BAFF与TACI相互作用功能结构域及关键氨基酸的研究
发布时间：2019-06-19 09:00
【摘要】：B细胞激活因子(B cell activating factor, BAFF)是1999年发现的TNF超家族成员之一,它可特异性的刺激B淋巴细胞增殖、分化和分泌抗体,对B细胞的存活和发育起到重要的调控作用。BAFF-R(BR3)、TACI、BCMA分别是BAFF的三个受体,它们分布在B细胞分化发育的不同时期,BAFF正是通过这些受体在B细胞分化发育的各个阶段及免疫调节中发挥的作用。研究表明：BAFF缺乏会导致外周B细胞成熟缺陷和降低免疫球蛋白的水平；而BAFF过表达则会破坏免疫耐受,与许多自身免疫病如系统性红斑狼疮(SLE), sjogren氏综合症,类风湿关节炎(RA)的发生、发展密切相关。因此,BAFF及其受体作为治疗自身免疫疾病的特异性靶标,受到广泛的关注。 受体TACI与BAFF具有高亲和力,它可以特异性识别BAFF并与之结合。因此,可溶性受体融合蛋白(TACI-Fc)可作为BAFF抑制剂治疗自身免疫疾病,目前该抑制剂已经进入临床试验阶段并取得初步成效。同时,新型拮抗剂开发已成为目前的研究热点。然而,BAFF是如何识别TACI发挥功能尚不十分清楚,确定BAFF与TACI相互作用的关键功能位点对于研制新型拮抗剂具有重要的指导意义。 本研究基于配体(BAFF)和受体(TACI)晶体结构以及理论模拟获得的三维构象,借助计算机辅助分子对接技术、动力学模拟技术构建配体-受体相互作用的复合物模型,通过计算机模拟的方式分析TACI与BAFF的结合模式以及BAFF被TACI识别的关键区域。利用分子生物学突变实验以及功能评价方法对理论预测的抗原表位进行实验验证。 具体工作如下： 一、通过分子生物学、免疫学等方法完成人BAFF蛋白胞外段的表达纯化及活性鉴定。 (1)将实验室保存的pET32a(+)/BAFF、pET32a(+)/TACI质粒进行原核表达、通过融合蛋白上携带的His标签进行镍离子亲和纯化,将获得的蛋白(rhBAFF、rhTACI)通过SDS-PAGE及Western Blot完成特异性鉴定； (2)通过非还原电泳、Western Blot、HPLC对rhBAFF在PBS缓冲液中的存在形式做了分析； (3)用ELISA方法检测了rhBAFF与rhTACI的结合活性,并与商品化蛋白进行了比较； (4)通过小鼠B细胞增殖实验在体外验证了rhBAFF的功能活性； 二、借助计算机模拟的方法、通过生物学实验验证确定rhBAFF与TACI相互作用的重要区域。 (1)借助BAFF以及受体TACI晶体结构,选择合适的力场参数,在考虑溶剂效应的情况下,利用计算机辅助分子对接技术经动力学模拟构建BAFF与受体相互作用空间构象； (2)利用分子生物学方法构建rhBAFF区域突变体的原核表达载体,在体外表达纯化及鉴定并通过非还原电泳和HPLC的方法鉴定三聚体结构； (3)通过ELISA及Octet蛋白相互作用动力学分析比较rhBAFF、rhBAFF区域突变体与TACI的结合能力的差异； (4)通过小鼠B细胞增殖实验以及经典NF-κB的激活实验在体外评判rhBAFF与突变体功能活性的差异,确定TACI识别BAFF的功能域； 三、在确定rhBAFF功能域的基础上进一步对功能域中每个氨基酸位点进行理论模拟,选择关键位点进行研究,经实验评价确定了rhBAFF与TACI相互作用功能域的重要位点。 (1)利用获得的TACI与BAFF作用复合物空间结构,通过计算机辅助虚拟突变技术对203～211、233～238结构域进行逐点虚拟扫描突变,通过相互作用能量的变化进而预测TACI识别BAFF的关键位点。 (2)利用分子生物学方法构建rhBAFF单点突变体的原核表达载体,完成突变体的表达纯化及鉴定； (3)通过ELISA及Octet蛋白相互作用动力学分析比较rhBAFF、rhBAFF单点突变体与TACI的结合能力的差异； (4)通过小鼠B细胞增殖实验以及经典NF-KB的激活实验在体外评判rhBAFF与单点突变体的功能活性差异确定TACI识别BAFF的关键功能位点； 具体结果如下： 一、获得了高纯度有活性的人BAFF胞外段及受体TACI胞外段的重组融合蛋白 (1)经原核表达、亲和纯化,获得rhBAFF、rhTACI的可溶表达蛋白,经SDS-PAGE及Western Blot鉴定,所表达蛋白的纯度高、特异性强； (2)经非还原电泳、Western Blot、HPLC分析rhBAFF在PBS缓冲液中的确为三聚体形式存在且纯度超过90%； (3)用ELISA双夹心法证实rhBAFF能够特异性的结合rhTACI,与商品化蛋白相比无明显差异； (4)通过小鼠B细胞增殖实验在体外验证了rhBAFF的促增殖活性； 二、明确了BAFF与TACI相互作用的关键功能区域 (1)利用获得的BAFF与受体作用复合物空间构象,通过分子间氢键形成理论、距离几何学以及计算机图形学技术从理论上合理判别BAFF受体TACI识别BAFF的功能位点；在合理考虑BAFF二级结构以及分子内氢键的条件下,通过计算机辅助分子模拟合理设计BAFF的五个区域突变体； (2)构建了rhBAFF(突变体)／pET32a(+)的原核表达载体；成功表达rhBAFF的片段突变体：M1(158-165)、M2(203-211)、M3(225-231)M4(233-238)、M5(264-269),并通过镍离子亲和介质纯化得到高纯度的目的蛋白,经SDS-PAGE和Western blot鉴定正确,Western印迹和HPLC的结果证实在非还原条件下突变体的分子量约为110bp,均为三聚体形式； (3) ELISA与蛋白相互作用动力学分析结果表明突变体M2、M4、M5的结合TACI的能力显著降低。小鼠B淋巴细胞增殖试验和经典NF-κB激活实验结果显示,突变体M2、M4与rhBAFF相比的生物学活性明显降低,初步确定rhBAFF的突变体M2、M4突变的区域203-211,233-238为BAFF的功能区域； 三、明确了rhBAFF与TACI相互作用功能域的关键位点 (1)通过虚拟单点突变对BAFF与TACI相互识别的关键位置(M2、M4)进行理论预测,经相互作用能的变化动态分析了单点突变对BAFF构象以及BAFF与TACI作用的影响,得出M208、G209、H210、Q234、M236、P237是TACI识别BAFF的关键位点。 (2)通过分子生物学定点诱变技术将理论模拟的重要位点构建为原核表达载体；体外表达纯化得到高纯度的目的蛋白,同样的方法证实单点突变体在溶液中仍以三聚体形式存在； (3)经ELISA、蛋白相互作用动力学分析评价突变体的结合能力,发现M208、G209、H210、M236、P237突变后rhBAFF与TACI的相互作用能明显降低。同时,活性分析实验的结果与结合实验相符,证实M208、G209、M236、P237在BAFF结合TACI介导的生物功能中作用明确,并确定P237为BAFF的关键功能位点； 结论：基于计算机辅助分子模拟技术与生物学实验有机结合,通过预测BAFF与受体相互作用复合物空间构象的结构特征以及动态识别模式,合理设计BAFF突变体并进行体外生物学活性评价,成功确定了BAFF与受体相互识别的功能位点,为进一步合理设计、改造BAFF拮抗剂奠定了基础。
[Abstract]:The B cell activating factor (BAFF) is one of the members of the TNF superfamily, which is found in 1999. It can stimulate the proliferation, differentiation and secretion of B-lymphocyte, and play an important role in regulating the survival and development of B-cells. BAFF-R (BR3), TACI and BAFA are the three receptors of BAFF, which are distributed in the different stages of B cell differentiation and development. The results showed that the lack of BAFF could lead to the maturation of peripheral B cells and the level of immunoglobulin, while the overexpression of BAFF could destroy the immune tolerance, and be associated with many autoimmune diseases, such as systemic lupus erythematosus (SLE), sjogren's syndrome, and rheumatoid arthritis (RA). Development is closely related. Therefore, BAFF and its receptor are widely concerned as the specific targets for the treatment of autoimmune diseases. The receptor TACI has a high affinity for BAFF, which can specifically identify the BAFF and bind to the BAFF In conclusion, the soluble receptor fusion protein (TACI-Fc) can be used as a BAFF inhibitor for the treatment of autoimmune diseases, and at present the inhibitor has entered the clinical trial phase and made preliminary In addition, the development of novel antagonists has become the current research heat. However, it is not clear how the BAFF can recognize the function of the TACI, and it is important to determine the key functional site of the interaction between the BAFF and the TACI. This study is based on the three-dimensional conformation obtained by the crystal structure of the ligand (BAFF) and the receptor (TACI) and the theoretical simulation. Object model, through computer simulation, the combined mode of TACI and BAFF and the correlation of BAFF by TACI by using the molecular biology mutation experiment and the function evaluation method, the epitope of the antigen which is predicted by the theory is real Verification and verification The work of the body is as follows: firstly, the expression of the extracellular segment of the human BAFF protein is completed by molecular biology, immunology and the like And (1) carrying out prokaryotic expression on the pET32a (+)/ BAFF, pET32a (+)/ TACI plasmid stored in the laboratory, carrying out nickel ion affinity purification on the His tag carried on the fusion protein, and passing the obtained protein (rhBAFF, rhTACI) through SDS-PAGE and Western Bl. (2) performing specific identification; (2) performing non-reduction electrophoresis, Western Blot and HPLC to buffer rhBAFF in PBS The binding activity of rhBAFF and rhTACI was detected by ELISA. and compared with the commercial protein; and (4) in vivo, the mouse B cell proliferation experiment external inspection the functional activity of rhBAFF is verified; secondly, by means of computer simulation, the determination of rhB by biological experiment The important area of the interaction between AFF and TACI. (1) With the help of BAFF and the crystal structure of the receptor TACI, the appropriate force field parameters are selected, and in the case of the solvent effect, the computer-aided molecular docking technology is used to solve the problem. in that method, a prokaryotic expression vector of the rhBAFF region mutant is constructed by a molecular biological method, Identification of trimer structures by non-reduced electrophoresis and HPLC; (3) by ELISA and O comparative analysis of the interaction kinetics of cet protein by rhBAFF, r The difference of the binding capacity of the hBAFF region mutant and the TACI; (4) the rhBAFF and mutation were evaluated in vitro by the mouse B cell proliferation assay and the activation of the classical NF-B body work The functional domain of the BAFF is identified by the difference of the activity, the functional domain of the BAFF is identified by the TACI, the theoretical simulation of each amino acid site in the functional domain is further carried out on the basis of the determination of the functional domain of the rhBAFF, the key sites are selected for research, and the experimental evaluation The important sites of the functional domains of the interaction of rhBAFF and TACI were determined. (1) The spatial structure of the complex structure of the combination of TACI and BAFF was used to make a point-by-point virtual scan mutation of 203-211,233-238 domains by computer-assisted virtual mutation technique. The change of the over-interaction energy further predicts that the TACI identifies the key site of the BAFF. (2) The method of molecular biology is used to construct r Prokaryotic expression vector of hBAFF single-point mutant, expression and purification of mutant and identification; (3) dynamic analysis of the interaction between ELISA and Octet protein Comparison of the difference of the binding capacity of rhBAFF, rhBAFF single-point mutant and TACI; (4) in vitro evaluation of rhB by the mouse B cell proliferation experiment and the activation of the classical NF-KB AFF and single-point process Variants The functional activity difference of the functional activity of the TACI is determined to identify the key functional site of the BAFF; the specific results are as follows 1. obtaining the soluble expression of rhBAFF and rhTACI by prokaryotic expression, affinity purification of the recombinant fusion protein (1) of the human BAFF extracellular section and the receptor TACI extracellular section of the high-purity active human BAFF; the protein is identified by SDS-PAGE and Western Blot, and the expressed protein has high purity and strong specificity; (2) non-reduced electrophoresis, Western blotting and Western Blot identification, Blot, HPLC analysis of rhBAFF was indeed in the form of trimer in PBS buffer with a purity of more than 90%; (3) with The binding of rhBAFF to rhTACI was confirmed by ELISA. No significant difference in commercial protein; (4) through mouse B cells Proliferative activity of rhBAFF was verified in vitro, and BAFF and TA were determined. The key functional area of CI interaction (1) uses the obtained BAFF and the receptor acting compound space conformations, and the functional site of BAFF is theoretically and reasonably determined by the intermolecular hydrogen bond formation theory, the distance geometry and the computer graphics technology; and in the present invention, the BAFF receptor TACI is used to identify the functional site of the BAFF by the theory of intermolecular hydrogen bond formation theory, distance geometry and computer graphics technology. Five regional mutants of BAFF were designed by computer-assisted molecular simulation with reasonable consideration of the secondary structure of BAFF and intramolecular hydrogen bonding; (2) a prokaryotic expression vector of rhBAFF (mutant)/ pET32a (+) was constructed; the fragment mutants of rhBAFF were successfully expressed: M1 (158-165), M2 (203-211), M3 (225-231) M4 (233-238), M5 (264- 269) and purifying with nickel ion affinity medium to obtain the high-purity target protein, The identification of the correct, Western blot and HPLC results confirmed that the molecular weight of the mutant was about 110 bp in the non-reducing condition, both of which were trimer. (3) The dynamic analysis of the interaction between the ELISA and the protein showed that the ability of the mutant M2, M4 and M5 to bind to the TACI was significantly reduced. The proliferation test of B-lymphocytes in mice and the activation of the classical NF-B-B showed the biological activity of the mutants M2, M4 compared with the rhBAFF. property The mutant M2 and M4 of the rhBAFF were initially determined to be mutated. Area 203-211,233-238 is the functional area of BAFF; III. It is clear that the key site (1) of the functional domain of the rhBAFF and the TACI is mutually recognized by the virtual single point mutation to the BAFF and the TACI The key position (M2, M4) is theoretically predicted. The change of the interaction energy is a dynamic analysis of the single point mutation to the BAFF conformation and BAF. The effect of F and TACI is that M208, G209, H210, Q234, M236 and P237 are the key sites for TACI to identify BAFF. important site structure of theoretical simulation Construction of the prokaryotic expression vector; in vitro expression and purification to obtain the high-purity target protein; the same method proves that the single-point mutant is still in the form of trimer in the solution; (3) the interaction kinetics of the protein by ELISA and the protein It was found that the interaction between rhBAFF and TACI could be significantly reduced after the mutation of M208, G209, H210, M236 and P237. real M208, G209, M236, P237 in combination with BAFF The function of the biological function mediated by the TACI is clear and the key functional site of the P237 is determined as BAFF. Conclusion: The structure of the space conformations of the complex of the BAFF and the receptor is predicted based on the organic combination of the computer-assisted molecular simulation and the biological experiment. Features and dynamic recognition mode, reasonable design
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363
本文编号：2502235
[Abstract]:The B cell activating factor (BAFF) is one of the members of the TNF superfamily, which is found in 1999. It can stimulate the proliferation, differentiation and secretion of B-lymphocyte, and play an important role in regulating the survival and development of B-cells. BAFF-R (BR3), TACI and BAFA are the three receptors of BAFF, which are distributed in the different stages of B cell differentiation and development. The results showed that the lack of BAFF could lead to the maturation of peripheral B cells and the level of immunoglobulin, while the overexpression of BAFF could destroy the immune tolerance, and be associated with many autoimmune diseases, such as systemic lupus erythematosus (SLE), sjogren's syndrome, and rheumatoid arthritis (RA). Development is closely related. Therefore, BAFF and its receptor are widely concerned as the specific targets for the treatment of autoimmune diseases. The receptor TACI has a high affinity for BAFF, which can specifically identify the BAFF and bind to the BAFF In conclusion, the soluble receptor fusion protein (TACI-Fc) can be used as a BAFF inhibitor for the treatment of autoimmune diseases, and at present the inhibitor has entered the clinical trial phase and made preliminary In addition, the development of novel antagonists has become the current research heat. However, it is not clear how the BAFF can recognize the function of the TACI, and it is important to determine the key functional site of the interaction between the BAFF and the TACI. This study is based on the three-dimensional conformation obtained by the crystal structure of the ligand (BAFF) and the receptor (TACI) and the theoretical simulation. Object model, through computer simulation, the combined mode of TACI and BAFF and the correlation of BAFF by TACI by using the molecular biology mutation experiment and the function evaluation method, the epitope of the antigen which is predicted by the theory is real Verification and verification The work of the body is as follows: firstly, the expression of the extracellular segment of the human BAFF protein is completed by molecular biology, immunology and the like And (1) carrying out prokaryotic expression on the pET32a (+)/ BAFF, pET32a (+)/ TACI plasmid stored in the laboratory, carrying out nickel ion affinity purification on the His tag carried on the fusion protein, and passing the obtained protein (rhBAFF, rhTACI) through SDS-PAGE and Western Bl. (2) performing specific identification; (2) performing non-reduction electrophoresis, Western Blot and HPLC to buffer rhBAFF in PBS The binding activity of rhBAFF and rhTACI was detected by ELISA. and compared with the commercial protein; and (4) in vivo, the mouse B cell proliferation experiment external inspection the functional activity of rhBAFF is verified; secondly, by means of computer simulation, the determination of rhB by biological experiment The important area of the interaction between AFF and TACI. (1) With the help of BAFF and the crystal structure of the receptor TACI, the appropriate force field parameters are selected, and in the case of the solvent effect, the computer-aided molecular docking technology is used to solve the problem. in that method, a prokaryotic expression vector of the rhBAFF region mutant is constructed by a molecular biological method, Identification of trimer structures by non-reduced electrophoresis and HPLC; (3) by ELISA and O comparative analysis of the interaction kinetics of cet protein by rhBAFF, r The difference of the binding capacity of the hBAFF region mutant and the TACI; (4) the rhBAFF and mutation were evaluated in vitro by the mouse B cell proliferation assay and the activation of the classical NF-B body work The functional domain of the BAFF is identified by the difference of the activity, the functional domain of the BAFF is identified by the TACI, the theoretical simulation of each amino acid site in the functional domain is further carried out on the basis of the determination of the functional domain of the rhBAFF, the key sites are selected for research, and the experimental evaluation The important sites of the functional domains of the interaction of rhBAFF and TACI were determined. (1) The spatial structure of the complex structure of the combination of TACI and BAFF was used to make a point-by-point virtual scan mutation of 203-211,233-238 domains by computer-assisted virtual mutation technique. The change of the over-interaction energy further predicts that the TACI identifies the key site of the BAFF. (2) The method of molecular biology is used to construct r Prokaryotic expression vector of hBAFF single-point mutant, expression and purification of mutant and identification; (3) dynamic analysis of the interaction between ELISA and Octet protein Comparison of the difference of the binding capacity of rhBAFF, rhBAFF single-point mutant and TACI; (4) in vitro evaluation of rhB by the mouse B cell proliferation experiment and the activation of the classical NF-KB AFF and single-point process Variants The functional activity difference of the functional activity of the TACI is determined to identify the key functional site of the BAFF; the specific results are as follows 1. obtaining the soluble expression of rhBAFF and rhTACI by prokaryotic expression, affinity purification of the recombinant fusion protein (1) of the human BAFF extracellular section and the receptor TACI extracellular section of the high-purity active human BAFF; the protein is identified by SDS-PAGE and Western Blot, and the expressed protein has high purity and strong specificity; (2) non-reduced electrophoresis, Western blotting and Western Blot identification, Blot, HPLC analysis of rhBAFF was indeed in the form of trimer in PBS buffer with a purity of more than 90%; (3) with The binding of rhBAFF to rhTACI was confirmed by ELISA. No significant difference in commercial protein; (4) through mouse B cells Proliferative activity of rhBAFF was verified in vitro, and BAFF and TA were determined. The key functional area of CI interaction (1) uses the obtained BAFF and the receptor acting compound space conformations, and the functional site of BAFF is theoretically and reasonably determined by the intermolecular hydrogen bond formation theory, the distance geometry and the computer graphics technology; and in the present invention, the BAFF receptor TACI is used to identify the functional site of the BAFF by the theory of intermolecular hydrogen bond formation theory, distance geometry and computer graphics technology. Five regional mutants of BAFF were designed by computer-assisted molecular simulation with reasonable consideration of the secondary structure of BAFF and intramolecular hydrogen bonding; (2) a prokaryotic expression vector of rhBAFF (mutant)/ pET32a (+) was constructed; the fragment mutants of rhBAFF were successfully expressed: M1 (158-165), M2 (203-211), M3 (225-231) M4 (233-238), M5 (264- 269) and purifying with nickel ion affinity medium to obtain the high-purity target protein, The identification of the correct, Western blot and HPLC results confirmed that the molecular weight of the mutant was about 110 bp in the non-reducing condition, both of which were trimer. (3) The dynamic analysis of the interaction between the ELISA and the protein showed that the ability of the mutant M2, M4 and M5 to bind to the TACI was significantly reduced. The proliferation test of B-lymphocytes in mice and the activation of the classical NF-B-B showed the biological activity of the mutants M2, M4 compared with the rhBAFF. property The mutant M2 and M4 of the rhBAFF were initially determined to be mutated. Area 203-211,233-238 is the functional area of BAFF; III. It is clear that the key site (1) of the functional domain of the rhBAFF and the TACI is mutually recognized by the virtual single point mutation to the BAFF and the TACI The key position (M2, M4) is theoretically predicted. The change of the interaction energy is a dynamic analysis of the single point mutation to the BAFF conformation and BAF. The effect of F and TACI is that M208, G209, H210, Q234, M236 and P237 are the key sites for TACI to identify BAFF. important site structure of theoretical simulation Construction of the prokaryotic expression vector; in vitro expression and purification to obtain the high-purity target protein; the same method proves that the single-point mutant is still in the form of trimer in the solution; (3) the interaction kinetics of the protein by ELISA and the protein It was found that the interaction between rhBAFF and TACI could be significantly reduced after the mutation of M208, G209, H210, M236 and P237. real M208, G209, M236, P237 in combination with BAFF The function of the biological function mediated by the TACI is clear and the key functional site of the P237 is determined as BAFF. Conclusion: The structure of the space conformations of the complex of the BAFF and the receptor is predicted based on the organic combination of the computer-assisted molecular simulation and the biological experiment. Features and dynamic recognition mode, reasonable design
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363
【参考文献】
相关期刊论文 前2条
1 李东升;王玮蓁;孙春艳;戴明;朱林学;段逸群;;系统性红斑狼疮患者B淋巴细胞刺激因子及其受体BAFF-R的表达[J];中华皮肤科杂志;2006年01期
2 陆进明;李志;陈兰芳;宣丹;徐亮;;类风湿关节炎中B细胞活化因子的研究[J];中国现代药物应用;2007年09期
，本文编号：2502235
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