摘要
干扰素(interferon,IFN)是一类重要的细胞因子,作为抗病毒和抗肿瘤的药物已广泛用于临床。IFN通过与靶细胞表面的受体结合后,激活信号通路,诱导相关抗病毒、抗肿瘤基因的转录和表达,发挥其生物学作用。而蛋白质之间的相互作用或识别是通过局部肽段间的少量氨基酸残基相互作用来实现的,合理设计的小肽可以完全或部分模拟完整蛋白分子的功能。通常完整的蛋白分子由于对蛋白酶敏感、具有宿主免疫原性以及成本高等原因并不是最佳的候选药物。IFN除了具有以上不足外,在临床使用时还会出现副作用,限制了其使用。而小分子肽类细胞因子模拟物或拮抗剂由于其相对分子质量小、易于合成、无抗原性和毒副作用较小等特点,在基础研究以及临床治疗等领域逐渐显现出其优势。本论文以IFN-α2b为研究对象,以Anti-IFN-α2b多克隆抗体、天然表达IFN受体的细胞为靶标,利用噬菌体随机肽库技术,研究IFN-α2b的抗原表位、筛选IFN-α2b的拮抗肽和模拟肽,对研究IFN的分子机制以及设计和开发IFN小分子模拟肽药物有重要的意义。
本文首先以结合有Anti-IFN-α2b多克隆抗体的免疫磁性微球为靶标,筛选噬菌体随机12肽库。经ELISA鉴定,四轮筛选后同抗体结合的噬菌体得到明显的富集,其阳性率达到53%(49/92)。随机挑取12个阳性噬菌体进行序列分析并与IFN-α2b序列进行同源比对。结果显示:12个阳性克隆可分为三组,分别对应IFN-α2b N端(1～13)、AB环附近(35～47)和CD环附近(98～110)的三组高抗原性表位区,与IFN抗原预测分析的数据相吻合。特异性抗体封闭试验和噬菌体免疫试验表明,所得到的噬菌体展示肽在免疫反应性和免疫原性上均可以一定程度地模拟IFN-α2b不同的表位区。结合IFN的三维结构进行分析,三组模拟表位均暴露在IFN分子结构的外部,更容易被抗原呈递细胞上的抗原识别受体所识别。其中第二组表位AB环(29～35)直接参与与IFN受体的结合,推测在使用IFN治疗疾病过程中,针对该区域所产生的中和抗体可能直接影响IFN的生物学活性。
采用基因重组技术,本文成功构建了原核表达载体pET32a(+)/GFP- IFN -α2b,并在大肠杆菌E.coli BL21中成功地进行了表达。SDS-PAGE和Western blot实验显示该融合蛋白GFP-IFN-α2b保持了良好的IFN抗原性;受体结合实验表明,该融合蛋白能够与WISH细胞上的IFN受体结合,并在488nm激发光下呈现亮绿色荧光;抗病毒实验表明,该融合蛋白具有一定的抗病毒活性,比活力为1.0×107IU/mg,证明该融合蛋白为具有绿色荧光和IFN抗病毒活性的双功能蛋白。本研究为在细胞及分子水平研究IFN与靶细胞相互作用提供了简便、快捷、直观的研究材料,同时也为IFN的代谢及功能研究建立了方法学基础。
以天然表达IFN受体的WISH细胞并结合Anti-IFN-α2b多克隆抗体为靶分子,本文采用竞争性洗脱的方法筛选噬菌体随机7肽库。经鉴定六轮筛选后同细胞和抗体结合的噬菌体得到明显的富集,最后一轮筛选的阳性率达到51.1%(23/45)。随机挑取10个阳性克隆进行序列分析并与IFN序列进行同源比对。结果显示:10个克隆可分为三组,分别对应IFN AB环的24～41和43～49以及E螺旋的148～158位氨基酸残基。分别选取对应AB环的第一组的No.26,以及E螺旋第三组的No.35两个噬菌体克隆为例做进一步研究。竞争性ELISA和细胞免疫组化实验显示,No.26和No.35能与IFN竞争性的同IFN受体或抗体结合。以其展示的序列分别合成SP-7(SLSPGLP)和FY-7(FSAPVRY)两个小肽。细胞受体竞争实验显示,SP-7和FY-7能够有效地模拟IFN的部分受体结合表位,特异性地抑制IFN与其受体的结合,其IC50值分别为8.90μg和3.22μg。采用细胞病变抑制法分析SP-7和FY-7对IFN抗病毒活性的影响,结果显示当两个合成肽的加入量在6.25～100μg之间时,合成肽对IFN抗病毒活性均具有抑制作用,并且存在剂量依赖关系,其IC50约为25μg,证明SP-7和FY-7为两个IFN拮抗肽。
为了得到具有抗病毒活性的IFN模拟肽,本文在上述细胞筛选的基础上,对上述同细胞结合的噬菌体,又增加了三轮功能筛选。经鉴定具有抗病毒活性的噬菌体克隆得到了一定的富集,其阳性率为1.98%(20/1012)。随机挑取10个阳性克隆进行序列分析并与IFN-α2b的序列进行比对。结果显示:10个克隆可分为四组,分别对应IFN的31～37、68～74、93～121和132～161位氨基酸残基。其中第一组的No.T9(31～37)与IFN中AB环(26～35)有部分重叠;第四组中的No.T3(143～149)与IFN中E螺旋(141～158)有部分重叠,并且其序列中144R和149R与IFN分子中参与受体结合并激活信号通路的“热点”氨基酸一致。竞争ELISA和细胞免疫组化实验显示,No.T9和No.T3能与IFN竞争性的同IFN受体结合。以其展示的序列分别合成KP-7(KNVHPPP)和IR-7(IRPDTPR)两个小肽。
细胞受体竞争实验显示,KP-7和IR-7能有效地模拟IFN的部分受体结合表位,特异性地抑制IFN与其受体的结合,其IC50值分别为13.43μg和9.49μg。细胞病变抑制法的抗病毒实验表明:KP-7和IR-7在1.25～5μg范围内,表现出一定的抗病毒活性,并呈现剂量依赖关系,证明KP-7和IR-7为两个模拟IFN不同表位具有抗病毒作用的模拟肽。其中KP-7抗病毒活性略高于IR-7,并在发挥抗病毒作用时两者呈现出一定的协同作用,提示IFN的抗病毒作用是多位点共同作用其受体的结果。
本文较全面地了解了IFN-α2b的抗原结合价,以及其特定的抗原决定簇的特性,为进一步探讨IFN分子与受体的作用机理,制备抗特定表位的单克隆抗体奠定了基础。同时本文开发了全细胞结合抗体或功能性筛选的淘筛系统,为细胞因子的拮抗肽和模拟肽的筛选,研究IFN的功能和分子机制,以及小肽类药物的开发提供了一条新的途径。
Interferons (IFNs) are important cytokines which are used clinically as antiviral and antitumor agents. Binding of IFN to a cell surface receptor mediates activation of the signal transduction pathway that elicits the biological functions by inducing the transcription and expression of IFN-related genes. However, the recognition and interaction of proteins with others are finished through the interaction among several amino acids in the peptide fragments. Many researches demonstrate that short peptide ligands can mimicry the function of the corresponding whole protein molecules. In general, whole proteins are not suitable drug candidates for a number of reasons, including susceptibility to proteolytic degeneration, antigenicity and high cost. Beside those shortcomings, efficient treatment by IFNs is also hampered by various side effects. Since the peptide agonist or antagonist of cytokines has the advantages including low molecular weight, easy to synthesize, without antigenicity as well as reduced side effects, it has attract great attention of both researchers and investors. In this paper, using phage random peptide library technology, the polyclonal anti-IFN-α2b antibodies and WISH cells naturally carrying IFN receptors on the cell membranes were chosen as targets to study the antigen epitope of IFN-α2b and isolate the mimetic or antagonist peptide of IFN-α2b, which is significant in researching on the functional mechanism of IFN-α2b as well as the minimolecular mimic design and development of IFN-α2b.
The polyclonal anti-IFN-α2b antibodies binding to immunomagnetic microspheres were chosen as targets and the biopanning of a 12-mer phage random peptide library was carried out. After 4 rounds of effective screening, the positive clones were characterized by ELISA and the positive rate was 53 % (49/92), which demonstrated that the positive clones specifically binding to antibodies were enriched. 12 clones randomly picked from the positive clones were sequenced. The homologous analysis of amino acid sequences of positive clones with IFN-α2b showed that 3 groups homologous to the 3 epitopes with strong antigenicity of IFN-α2b were obtained, defined by residues 1~13, 35~47 and 98~110 of IFN-α2b respectively. The results were anatomizing to the antigenicity predicting of IFN-α2b. Antibody blocking and phage immunity tests suggested peptides displayed on the positive phage could simulate, partly, the different epitopes of IFN-α2b in aspect of immune and antigen reactivity. Analysis on the three-dimensional structure of IFN-α2b showed that all 3 mimetic epitopes exposed to outsides of the whole molecular, which was easy to recognize by the antigen receptor of the antigen presenting cell. Especially the sequences of group II exhibited structural homology with Loop AB (29-35) of IFN-α2b involving binding to IFN receptor. It was supposed that neutralizing antibodies against AB loop produced after using IFN affected its biological function directly.
Using gene recombinant technology, the prokaryotic expression vector pET32a(+)/GFP-IFN-α2b was successfully constructed and the fusion protein was expressed in E.coli BL21. SDS-PAGE and Western blot analysis showed that the fusion protein GFP-IFN-α2b retained the antigenicity of IFN-α2b. The receptor binding experiments indicated that the fusion protein with GFP activity could bind especially to WISH cells containing IFN receptors. The antiviral assay showed that the fusion protein possessed the IFN functions of antiviral activity and the antiviral activity was 1.0×107IU/mg. All the results demonstrated that the fusion protein was a bio-functional one which had not only the green fluorescence but also the IFN-α2b activity. As a simple and visualized material, the fusion protein we prepared here could be used to study the interaction of IFN-α2b with its receptor at the cell or molecular level. Meanwhile, the results laid the foundation for the research on the metabolism and function of IFN-α2b in methodology.
WISH cells naturally carrying IFN receptors and polyclonal anti-IFN-α2b antibodies were chosen as targets and the biopanning of a 7-mer phage random peptide library was carried out by competitive elution method. The positive clones specifically binding to both WISH cells and antibodies were enriched and the positive rate of the last round was 51.1 % (23/45). 10 clones randomly picked from the positive clones were sequenced and the homologous analysis of amino acid sequences of positive clones with IFN-α2b was done. The corresponding amino acid sequences suggested 3 groups homologous to the 3 domains of IFN-α2b, defined by residues 24~41, 43~49, and 148~158 of IFN-α2b respectively. As represents corresponding to IFN receptor-binding domains, AB loop and E helix, clone No 26 and 35 were chosen for further characterization. Competitive ELISA and immunohistochemistry tests showed that clone No 26 and 35 could compete with IFN for binding to WISH cells and antibodies. Two peptides corresponding to clone No 26 and 35, designated SP-7(SLSPGLP) and FY-7(FSAPVRY) were synthesized. The receptor binding experiments showed that the two peptides could compete with GFP-IFN-α2b for binding to its receptor and the IC50 value was 8.90μg and 3.22μg respectively, which demonstrated that they could mimicry, partly, epitopes of IFN-α2b in charge of binding to receptor. The effects of two synthesized peptides to the antiviral activity induced by IFN were analyzed through CPE method. As a result, when the added amount was from 6.25μg to 100μg, both peptides could inhibit the IFN-induced antiviral activity in a dose-dependent manner. The IC50 values of both peptides were approximate 25μg, which suggested that they were antagonist peptides of IFN-α2b suppressing the antiviral activity mediated by IFN-α2b.
In order to obtain the peptides mimicking IFN activity, on the basis of cell selection described above, additional 3 rounds of functional screening of the peptide library binding to WISH cells were proceed. The positive clones possessing the IFN functions of antiviral activity were enriched and the positive rate of the last round was 1.98 %( 20/1012). 10 clones randomly picked from the positive clones were sequenced and the homologous analysis of amino acid sequences of positive clones with IFN-α2b was done. The corresponding amino acid sequences suggested 4 groups homologous to the 4 domains of IFN-α2b, defined by residues 31~37, 68~74, 93~121 and 132~161 of IFN-α2b respectively. Of these positive clones, No.T9 (31~37) in group I and No.T3 (143~149) in group III overlapped, partly, with AB Loop (26~35) and E helix (141~158) of IFN-α2b respectively. Especially 2R and 7R of IR-7 were identical to 144R and 149R of IFN-α2b which were so called“hot-spot”residues very important for binding of IFN to its receptor. Competitive ELISA and immunohistochemistry tests showed that No T9 and T3 could compete with IFN for binding to its receptor. Two peptides corresponding to clone No T9 and T3, designated KP-7(KNVHPPP) and IR-7(IRPDTPR) were synthesized. The receptor binding experiments showed that the two peptides could compete with GFP-IFN-α2b for binding to its receptor and the IC50 value was 13.43μg and 9.49μg respectively, which demonstrated that they could mimicry, partly, epitopes of IFN-α2b in charge of binding to receptor. The antiviral assay showed that when the added amount was from 1.25μg to 5μg, both peptides could have the IFN-induced antiviral activity in a dose-dependent manner, which suggested that they were mimetic peptides of IFN-α2b. Furthermore, the antiviral activity of KP-7 was a little higher than IR-7 and there was a cooperation when both peptides were added together. It assumed that multi-sites interaction of IFN to its receptor caused to the IFN-induced antiviral activity.
In summary, the valences and characteristics of IFN-α2b antigen epitopes had been understood comprehensively. It is helpful in laying the foundation for the molecular mechanism of the interaction of IFN with its receptor and the preparation of monoclonal antibody against specific epitopes. Furthermore, the screening systems such as whole cell together with antibodies or functional screening strategies had been developed, which presented a new method in studying the molecular mechanism of IFN function and screening the mimetic or antagonist peptides of cytokines as well as developing the minimolecular mimics of IFN-α2b.
引文
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