从噬菌体肽库中筛选与人转铁蛋白受体特异性结合的肽
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摘要
人转铁蛋白受体(human transferrin receptor，hTfR)是一类Ⅱ型跨膜蛋白，其N端位于细胞内，C端位于细胞外，主要功能是以内化的方式介导细胞对铁离子的吸收。由于hTfR在肿瘤细胞表面及组成血脑屏障的脑毛细血管内皮细胞的表面高密度分布，而且其介导的内化途径可准许较大的分子进入细胞，故hTfR是药物传递系统中十分具有吸引力的靶点之一。大量的研究结果证明了以转铁蛋白受体介导的内化途径来进行药物设计这一思路的可行性，其适用性很广，可介导的药物不仅包括金属离子，化学药物，还包括蛋白、肽以及寡核苷酸等。被改建的药物具有更强的组织分布特异性，更长的半衰期及更好的可控性，在疾病的诊断和治疗中，尤其是在肿瘤治疗和中枢神经疾病的治疗方面具有良好的前景。
噬菌体呈现肽库是近年来发展很快的一项技术，它已成为为已知分子寻找相应配体的有效工具。
本实验中，我们以hTfR为靶蛋白，从噬菌体呈现随
第四军医大学硕士学位论文
机12肤库中筛选与之结合的肤，经过三轮生物淘筛和噬
菌体ELISA，我们获得了8个阳性重组噬菌体克隆。DNA
测序及根据DNA序列推导出的短肤的一级结构表明8个
克隆中含有两个共同的短肤序列RXXXR和RXR。在噬菌
体ELISA实验中，A410nm吸光值最高的噬菌体被命名为
B18(其所呈现的肤命名为P18)。经网上蛋白BLAST分
析，未发现P18与包括转铁蛋白在内的任何已知蛋白有
同源性，竞争抑制实验也表明转铁蛋白不能抑制B18与
转铁蛋白受体的结合，说明B18与转铁蛋白受体的结合
位点不同于转铁蛋白。流式细胞术和细胞免疫组化均表
明B18可以与高表达hTfR的细胞SMMC一7721『结合。噬菌
体在荷瘤小鼠的体内分布实验表明，归巢于肿瘤组织的
噬菌体B18是对照噬菌体的5.23倍，说明B18在肿瘤组
织中有一定程度的特异性富集。
为了进一步证明P18在脱离了噬菌体之后仍具有介
导其它分子与活细胞上的hTfR结合的能力，我们用基因
工程的方法构建了表达GST一P18的融合基因，GST一P18
经大肠杆菌表达、亲和层析纯化后，进行流式细胞术分
析和激光共聚焦显微镜观察，实验结果表明GST一P18可
以与SMMC·7721结合并且主要结合于细胞膜上。
Human transferrin receptor (hTfR) is a II type transmembrane protein of which N terminal is in the cytoplasm and C terminal is out of the cytoplasm. It plays an important role in mediating the iron ion being uptaked by cells through endocytosis way. On the surfaces of tumor cells and the brain capillary endothelia cells, which consist of the brain blood barrier, the expressione levels of hTfR are high. And it has been proved that endocytosis is an efficient way to transport big molecules into cells. The two features mentioned above make hTfR an attractive target in drug delivery system and many researches have verified the feasibility of such strategy. Many therapeutic molecules such as anti-cancer drugs, proteins, peptides, DNA, PNA and so on can be delivered into cells with higher specificity through this method.
hTfR has great potential in medical diagnosis and therapy, especially in treatment of cancer and central nervous system diseases.
Phage-display technology is a powerful tool in finding the ligands for proteins, enzymes, antibodies and et al.
In our study a phage-display 12 peptides library was screened with hTfR. After three cycles biopanning and phage ELISA, 8 positive phages were selected. DNA sequencing revealed that two consensus peptides sequences were included in the 8 positive phages, one is RXR and the other is RXXXR. The phage, which had highest A410nm absorbtion in phage ELISA was named as B18, and the peptide displayed was named as P18.
The binding of B18 to hTfR is dose-dependent. Blast online analysis indicated that none homogeneouse sequence with PI 8 was found in proteins including Tf. Competition assay also demonstrated that the binding between B18 and hTfR could not be blocked by Tf which indicated that the binding site of B18 to hTfR is distinct from Tf, so the binding between Tf and hTfR will not be interfered with by B18. The assays of flow cytometry and
cell immochemistry indicated that B18 has the ability to bind to SMMC-7721 on which the hTfR were highly expressed.
When phage B18 and B26, a control phages were injected into the nude mice from tail vein, B18 recovered from tumor was as 5.23 times as B26, which indicated that B18 could home to tumor tissues specifically.
In order to study whether the peptide still has the ability to direct other molecules to bind to hTfR when it was departed from phage, GST-P18 fusion gene was constructed and expressed in E.coli. The fusion protein was purified by affinity chromatography. The flow cytometry assay indicated that the fusion protein could bind to SMMC-7721 and when it was observed with a confocal laser-scanning microscopy, the imagines showed that the fusion protein was mainly binded on the cell membrane.
噬菌体呈现肽库是近年来发展很快的一项技术，它已成为为已知分子寻找相应配体的有效工具。
本实验中，我们以hTfR为靶蛋白，从噬菌体呈现随
第四军医大学硕士学位论文
机12肤库中筛选与之结合的肤，经过三轮生物淘筛和噬
菌体ELISA，我们获得了8个阳性重组噬菌体克隆。DNA
测序及根据DNA序列推导出的短肤的一级结构表明8个
克隆中含有两个共同的短肤序列RXXXR和RXR。在噬菌
体ELISA实验中，A410nm吸光值最高的噬菌体被命名为
B18(其所呈现的肤命名为P18)。经网上蛋白BLAST分
析，未发现P18与包括转铁蛋白在内的任何已知蛋白有
同源性，竞争抑制实验也表明转铁蛋白不能抑制B18与
转铁蛋白受体的结合，说明B18与转铁蛋白受体的结合
位点不同于转铁蛋白。流式细胞术和细胞免疫组化均表
明B18可以与高表达hTfR的细胞SMMC一7721『结合。噬菌
体在荷瘤小鼠的体内分布实验表明，归巢于肿瘤组织的
噬菌体B18是对照噬菌体的5.23倍，说明B18在肿瘤组
织中有一定程度的特异性富集。
为了进一步证明P18在脱离了噬菌体之后仍具有介
导其它分子与活细胞上的hTfR结合的能力，我们用基因
工程的方法构建了表达GST一P18的融合基因，GST一P18
经大肠杆菌表达、亲和层析纯化后，进行流式细胞术分
析和激光共聚焦显微镜观察，实验结果表明GST一P18可
以与SMMC·7721结合并且主要结合于细胞膜上。
Human transferrin receptor (hTfR) is a II type transmembrane protein of which N terminal is in the cytoplasm and C terminal is out of the cytoplasm. It plays an important role in mediating the iron ion being uptaked by cells through endocytosis way. On the surfaces of tumor cells and the brain capillary endothelia cells, which consist of the brain blood barrier, the expressione levels of hTfR are high. And it has been proved that endocytosis is an efficient way to transport big molecules into cells. The two features mentioned above make hTfR an attractive target in drug delivery system and many researches have verified the feasibility of such strategy. Many therapeutic molecules such as anti-cancer drugs, proteins, peptides, DNA, PNA and so on can be delivered into cells with higher specificity through this method.
hTfR has great potential in medical diagnosis and therapy, especially in treatment of cancer and central nervous system diseases.
Phage-display technology is a powerful tool in finding the ligands for proteins, enzymes, antibodies and et al.
In our study a phage-display 12 peptides library was screened with hTfR. After three cycles biopanning and phage ELISA, 8 positive phages were selected. DNA sequencing revealed that two consensus peptides sequences were included in the 8 positive phages, one is RXR and the other is RXXXR. The phage, which had highest A410nm absorbtion in phage ELISA was named as B18, and the peptide displayed was named as P18.
The binding of B18 to hTfR is dose-dependent. Blast online analysis indicated that none homogeneouse sequence with PI 8 was found in proteins including Tf. Competition assay also demonstrated that the binding between B18 and hTfR could not be blocked by Tf which indicated that the binding site of B18 to hTfR is distinct from Tf, so the binding between Tf and hTfR will not be interfered with by B18. The assays of flow cytometry and
cell immochemistry indicated that B18 has the ability to bind to SMMC-7721 on which the hTfR were highly expressed.
When phage B18 and B26, a control phages were injected into the nude mice from tail vein, B18 recovered from tumor was as 5.23 times as B26, which indicated that B18 could home to tumor tissues specifically.
In order to study whether the peptide still has the ability to direct other molecules to bind to hTfR when it was departed from phage, GST-P18 fusion gene was constructed and expressed in E.coli. The fusion protein was purified by affinity chromatography. The flow cytometry assay indicated that the fusion protein could bind to SMMC-7721 and when it was observed with a confocal laser-scanning microscopy, the imagines showed that the fusion protein was mainly binded on the cell membrane.
引文
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