炭疽毒素保护性抗原中和性表位的研究

发布时间：2018-06-13 14:17

本文选题：炭疽芽孢杆菌 + 炭疽毒素　；参考：《中国人民解放军军事医学科学院》2008年博士论文

【摘要】： 本研究拟利用炭疽毒素保护性抗原(PA)的中和性单克隆抗体,结合毒素的结构和功能研究,确定PA的主要中和性表位,分析中和性表位与毒素作用分子机理的关系,观察这些表位在疫苗免疫过程中的意义。研究中用rPA免疫BALB/c小鼠,进行杂交瘤细胞融合和筛选,获得了9株具有中和活性的单克隆抗体。利用PA不同结构域重组蛋白进行western blot以及竞争抑制ELISA实验,发现这9株中和性单抗分别识别PA3个结构域的5个不同中和表位区,其中3株中和活性强于抗PA多抗的单抗均识别PA结构域2。结果表明PA具有多个中和表位,分别位于其不同结构域,其中结构域2、4包含主要中和表位。虽然无法与国外获得的单抗的中和活性进行直接比较,但与抗PA多抗的比较结果表明本研究获得的针对结构域2的单抗5E12、2A8、5E1具有较强的中和活性,不仅强于抗PA多抗,也强于其他中和性单抗,并且能够保护Fisher344大鼠免受毒素的攻击,这再次提示结构域2包含PA的重要中和性表位。首先对结合于PA结构域2同一个表位区并且中和活性比较高的三株单抗5E1、2A8和5E12使用NEB公司的线性12肽库和环7肽库进行了筛选,结果得到了一致性的序列“SFFD”,这与PA中312-315位氨基酸完全一致。这段序列位于PA结构域2的2β_2-2β_3 loop之中,并且是胰凝乳蛋白酶的识别位点。针对这一特性设计了蛋白酶切实验,蛋白酶切保护实验,并且构建了“SFFD”缺失的突变体。进一步证实了“SFFD”是这3株单抗识别的关键氨基酸位点。在动物免疫试验中,以筛选到的特异性的噬菌体克隆作为抗原,结果发现,在小鼠体内能产生针对PA的抗体,进一步证明该表位的特异性。但获得的抗体未显示明显的毒素中和活性,分析可能于表位序列较短,免疫原性差有关。以上的研究结果显示PA结构域2的2β_2-2β_3 loop中存在一个主要的中和性表位。在毒素作用的过程中,PA domain2的主要功能是形成跨膜孔道,为LF和EF进入胞质提供途径。在由prepore到pore的转变过程中,PA domain2的2β_2-2β_3 loop起着重要的作用,它从单体中的无序结构变成一个跨膜的β折叠结构。在PA七聚体形成过程中,2β_2-2β_3 loop伸到单体结构外,与邻近单体的domain2和4相互作用,这种作用可能对七聚体的稳定性有着一定的作用。利用软件Accelrys Discovery Stdio分析,单抗5E1识别的表位“SFFD”正好位于2β_2-2β_3 loop的中间位置,通过分析与表位序列相距7A的邻近PA单体的氨基酸位点发现,邻近PA单体的410、414、496、498、633、637、668、670、672、673位氨基酸与该表位可能存在着相互作用,这包含了与2β_2-2β_3 loop邻近的氨基酸,这一结果也证明2β_2-2β_3 loop在毒素作用过程中起的作用,可能全部或大部分由312-315位氨基酸来完成。同时单抗5E1能够抑制SDS-resistant七聚体的形成也证明这一中和性表位,在PA由prepore到pore的转变过程中起着重要的作用。很多研究表明rPA为基础的新型疫苗能够激起保护性免疫。本研究利用rPA疫苗作为免疫原,免疫动物。观察在疫苗免疫状态下,该中和性表位激发机体免疫应答,诱导PA特异性抗体、表位特异性抗体及中和性抗体产生的变化特点及规律。结果显示,在两种动物模型中,抗PA特异性抗体与中和性抗体的滴度变化趋势基本一致,不同的是在豚鼠模型中中和性抗体滴度在加强免疫后才开始升高。对于表位特异性抗体,在小鼠模型中,出现的时间晚,升高的速度慢。而在豚鼠模型中,它的变化趋势与抗PA特异性抗体基本一致,出现早,升高快,与中和性抗体滴度的变化也一致。同样对单抗4D10也利用噬菌体随机肽库对其表位进行筛选,结果获得一致序列“NETNI”,这段序列与PA结构域3的570-574位“NATNI”基本一致。同时采取了表达PA截断突变体,分别与单抗结合的方法,确定556-579位氨基酸间包含4D10结合的位点,这与肽库筛选的结果吻合。根据肽库筛选的结果构建缺失突变体PAdelNATNI,PAdelTNI,PAdelI,结果发现其中只有PAdelI能够与4D10呈微弱结合。进一步验证“NATNI”是这株单抗识别的关键氨基酸位点。本研究中并没有发现这株单抗在毒素作用过程中具体干涉的步骤,但是单抗协同实验的结果表明它能够增强其他单抗的中和活性。利用软件Accelrys Discovery Stdio对该表位在PA prepore晶体结构中的位置进行分析。结果显示,与表位序列相距7A的氨基酸均位于自身单体结构中,主要分布在548-581位氨基酸之间。说明该表位较独立与其他结构域无明显关系。虽然并没有找到有效的方法验证这种协同作用的原因,但是单抗协同实验的结果说明4D10所识别的表位在毒素作用过程中确实有着一定的意义。针对PA结构域4的单抗选择中和活性较高的4B2进行了噬菌体随机肽库筛选。结果获得了一致性的序列“PLYISN#N”,这与PA中686-693位氨基酸“PLYISNPN”基本一致。为了进一步证实,于是用表达不同长度的PA截断突变体,分析单抗的结合位点,结果发现所有的截断突变体都不能与单抗4B2结合,这与之前噬菌体随机肽库筛选结果并不一致,说明这株单抗的识别表位也存在构象部分。根据噬菌体筛选结果设计了缺失突变体PAdelPL,PAdelYI,PAdelSNPN,进一步分析突变体的单抗结合活性,结果显示686-689位氨基酸“PLYI”是两种单抗结合表位的关键氨基酸,而690-693位氨基酸“SNPN”仅是单抗4B2识别表位的关键位点。国内外的研究表明PA的682N和683D在PA与受体的结合中起着至关重要的作用,因此利用实验室已经构建的突变体PAD683A,PAD683N,PAN682D,PAN683A,检测其单抗结合活性,结果发现PAN682D和PAN683A均不能和单抗482、4F12结合,由此推断682位氨基酸可能是这两株单抗结合表位的关键位点。因此推测PA结构域4的4β_8-4β_9 loop中也存在一个主要的中和性表位。通过PA与受体结合实验证实识别结构域4的单抗482和4F12能够抑制PA与受体的结合,切断了毒素致病途径中的起点。从PA的晶体结构来看,PA domain4是一个相对独立的球状实体,与其他结构域的相互作用较少。目前已经明确PA domain4的4β_8-4β_9 loop在PA与受体结合的过程中起至关重要的作用,因此可以推断该中和性表位在毒素作用的起始步骤中有着重要的意义。本研究获得了多株针对PA不同结构域的中和性单抗,一方面可以发展成为抗毒素药物,另一方面也为研究毒素作用机理提供了有力工具;在PA结构域2、3、4均存在优势中和性表位,分析表明这些中和性表位与毒素作用机制密切相关;探讨了中和性表位特异性抗体的动态变化对PA保护性免疫的意义,为发展新型疫苗(表位疫苗)提供了依据。
[Abstract]:The aim of this study is to use the neutralizing monoclonal antibodies (McAbs) of the protective antigen of anthrax toxin (PA), to combine the structure and function of the toxin, to determine the main neutralization epitopes of PA, to analyze the relationship between the neutralization epitopes and the molecular mechanism of the toxin, and to observe the significance of these epitopes in the immunization process of the vaccine.
In the study, rPA immune BALB/c mice were immunized with the fusion and screening of hybridoma cells. 9 monoclonal antibodies with neutralization activity were obtained. Western blot and competitive inhibition ELISA experiments were carried out with the recombinant protein of PA domain, and the 9 neutralizing McAbs identified 5 different neutralization regions of the PA3 domains, 3 of them. The neutralization activity stronger than the anti PA polyclonal antibody can identify the PA domain 2., which indicates that PA has multiple epitopes and is located in its different domains, respectively. The domain 2,4 contains the main neutralization epitopes. Although the neutralization activity of the mAb can not be directly compared with those obtained from abroad, the comparison results with anti PA polyclonal results show that this study obtained this study. The monoclonal antibody 5E12,2A8,5E1 for domain 2 has strong neutralization activity, not only stronger than anti PA polyanti, but also stronger than other neutralization McAbs, and can protect Fisher344 rats from the attack of toxins. This again suggests that the domain 2 contains important neutralization epitopes of PA.
First, we screened the linear 12 peptide library and ring 7 peptide library of three strains of monoclonal antibody 5E1,2A8 and 5E12 with high neutralization activity in the same epitope region of the PA domain and the high neutralization activity. The results obtained the consistent sequence "SFFD", which is exactly the same as the 312-315 amino acids in PA. This sequence is located in the 2 beta _2-2 beta _3 lo of the PA domain 2. OP, and the identification site of chymotrypsin, designed the protease cutting experiment, the protease cutting protection experiment, and the construction of the "SFFD" deletion mutant. Further confirmed that "SFFD" is the key amino acid site of the 3 monoclonal antibodies. In the dynamic immune test, the specific phage is screened. As an antigen, it was found that the antibody against PA could be produced in mice and further demonstrated the specificity of the epitopes. However, the obtained antibodies did not show obvious toxin neutralization activity. The analysis may be short of the epitope sequence and poor immunogenicity. The above results show that there is one in the 2 beta _2-2 beta _3 loop of 2 of the PA domain. In the process of toxin action, the main function of PA domain2 is to form a transmembrane channel, which provides a way for LF and EF to enter the cytoplasm. During the transition from prepore to pore, the 2 beta _2-2 beta _3 loop of PA domain2 plays an important role in transforming from the disorder structure in the monomer into a transmembrane beta folding structure. During the formation of the seven polymer, 2 beta _2-2 beta _3 loop extended to the monomer structure and interacted with the adjacent monomer domain2 and 4. This action may have a certain effect on the stability of the seven polymer. Using the software Accelrys Discovery Stdio, the epitope "SFFD" identified by the monoclonal antibody 5E1 is just located in the middle of the 2 beta _2-2 beta _3 loop. Analysis of the amino acid loci of the adjacent PA monomers of the epitope sequence 7A found that the 410414496498633637668670672673 bit amino acid of the adjacent PA monomer may interact with the epitope, which contains the amino acid adjacent to 2 beta _2-2 beta _3 loop, which also proves the role of the 2 beta _2-2 beta _3 loop in the process of the toxin action. All or most of it may be done by 312-315 amino acids. At the same time, mAb 5E1 can inhibit the formation of SDS-resistant seven polymer, which also proves that the neutralization epitope plays an important role in the transformation of PA from prepore to pore. Many studies have shown that the new vaccine based on rPA can stimulate protective immunity. This study uses rPA. The vaccine was used as immunogen, immune animal. It was observed that the neutralization epitopes stimulated the immune response, induced PA specific antibodies, epitopes specific antibodies and neutralizing antibodies in the immune state of the two animal models. The results showed that in the two animal models, the titer of anti PA specific antibody and neutralizing antibody was changed. The potential of neutralizing antibody titers in guinea pig models began to rise after strengthening immunity. For epitopes specific antibodies, the time was late and increased slowly in the mouse model. In the guinea pig model, its change trend was basically consistent with the anti PA specific antibody, early, fast, and neutralization resistance. The change of body titer is also the same.
The monoclonal antibody 4D10 also used phage random peptide library to screen its epitopes and obtained a consistent sequence "NETNI". This sequence was basically consistent with the 570-574 bit "NATNI" of PA domain 3. At the same time, the PA truncated mutant was expressed, and the method of combining with monoclonal antibody was used to determine the loci of 4D10 binding among the 556-579 amino acids. This was in accordance with the results of peptide library screening. Based on the results of peptide library screening, deletion mutants PAdelNATNI, PAdelTNI, PAdelI were constructed, and only PAdelI was found to be a weak combination with 4D10. Further verification that "NATNI" was a key amino acid site for the identification of the monoclonal antibody. The steps of specific interference in the process, but the results of the monoclonal antibody synergistic experiment show that it can enhance the neutralization activity of other mAbs. Using the software Accelrys Discovery Stdio, the location of the epitope in the PA prepore crystal structure is analyzed. The results show that the amino acid from the epitope sequence 7A is located in the single structure of its own, and the main distribution is in its own single structure. It was found that the epitopes were independent of the other domains between 548-581 amino acids. Although there was no effective method to verify the synergistic effect, the results of the monoclonal antibody synergy showed that the epitopes identified by 4D10 did have a certain significance in the process of toxin action.
The screening of phage random peptide library was carried out for the selection of monoclonal antibodies and high activity of 4B2 in PA domain 4. The results obtained a consistent sequence "PLYISN#N", which was basically consistent with the 686-693 bit amino acid "PLYISNPN" in PA. In order to further confirm, the binding sites of monoclonal antibodies were analyzed by the expression of PA truncated mutants with different lengths. The results showed that all the truncated mutants could not be combined with the monoclonal antibody 4B2, which was not consistent with the previous screening results of the phage random peptide library, indicating that the identification epitopes of the mAb also had conformation parts. The deletion mutant PAdelPL, PAdelYI, and PAdelSNPN were designed according to the phage screening results, and the monoclonal antibody binding activity of the mutant was further analyzed. The results show that the 686-689 - bit amino acid "PLYI" is the key amino acid of the two mAb binding epitopes, and the 690-693 - bit amino acid "SNPN" is only the key site of the mAb 4B2 recognition epitope. Research at home and abroad shows that PA's 682N and 683D play a vital role in the binding of PA to the receptor, thus using the mutations that have been constructed in the laboratory. Body PAD683A, PAD683N, PAN682D, PAN683A, detection of its monoclonal antibody binding activity, it is found that PAN682D and PAN683A can not bind to the monoclonal antibody 482,4F12, thus infer that the 682 bit amino acid may be the key site of the two monoclonal antibody binding epitopes. Therefore, there is a major neutralization epitopes in the 4 beta _8-4 beta _9 loop of the PA domain 4. The binding assay with the receptor confirmed that the monoclonal antibody 482 and 4F12 in the recognition domain 4 could inhibit the binding of PA to the receptor and cut off the starting point in the pathogenicity pathway of the toxin. From the crystal structure of PA, PA domain4 is a relatively independent spherical entity, and the interaction with other domains is less. At present, the 4 beta _8-4 beta _9 loop of PA domain4 has been clearly defined at present. It is concluded that the neutralization epitope plays an important role in the initial steps of PA.
In this study, a number of neutralizing monoclonal antibodies against PA domains were obtained. On the one hand, they can be developed into antitoxin drugs. On the other hand, it also provides a powerful tool for the study of the mechanism of toxin action. There are dominant neutralization epitopes in the PA domain 2,3,4. The analysis shows that these neutralization epitopes are closely related to the mechanism of toxic action. The significance of the dynamic changes of neutralizing epitope specific antibodies to PA protective immunity provides a basis for developing new vaccines (epitope vaccines).
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R392

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