本文選題：炭疽芽孢桿菌 + 炭疽毒素�。� 參考：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2008年博士論文

【摘要】： 本研究擬利用炭疽毒素保護(hù)性抗原(PA)的中和性單克隆抗體,結(jié)合毒素的結(jié)構(gòu)和功能研究,確定PA的主要中和性表位,分析中和性表位與毒素作用分子機(jī)理的關(guān)系,觀察這些表位在疫苗免疫過程中的意義。 研究中用rPA免疫BALB/c小鼠,進(jìn)行雜交瘤細(xì)胞融合和篩選,獲得了9株具有中和活性的單克隆抗體。利用PA不同結(jié)構(gòu)域重組蛋白進(jìn)行western blot以及競爭抑制ELISA實(shí)驗(yàn),發(fā)現(xiàn)這9株中和性單抗分別識(shí)別PA3個(gè)結(jié)構(gòu)域的5個(gè)不同中和表位區(qū),其中3株中和活性強(qiáng)于抗PA多抗的單抗均識(shí)別PA結(jié)構(gòu)域2。結(jié)果表明PA具有多個(gè)中和表位,分別位于其不同結(jié)構(gòu)域,其中結(jié)構(gòu)域2、4包含主要中和表位。雖然無法與國外獲得的單抗的中和活性進(jìn)行直接比較,但與抗PA多抗的比較結(jié)果表明本研究獲得的針對(duì)結(jié)構(gòu)域2的單抗5E12、2A8、5E1具有較強(qiáng)的中和活性,不僅強(qiáng)于抗PA多抗,也強(qiáng)于其他中和性單抗,并且能夠保護(hù)Fisher344大鼠免受毒素的攻擊,這再次提示結(jié)構(gòu)域2包含PA的重要中和性表位。 首先對(duì)結(jié)合于PA結(jié)構(gòu)域2同一個(gè)表位區(qū)并且中和活性比較高的三株單抗5E1、2A8和5E12使用NEB公司的線性12肽庫和環(huán)7肽庫進(jìn)行了篩選,結(jié)果得到了一致性的序列“SFFD”,這與PA中312-315位氨基酸完全一致。這段序列位于PA結(jié)構(gòu)域2的2β_2-2β_3 loop之中,并且是胰凝乳蛋白酶的識(shí)別位點(diǎn)。針對(duì)這一特性設(shè)計(jì)了蛋白酶切實(shí)驗(yàn),蛋白酶切保護(hù)實(shí)驗(yàn),并且構(gòu)建了“SFFD”缺失的突變體。進(jìn)一步證實(shí)了“SFFD”是這3株單抗識(shí)別的關(guān)鍵氨基酸位點(diǎn)。在動(dòng)物免疫試驗(yàn)中,以篩選到的特異性的噬菌體克隆作為抗原,結(jié)果發(fā)現(xiàn),在小鼠體內(nèi)能產(chǎn)生針對(duì)PA的抗體,進(jìn)一步證明該表位的特異性。但獲得的抗體未顯示明顯的毒素中和活性,分析可能于表位序列較短,免疫原性差有關(guān)。以上的研究結(jié)果顯示PA結(jié)構(gòu)域2的2β_2-2β_3 loop中存在一個(gè)主要的中和性表位。在毒素作用的過程中,PA domain2的主要功能是形成跨膜孔道,為LF和EF進(jìn)入胞質(zhì)提供途徑。在由prepore到pore的轉(zhuǎn)變過程中,PA domain2的2β_2-2β_3 loop起著重要的作用,它從單體中的無序結(jié)構(gòu)變成一個(gè)跨膜的β折疊結(jié)構(gòu)。在PA七聚體形成過程中,2β_2-2β_3 loop伸到單體結(jié)構(gòu)外,與鄰近單體的domain2和4相互作用,這種作用可能對(duì)七聚體的穩(wěn)定性有著一定的作用。利用軟件Accelrys Discovery Stdio分析,單抗5E1識(shí)別的表位“SFFD”正好位于2β_2-2β_3 loop的中間位置,通過分析與表位序列相距7A的鄰近PA單體的氨基酸位點(diǎn)發(fā)現(xiàn),鄰近PA單體的410、414、496、498、633、637、668、670、672、673位氨基酸與該表位可能存在著相互作用,這包含了與2β_2-2β_3 loop鄰近的氨基酸,這一結(jié)果也證明2β_2-2β_3 loop在毒素作用過程中起的作用,可能全部或大部分由312-315位氨基酸來完成。同時(shí)單抗5E1能夠抑制SDS-resistant七聚體的形成也證明這一中和性表位,在PA由prepore到pore的轉(zhuǎn)變過程中起著重要的作用。很多研究表明rPA為基礎(chǔ)的新型疫苗能夠激起保護(hù)性免疫。本研究利用rPA疫苗作為免疫原,免疫動(dòng)物。觀察在疫苗免疫狀態(tài)下,該中和性表位激發(fā)機(jī)體免疫應(yīng)答,誘導(dǎo)PA特異性抗體、表位特異性抗體及中和性抗體產(chǎn)生的變化特點(diǎn)及規(guī)律。結(jié)果顯示,在兩種動(dòng)物模型中,抗PA特異性抗體與中和性抗體的滴度變化趨勢基本一致,不同的是在豚鼠模型中中和性抗體滴度在加強(qiáng)免疫后才開始升高。對(duì)于表位特異性抗體,在小鼠模型中,出現(xiàn)的時(shí)間晚,升高的速度慢。而在豚鼠模型中,它的變化趨勢與抗PA特異性抗體基本一致,出現(xiàn)早,升高快,與中和性抗體滴度的變化也一致。 同樣對(duì)單抗4D10也利用噬菌體隨機(jī)肽庫對(duì)其表位進(jìn)行篩選,結(jié)果獲得一致序列“NETNI”,這段序列與PA結(jié)構(gòu)域3的570-574位“NATNI”基本一致。同時(shí)采取了表達(dá)PA截?cái)嗤蛔凅w,分別與單抗結(jié)合的方法,確定556-579位氨基酸間包含4D10結(jié)合的位點(diǎn),這與肽庫篩選的結(jié)果吻合。根據(jù)肽庫篩選的結(jié)果構(gòu)建缺失突變體PAdelNATNI,PAdelTNI,PAdelI,結(jié)果發(fā)現(xiàn)其中只有PAdelI能夠與4D10呈微弱結(jié)合。進(jìn)一步驗(yàn)證“NATNI”是這株單抗識(shí)別的關(guān)鍵氨基酸位點(diǎn)。本研究中并沒有發(fā)現(xiàn)這株單抗在毒素作用過程中具體干涉的步驟,但是單抗協(xié)同實(shí)驗(yàn)的結(jié)果表明它能夠增強(qiáng)其他單抗的中和活性。利用軟件Accelrys Discovery Stdio對(duì)該表位在PA prepore晶體結(jié)構(gòu)中的位置進(jìn)行分析。結(jié)果顯示,與表位序列相距7A的氨基酸均位于自身單體結(jié)構(gòu)中,主要分布在548-581位氨基酸之間。說明該表位較獨(dú)立與其他結(jié)構(gòu)域無明顯關(guān)系。雖然并沒有找到有效的方法驗(yàn)證這種協(xié)同作用的原因,但是單抗協(xié)同實(shí)驗(yàn)的結(jié)果說明4D10所識(shí)別的表位在毒素作用過程中確實(shí)有著一定的意義。 針對(duì)PA結(jié)構(gòu)域4的單抗選擇中和活性較高的4B2進(jìn)行了噬菌體隨機(jī)肽庫篩選。結(jié)果獲得了一致性的序列“PLYISN#N”,這與PA中686-693位氨基酸“PLYISNPN”基本一致。為了進(jìn)一步證實(shí),于是用表達(dá)不同長度的PA截?cái)嗤蛔凅w,分析單抗的結(jié)合位點(diǎn),結(jié)果發(fā)現(xiàn)所有的截?cái)嗤蛔凅w都不能與單抗4B2結(jié)合,這與之前噬菌體隨機(jī)肽庫篩選結(jié)果并不一致,說明這株單抗的識(shí)別表位也存在構(gòu)象部分。根據(jù)噬菌體篩選結(jié)果設(shè)計(jì)了缺失突變體PAdelPL,PAdelYI,PAdelSNPN,進(jìn)一步分析突變體的單抗結(jié)合活性,結(jié)果顯示686-689位氨基酸“PLYI”是兩種單抗結(jié)合表位的關(guān)鍵氨基酸,而690-693位氨基酸“SNPN”僅是單抗4B2識(shí)別表位的關(guān)鍵位點(diǎn)。國內(nèi)外的研究表明PA的682N和683D在PA與受體的結(jié)合中起著至關(guān)重要的作用,因此利用實(shí)驗(yàn)室已經(jīng)構(gòu)建的突變體PAD683A,PAD683N,PAN682D,PAN683A,檢測其單抗結(jié)合活性,結(jié)果發(fā)現(xiàn)PAN682D和PAN683A均不能和單抗482、4F12結(jié)合,由此推斷682位氨基酸可能是這兩株單抗結(jié)合表位的關(guān)鍵位點(diǎn)。因此推測PA結(jié)構(gòu)域4的4β_8-4β_9 loop中也存在一個(gè)主要的中和性表位。通過PA與受體結(jié)合實(shí)驗(yàn)證實(shí)識(shí)別結(jié)構(gòu)域4的單抗482和4F12能夠抑制PA與受體的結(jié)合,切斷了毒素致病途徑中的起點(diǎn)。從PA的晶體結(jié)構(gòu)來看,PA domain4是一個(gè)相對(duì)獨(dú)立的球狀實(shí)體,與其他結(jié)構(gòu)域的相互作用較少。目前已經(jīng)明確PA domain4的4β_8-4β_9 loop在PA與受體結(jié)合的過程中起至關(guān)重要的作用,因此可以推斷該中和性表位在毒素作用的起始步驟中有著重要的意義。 本研究獲得了多株針對(duì)PA不同結(jié)構(gòu)域的中和性單抗,一方面可以發(fā)展成為抗毒素藥物,另一方面也為研究毒素作用機(jī)理提供了有力工具;在PA結(jié)構(gòu)域2、3、4均存在優(yōu)勢中和性表位,分析表明這些中和性表位與毒素作用機(jī)制密切相關(guān);探討了中和性表位特異性抗體的動(dòng)態(tài)變化對(duì)PA保護(hù)性免疫的意義,為發(fā)展新型疫苗(表位疫苗)提供了依據(jù)。
[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).
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392

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