分子印迹聚合物的应用
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摘要
本博士论文以分子印迹技术为基础,在我组前期引入了辅助识别聚合物链(Assistant recognition polymer chains, ARPCs)探讨印迹技术的基础上进一步研究。本论文共分两部分,第一部分用ARPC合成的蛋白质印迹聚合物(Protein-imprinted polymer, PIP)作为人工抗体来纯化天然高分子量微量蛋白、同时PIP还可以用作人工抗体研究蛋白质之间的相互作用;第二部分探讨了利用分子印迹制备可以识别肿瘤表面细胞的特征跨膜蛋白的纳米微球。
在论文的第一部分工作中,我们以克隆的(immunoglobulin binding protein, BiP) BiP蛋白为模板、用带有芳香性基团吡啶的ARPC来制备PIP。BiP是内质网中的一种高分子量微量蛋白,在内质网(Endoplasmic Reticulum, ER)中含量很低,ER提取液中的BiP只有0.0590%；而PIP的特异性吸附ER提取液后,BiP的含量可达2.620%,富集了45倍。这是我组首次利用印迹聚合物的方法富集“高分子量”的天然微量蛋白。我组曾利用免疫共沉淀法证明了BiP和FKBP23在ER中特异性结合,该结合同时还受钙离子浓度的调节。在本课题中,我们利用PIP来替代免疫共沉淀法研究BiP和FKBP23之间的相互作用,其结果与免疫共沉淀的结果一致,在钙离子浓度较高时(>3mmM)二者不结合,而在钙离子浓度较低时(<2mM)二者特异性结合。
在论文的第二部分工作中,我们以肿瘤(erythroblastosis B2, ErbB-2)表面的一段特异性多肽为研究对象,研究利用带有印迹功能的纳米微球。ErbB2所属的表皮生长因子受体家族具有广泛的生物活性,其基因的活化表达和多种癌症相关。首先,利用硅烷试剂把ErbB2膜外一段特异性多肽键合到玻璃板上然后,使用不同的方法在没有多肽的条件下合成了壳聚糖纳米微球,微球的尺度在50nm-150nm之间。最后,在载体玻璃板上多肽存在的情况下,在多肽的末端辅以ARPC合成了聚壳聚糖-丙烯酸微球。该研究为后续壳聚糖载锚定肿瘤多肽、以期载药治疗肿瘤奠定了基础。
This dissertation is composed of two parts:the first part is concerned to the using protein imprinted polymer (PIP) as artificial antibody to study in biochemistry; the second part is concerned to the study of synthesizing nano chitosan by imprinting the specific peptides on transmembrane protein erythroblastosis B2 (ErbB-2) of tumor.
In the firsr part, we used a new method for enrichment of the low-content cellular protein in high molecular weight by molecular imprinting. The template protein, bacterial cloned immunoglobulin binding protein (BiP), was selectively assembled with assistant recognition polymer chain (ARPC) from their library, which consisted of numerous limited length polymer chains with randomly distributed recognitions and immobilizing sites. The assemblies of protein and ARPC were adsorbed by polymeric beads and immobilized by cross-linking polymerization. After the template was removed, the synthesized imprinted polymer was used to adsorb authentic BiP from endoplasmic re-ticulum (ER) extract, whose proportional content was then enriched 45 times. It is the first time that the low-content cellular natural protein with relatively high molecular weight (78 kDa) was enriched by molecular imprinted polymer. In the previous study of our group, we found that FK506-binding protein (FKBP23) could bind to immunoglobulin-binding protein (BiP) by biological method co-immunoprecipitation. Here this result was confirmed again by chemical method using PIP instead of co-immunoprecipitation. In addition, these two methods both confirmed that the binding between BiP and FKBP23 was regulated by [Ca2+] and that the binding could not be detected at a high [Ca2+]. Therefore, this new type of PIP provided a feasible alternative to biological antibodies in biochemical research.
In the second part, specific peptides from ERBB-2, many different tumors were motivated by which, were bond onto glass sheet by using OTCS (Octadecyl trichlorosilane). Then, nano-chitosan was synthesized by using different methods without pepetide. The TEM of the particles showed that the polychitosan-acrylic acid microspheres was 50-150 nanometer, which meeted our demand. Finally, nano-chitosan microspheres in the presence of peptides on glass sheet and ARPC were synthesized. These will be a foundation for the further research on targeted therapy by nano-chitosan.
在论文的第一部分工作中,我们以克隆的(immunoglobulin binding protein, BiP) BiP蛋白为模板、用带有芳香性基团吡啶的ARPC来制备PIP。BiP是内质网中的一种高分子量微量蛋白,在内质网(Endoplasmic Reticulum, ER)中含量很低,ER提取液中的BiP只有0.0590%；而PIP的特异性吸附ER提取液后,BiP的含量可达2.620%,富集了45倍。这是我组首次利用印迹聚合物的方法富集“高分子量”的天然微量蛋白。我组曾利用免疫共沉淀法证明了BiP和FKBP23在ER中特异性结合,该结合同时还受钙离子浓度的调节。在本课题中,我们利用PIP来替代免疫共沉淀法研究BiP和FKBP23之间的相互作用,其结果与免疫共沉淀的结果一致,在钙离子浓度较高时(>3mmM)二者不结合,而在钙离子浓度较低时(<2mM)二者特异性结合。
在论文的第二部分工作中,我们以肿瘤(erythroblastosis B2, ErbB-2)表面的一段特异性多肽为研究对象,研究利用带有印迹功能的纳米微球。ErbB2所属的表皮生长因子受体家族具有广泛的生物活性,其基因的活化表达和多种癌症相关。首先,利用硅烷试剂把ErbB2膜外一段特异性多肽键合到玻璃板上然后,使用不同的方法在没有多肽的条件下合成了壳聚糖纳米微球,微球的尺度在50nm-150nm之间。最后,在载体玻璃板上多肽存在的情况下,在多肽的末端辅以ARPC合成了聚壳聚糖-丙烯酸微球。该研究为后续壳聚糖载锚定肿瘤多肽、以期载药治疗肿瘤奠定了基础。
This dissertation is composed of two parts:the first part is concerned to the using protein imprinted polymer (PIP) as artificial antibody to study in biochemistry; the second part is concerned to the study of synthesizing nano chitosan by imprinting the specific peptides on transmembrane protein erythroblastosis B2 (ErbB-2) of tumor.
In the firsr part, we used a new method for enrichment of the low-content cellular protein in high molecular weight by molecular imprinting. The template protein, bacterial cloned immunoglobulin binding protein (BiP), was selectively assembled with assistant recognition polymer chain (ARPC) from their library, which consisted of numerous limited length polymer chains with randomly distributed recognitions and immobilizing sites. The assemblies of protein and ARPC were adsorbed by polymeric beads and immobilized by cross-linking polymerization. After the template was removed, the synthesized imprinted polymer was used to adsorb authentic BiP from endoplasmic re-ticulum (ER) extract, whose proportional content was then enriched 45 times. It is the first time that the low-content cellular natural protein with relatively high molecular weight (78 kDa) was enriched by molecular imprinted polymer. In the previous study of our group, we found that FK506-binding protein (FKBP23) could bind to immunoglobulin-binding protein (BiP) by biological method co-immunoprecipitation. Here this result was confirmed again by chemical method using PIP instead of co-immunoprecipitation. In addition, these two methods both confirmed that the binding between BiP and FKBP23 was regulated by [Ca2+] and that the binding could not be detected at a high [Ca2+]. Therefore, this new type of PIP provided a feasible alternative to biological antibodies in biochemical research.
In the second part, specific peptides from ERBB-2, many different tumors were motivated by which, were bond onto glass sheet by using OTCS (Octadecyl trichlorosilane). Then, nano-chitosan was synthesized by using different methods without pepetide. The TEM of the particles showed that the polychitosan-acrylic acid microspheres was 50-150 nanometer, which meeted our demand. Finally, nano-chitosan microspheres in the presence of peptides on glass sheet and ARPC were synthesized. These will be a foundation for the further research on targeted therapy by nano-chitosan.
引文
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