【摘要】：B細(xì)胞激活因子(B cell activating factor, BAFF)是1999年發(fā)現(xiàn)的TNF超家族成員之一,它可特異性的刺激B淋巴細(xì)胞增殖、分化和分泌抗體,對(duì)B細(xì)胞的存活和發(fā)育起到重要的調(diào)控作用。BAFF-R(BR3)、TACI、BCMA分別是BAFF的三個(gè)受體,它們分布在B細(xì)胞分化發(fā)育的不同時(shí)期,BAFF正是通過這些受體在B細(xì)胞分化發(fā)育的各個(gè)階段及免疫調(diào)節(jié)中發(fā)揮的作用。研究表明：BAFF缺乏會(huì)導(dǎo)致外周B細(xì)胞成熟缺陷和降低免疫球蛋白的水平；而BAFF過表達(dá)則會(huì)破壞免疫耐受,與許多自身免疫病如系統(tǒng)性紅斑狼瘡(SLE), sjogren氏綜合癥,類風(fēng)濕關(guān)節(jié)炎(RA)的發(fā)生、發(fā)展密切相關(guān)。因此,BAFF及其受體作為治療自身免疫疾病的特異性靶標(biāo),受到廣泛的關(guān)注。 受體TACI與BAFF具有高親和力,它可以特異性識(shí)別BAFF并與之結(jié)合。因此,可溶性受體融合蛋白(TACI-Fc)可作為BAFF抑制劑治療自身免疫疾病,目前該抑制劑已經(jīng)進(jìn)入臨床試驗(yàn)階段并取得初步成效。同時(shí),新型拮抗劑開發(fā)已成為目前的研究熱點(diǎn)。然而,BAFF是如何識(shí)別TACI發(fā)揮功能尚不十分清楚,確定BAFF與TACI相互作用的關(guān)鍵功能位點(diǎn)對(duì)于研制新型拮抗劑具有重要的指導(dǎo)意義。 本研究基于配體(BAFF)和受體(TACI)晶體結(jié)構(gòu)以及理論模擬獲得的三維構(gòu)象,借助計(jì)算機(jī)輔助分子對(duì)接技術(shù)、動(dòng)力學(xué)模擬技術(shù)構(gòu)建配體-受體相互作用的復(fù)合物模型,通過計(jì)算機(jī)模擬的方式分析TACI與BAFF的結(jié)合模式以及BAFF被TACI識(shí)別的關(guān)鍵區(qū)域。利用分子生物學(xué)突變實(shí)驗(yàn)以及功能評(píng)價(jià)方法對(duì)理論預(yù)測(cè)的抗原表位進(jìn)行實(shí)驗(yàn)驗(yàn)證。 具體工作如下： 一、通過分子生物學(xué)、免疫學(xué)等方法完成人BAFF蛋白胞外段的表達(dá)純化及活性鑒定。 (1)將實(shí)驗(yàn)室保存的pET32a(+)/BAFF、pET32a(+)/TACI質(zhì)粒進(jìn)行原核表達(dá)、通過融合蛋白上攜帶的His標(biāo)簽進(jìn)行鎳離子親和純化,將獲得的蛋白(rhBAFF、rhTACI)通過SDS-PAGE及Western Blot完成特異性鑒定； (2)通過非還原電泳、Western Blot、HPLC對(duì)rhBAFF在PBS緩沖液中的存在形式做了分析； (3)用ELISA方法檢測(cè)了rhBAFF與rhTACI的結(jié)合活性,并與商品化蛋白進(jìn)行了比較； (4)通過小鼠B細(xì)胞增殖實(shí)驗(yàn)在體外驗(yàn)證了rhBAFF的功能活性； 二、借助計(jì)算機(jī)模擬的方法、通過生物學(xué)實(shí)驗(yàn)驗(yàn)證確定rhBAFF與TACI相互作用的重要區(qū)域。 (1)借助BAFF以及受體TACI晶體結(jié)構(gòu),選擇合適的力場(chǎng)參數(shù),在考慮溶劑效應(yīng)的情況下,利用計(jì)算機(jī)輔助分子對(duì)接技術(shù)經(jīng)動(dòng)力學(xué)模擬構(gòu)建BAFF與受體相互作用空間構(gòu)象； (2)利用分子生物學(xué)方法構(gòu)建rhBAFF區(qū)域突變體的原核表達(dá)載體,在體外表達(dá)純化及鑒定并通過非還原電泳和HPLC的方法鑒定三聚體結(jié)構(gòu)； (3)通過ELISA及Octet蛋白相互作用動(dòng)力學(xué)分析比較rhBAFF、rhBAFF區(qū)域突變體與TACI的結(jié)合能力的差異； (4)通過小鼠B細(xì)胞增殖實(shí)驗(yàn)以及經(jīng)典NF-κB的激活實(shí)驗(yàn)在體外評(píng)判rhBAFF與突變體功能活性的差異,確定TACI識(shí)別BAFF的功能域； 三、在確定rhBAFF功能域的基礎(chǔ)上進(jìn)一步對(duì)功能域中每個(gè)氨基酸位點(diǎn)進(jìn)行理論模擬,選擇關(guān)鍵位點(diǎn)進(jìn)行研究,經(jīng)實(shí)驗(yàn)評(píng)價(jià)確定了rhBAFF與TACI相互作用功能域的重要位點(diǎn)。 (1)利用獲得的TACI與BAFF作用復(fù)合物空間結(jié)構(gòu),通過計(jì)算機(jī)輔助虛擬突變技術(shù)對(duì)203～211、233～238結(jié)構(gòu)域進(jìn)行逐點(diǎn)虛擬掃描突變,通過相互作用能量的變化進(jìn)而預(yù)測(cè)TACI識(shí)別BAFF的關(guān)鍵位點(diǎn)。 (2)利用分子生物學(xué)方法構(gòu)建rhBAFF單點(diǎn)突變體的原核表達(dá)載體,完成突變體的表達(dá)純化及鑒定； (3)通過ELISA及Octet蛋白相互作用動(dòng)力學(xué)分析比較rhBAFF、rhBAFF單點(diǎn)突變體與TACI的結(jié)合能力的差異； (4)通過小鼠B細(xì)胞增殖實(shí)驗(yàn)以及經(jīng)典NF-KB的激活實(shí)驗(yàn)在體外評(píng)判rhBAFF與單點(diǎn)突變體的功能活性差異確定TACI識(shí)別BAFF的關(guān)鍵功能位點(diǎn)； 具體結(jié)果如下： 一、獲得了高純度有活性的人BAFF胞外段及受體TACI胞外段的重組融合蛋白 (1)經(jīng)原核表達(dá)、親和純化,獲得rhBAFF、rhTACI的可溶表達(dá)蛋白,經(jīng)SDS-PAGE及Western Blot鑒定,所表達(dá)蛋白的純度高、特異性強(qiáng)； (2)經(jīng)非還原電泳、Western Blot、HPLC分析rhBAFF在PBS緩沖液中的確為三聚體形式存在且純度超過90%； (3)用ELISA雙夾心法證實(shí)rhBAFF能夠特異性的結(jié)合rhTACI,與商品化蛋白相比無明顯差異； (4)通過小鼠B細(xì)胞增殖實(shí)驗(yàn)在體外驗(yàn)證了rhBAFF的促增殖活性； 二、明確了BAFF與TACI相互作用的關(guān)鍵功能區(qū)域 (1)利用獲得的BAFF與受體作用復(fù)合物空間構(gòu)象,通過分子間氫鍵形成理論、距離幾何學(xué)以及計(jì)算機(jī)圖形學(xué)技術(shù)從理論上合理判別BAFF受體TACI識(shí)別BAFF的功能位點(diǎn)；在合理考慮BAFF二級(jí)結(jié)構(gòu)以及分子內(nèi)氫鍵的條件下,通過計(jì)算機(jī)輔助分子模擬合理設(shè)計(jì)BAFF的五個(gè)區(qū)域突變體； (2)構(gòu)建了rhBAFF(突變體)／pET32a(+)的原核表達(dá)載體；成功表達(dá)rhBAFF的片段突變體：M1(158-165)、M2(203-211)、M3(225-231)M4(233-238)、M5(264-269),并通過鎳離子親和介質(zhì)純化得到高純度的目的蛋白,經(jīng)SDS-PAGE和Western blot鑒定正確,Western印跡和HPLC的結(jié)果證實(shí)在非還原條件下突變體的分子量約為110bp,均為三聚體形式； (3) ELISA與蛋白相互作用動(dòng)力學(xué)分析結(jié)果表明突變體M2、M4、M5的結(jié)合TACI的能力顯著降低。小鼠B淋巴細(xì)胞增殖試驗(yàn)和經(jīng)典NF-κB激活實(shí)驗(yàn)結(jié)果顯示,突變體M2、M4與rhBAFF相比的生物學(xué)活性明顯降低,初步確定rhBAFF的突變體M2、M4突變的區(qū)域203-211,233-238為BAFF的功能區(qū)域； 三、明確了rhBAFF與TACI相互作用功能域的關(guān)鍵位點(diǎn) (1)通過虛擬單點(diǎn)突變對(duì)BAFF與TACI相互識(shí)別的關(guān)鍵位置(M2、M4)進(jìn)行理論預(yù)測(cè),經(jīng)相互作用能的變化動(dòng)態(tài)分析了單點(diǎn)突變對(duì)BAFF構(gòu)象以及BAFF與TACI作用的影響,得出M208、G209、H210、Q234、M236、P237是TACI識(shí)別BAFF的關(guān)鍵位點(diǎn)。 (2)通過分子生物學(xué)定點(diǎn)誘變技術(shù)將理論模擬的重要位點(diǎn)構(gòu)建為原核表達(dá)載體；體外表達(dá)純化得到高純度的目的蛋白,同樣的方法證實(shí)單點(diǎn)突變體在溶液中仍以三聚體形式存在； (3)經(jīng)ELISA、蛋白相互作用動(dòng)力學(xué)分析評(píng)價(jià)突變體的結(jié)合能力,發(fā)現(xiàn)M208、G209、H210、M236、P237突變后rhBAFF與TACI的相互作用能明顯降低。同時(shí),活性分析實(shí)驗(yàn)的結(jié)果與結(jié)合實(shí)驗(yàn)相符,證實(shí)M208、G209、M236、P237在BAFF結(jié)合TACI介導(dǎo)的生物功能中作用明確,并確定P237為BAFF的關(guān)鍵功能位點(diǎn)； 結(jié)論：基于計(jì)算機(jī)輔助分子模擬技術(shù)與生物學(xué)實(shí)驗(yàn)有機(jī)結(jié)合,通過預(yù)測(cè)BAFF與受體相互作用復(fù)合物空間構(gòu)象的結(jié)構(gòu)特征以及動(dòng)態(tài)識(shí)別模式,合理設(shè)計(jì)BAFF突變體并進(jìn)行體外生物學(xué)活性評(píng)價(jià),成功確定了BAFF與受體相互識(shí)別的功能位點(diǎn),為進(jìn)一步合理設(shè)計(jì)、改造BAFF拮抗劑奠定了基礎(chǔ)。
[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
【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R363

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 李東升;王瑋蓁;孫春艷;戴明;朱林學(xué);段逸群;;系統(tǒng)性紅斑狼瘡患者B淋巴細(xì)胞刺激因子及其受體BAFF-R的表達(dá)[J];中華皮膚科雜志;2006年01期

2 陸進(jìn)明;李志;陳蘭芳;宣丹;徐亮;;類風(fēng)濕關(guān)節(jié)炎中B細(xì)胞活化因子的研究[J];中國(guó)現(xiàn)代藥物應(yīng)用;2007年09期

，

本文編號(hào)：2502235