BirA enzyme with bioactivity in E.coli BL-21 (DE3).Methods: The BirA gene was amplified from E.coli genome by PCR and it was cloned into pGEX-4T-2 to construct the recombinant plasmid pGEX-BirA. After being verified by DNA sequencing, the fusion protein was expressed under IPTG induction in the E.coli BL-21 (DE3). The expressed product was purified through Glutathione-agarose chromatography column. The enzyme activity of the expressed product was identified by ELISA and Western blot.Results: The recombinant prokaryotic expression vector pGEX-BirA was constructed and the fusion protein GST-BirA with Mr being 61300 and bioactivity was expressed successfully. After the expressed product was purified through Glutathione-agarose chromatography column, the results of ELISA and Western blot showed that the expressed product could make HLA-A2 peptide complex biotinylation.Conclusion: BirA enzyme with bioactivity is prepared successfully which provide an effective reagent for studying the interaction between protein and protein.更多还原