论文标题:幽门螺杆菌主要保护性抗原的基因克隆、表达、单克隆抗体制备及其B细胞表位的鉴定
Gene Cloning, Expression, Monoclonal Antibody Preparation and B Cell Epitope Determination of Major Protective Antigens of Helicobacter Pylori
论文作者
论文导师 李明,论文学位 博士,论文专业 免疫学
论文单位 第一军医大学,点击次数 78,论文页数 106页File Size10338K
2007-05-01论文网 http://www.lw23.com/lunwen_710445042/
Helicobacter pylori;; Major protective antigens;; Gene cloning;; Expression;; Monoclonal antibody;; Phage peptide library;; Epitope
幽门螺杆菌(Helicobacter pylori，H．pylori，Hp)是一种长期定植于人体胃黏膜的微需氧、革兰氏阴性螺旋形细菌。大量研究证实，Hp的长期感染是慢性消化道疾病如B型(胃窦)胃炎、消化性溃疡、胃粘膜相关淋巴组织(MALT)胃癌等发生、发展的重要原因，世界卫生组织(WHO)已将其列为Ⅰ级致癌物质，全世界约有50％的人口感染Hp。尽管抗生素仍然是当前治疗Hp感染最有效的方法，但由于感染范围广，药物的不良反应、耐药菌株逐年增多，且停药后易反复再感染等因素，使药物治疗的临床应用受到极大限制。随着人们对Hp基因组到蛋白质组全面深入的解析，通过疫苗来防治Hp的感染成为可能，并逐渐成为研究的热点。已有研究表明，免疫接种可预防甚至治疗Hp的感染，使人群获得持久和较强的免疫力，所以Hp疫苗的研究具有重要的意义。 在一般情况下，Hp自然感染时体内产生强烈的免疫应答，但是感染仍呈慢性持续化甚至终身感染，表明机体存在免疫耐受状态，产生的免疫应答不能起到免疫保护作用，因而研制Hp疫苗必须选择有保护性作用的抗原并对其进行改造，以激发更有效的免疫反应。随着Hp基因组、蛋白质组、免疫组及疫苗组等研究的深入，许多在抗Hp感染中具有重要功能的保护性抗原相继被发现，这为阐明Hp在人体中的入侵、致病、免疫机制及为研制新一代Hp疫苗提供了基础。研究表明，空泡毒素(Vacuolating cytotoxin，VacA)、脂蛋白(lipoprotein 20，Lpp20)、热休克蛋白(Heat shot protein A，HspA)、细胞毒素相关蛋白(cytotoxin-assosiated geneA，CagA)、尿素酶A(UreaseA)、尿素酶B(Urease B)、过氧化氢酶(Catalase)、中性粒细胞激活蛋白(Neutrophil-activating protein，NapA)及黏附素(Adhesion)等主要保护性抗原都可以单独作为抗原，在动物实验中均表现出一定的免疫保护作用，但单独使用上述保护性抗原作为疫苗时，其保护性具有一定的局限性，表现为免疫原性弱，不能激发有效的免疫应答。当机体再次感染Hp时，不能产生有效的免疫反应对抗感染。Telford等实验表明，Hp单一抗原的保护率几乎均低于80％，二种或二种以上Hp抗原组合的疫苗有理想的保护效果。虽然采用保护性抗原全分子的联合疫苗可显著提高疫苗的保护性，但同时不可避免对人体带来较严重的毒副作用。为了进一步提高疫苗的安全性和有效性，研制基于Hp主要保护性抗原的表位亚单位疫苗将成为研制Hp疫苗新的发展方向。 Hp感染宿主的免疫机制表明，宿主可能通过特异性和非特异性免疫反应对抗Hp的感染。在特异性免疫反应中，由于Hp的感染主要是胞外感染，抗原诱发的抗体反应可能在Hp的免疫保护中起主要作用，因此对Hp主要保护性抗原的B细胞表位的鉴定也就变的尤为重要。本研究利用噬菌体随机肽库技术对Hp主要保护性抗原的B细胞表位进行了鉴定和研究。 根据相关文献报道，本研究选取Lpp20、HspA、UreaseA、CagA、UreaseB、Catalase六种Hp主要保护性抗原作为侯选靶分子进行研究。首先，根据GenBank中登录的Hp标准株26695序列设计了针对Lpp20等六种抗原基因的特异性引物，利用PCR技术从Hp DNA染色体中扩增出Lpp20等六种抗原的编码基因片段，先将其T-A克隆和测序，结果显示扩增的Lpp20、HspA、UreaseA、Cae-A、UreaseB、Catalase基因的长度分别为528bp、351bp、675bp、855bp、1704bp、1515bp，并登录在GenBank上(登录号分别为DQ106902、DQ141574、DQ141577、DQ141575、DQ141576、DQ333889)，与GenBank公布的其它菌株的核酸和氨基酸序列进行比对，同源性很高。再将目的基因克隆至融合表达载体pGEX-4T-1上，并在E.coli TOP10中进行表达，表达的Lpp20、HspA、UreaseA、CagA、UreaseB、Catalase融合蛋白的相对分子量(Mr)分别约为48000Da、41000Da、52000Da、60000Da、91000Da、85000Da。对表达产物进行超声破菌，目的蛋白以可溶性的形式存在于上清中，利用Glutathione Sepharose 4B亲和层析柱对破菌后的上清进行了纯化。Western-blotting鉴定结果显示，六种抗原的纯化产物均可被Hp感染病人血清特异性识别，说明重组表达的Lpp20等六种抗原均具有较好的抗原性。 为制备抗Hp的单克隆抗体，将培养的Hp标准株和临床株混合物超声破菌后，以破菌后的上清和沉淀为免疫原分别免疫BALB／c小鼠，通过杂交瘤技术制备了34株单克隆抗体，目前已鉴定的部分单克隆抗体亚类均为IgG1κ型，细胞培养上清的效价为1：32～1：128，腹水效价为1：32000～1：128000，抗体亲和常数介于1×10~8L／M～1×10~(10)L／M。用重组表达的HpLpp20、HspA、UreaseA、CagA、UreaseB、Catalase六种蛋白通过ELISA结合Western-blotting实验对抗体进行了鉴定，获得了分别针对这六种蛋白的特异性的单克隆抗体。用辣根过氧化物酶(HRP)对抗体进行了标记，并用竞争ELISA法鉴定抗体表位是否为同一表位，3株抗Lpp20抗体为同一表位，2株抗HspA抗体为同一表位，4株抗UreaseA抗体为同一表位，6株抗UreaseB抗体为2个表位，2株Catalase抗体为同一表位。抗Hp主要保护性抗原单克隆抗体的制备和鉴定，为下一步用噬菌体随机肽库技术筛选和鉴定抗体结合抗原决定蔟的的位点奠定了基础。 为鉴定UreaseB抗原的B细胞表位，以抗UreaseB抗原的mAb U001筛选噬菌体随机12肽库，经过3轮的淘筛，结合抗体的噬菌体得到富集，随机挑取噬菌体克隆用ELISA方法检测其与抗体的反应。对15个阳性克隆的DNA单链进行测序并推导其所编码的氨基酸序列。阳性克隆大多数呈递EHWSHMFDSPGD序列，将此序列与UreaseB蛋白的氨基酸序列进行比对，12肽序列的第1、5、6和7位置上的E、H、D、M氨基酸与UreaseB蛋白的氨基酸残基(347-353位)有较高的同源性，说明E、H、D、M可能决定了抗原表位的性质，是构成该抗原表位的关键氨基酸。竞争ELISA实验表明这个噬菌体克隆能抑制UreaseB抗原与抗体的结合。用此噬菌体克隆免疫BALB／c小鼠，ELISA和Western-blotting实验均证实抗血清识别UreaseB抗原。有国外学者对UreaseB抗原中和抗体的表位进行鉴定，认为UreaseB抗原的321-339位氨基酸残基为中和表位位点，这一结果与我们获得的结果位置相近，说明在UreaseB抗原上可能存在优势抗原表位。 为鉴定Lpp20抗原的B细胞表位，以抗Lpp20抗原的mAb L001筛选噬菌体随机12肽库，经过3轮的淘筛，结合抗体的噬菌体得到富集，随机挑取噬菌体克隆用EHSA方法检测其与抗体的反应。对15个阳性克隆的DNA单链进行测序并推导其所编码的氨基酸序列。阳性克隆大多数呈递SWPLYSDASGLG序列，将此序列与Lpp20蛋白的氨基酸序列进行比对，12肽序列中的D、A、S、G氨基酸与Lpp20蛋白的氨基酸残基(114-117位)完全同源，并且这几个氨基酸完全连续在一起，说明D、A、S、G可能决定了抗原表位的性质，是构成该抗原表位的关键氨基酸，这个表位有可能是一个线性表位。竞争ELISA实验表明这个噬菌体克隆能抑制Lpp20抗原与抗体的结合。用此噬菌体克隆免疫BALB／c小鼠，ELISA和Western-blotting实验均证实抗血清识别Lpp20抗原。 为鉴定Catalase抗原的B细胞表位，以抗Catalase抗原的mAb C001筛选噬菌体随机12肽库，经过3轮的淘筛，结合抗体的噬菌体得到富集，随机挑取噬菌体克隆用ELISA方法检测其与抗体的反应。对15个阳性克隆的DNA单链进行测序并推导其所编码的氨基酸序列。阳性克隆大多数呈递SVSLPYANLAHI序列，将此序列与Catalase蛋白氨基酸序列进行比对，12肽序列中的1、5、8、9、10位置上的氨基酸S、P、N、L、A与Catalase氨基酸残基(394-405位)有较高的同源性，说明S、P、N、L、A决定了抗原表位的性质，可能是构成该抗原表位的关键氨基酸。竞争抑制ELISA表明这个噬菌体克隆能抑制Catalase抗原与抗体的结合。用此噬菌体克隆免疫BALB／c小鼠检，ELISA和Western-blotting均实验证实抗血清识别Catalase抗原。 本研究通过噬菌体随机肽库技术筛选和鉴定了Hp主要保护性抗原UreaseB、Lpp20、Catalase的B细胞表位，初步建立了Hp主要保护性抗原B细胞表位鉴定的研究平台，为Hp表位疫苗的研制奠定了一定的理论基础，同时还说明利用噬菌体表位也是研制疫苗的发展方向。比较从噬菌体中筛选到的模拟表位与相应抗原序列，可以进一步从分子水平上加深对Hp表位的认识，设计出疫苗所需的特异性短肽。
HeIicobacter pylori (H.pylori, Hp), a curved microaerophilic Gram-negativebacterium, colonizes long-term in the gastric mucosa of human. Many studiesdemonstrated that Hp infection was the main cause of various chronic gastroduodenaldiseases such gastritis, peptic ulceration, mucosa-associated lymphoid tissue (MALT)and peptic cancer. WHO has already considered it as classⅠcarcinogen.Approximately half of the world"s populations are infected with Hp. Even thoughantibiotics are still the most effective treatment for the Hp infection, the use ofdrugs for treatment is limited because of expansive infection, side-effects andbacterial résistance, the frequency of re-infection. With people comprehensivelyunderstanding of the genome and proteome of Hp, vaccination is an alternative wayto control Hp infection and become one of the most promising approaches. Previousstudies have proved that the immunization had prevented and treated Hp infection,providing people"s long-lasting and strong immunity to the disease. Thus, it is ofgreat interests to develop Hp vaccine. Commonly, Hp natural infection induces intensive immune responses in thehuman body, but the infection is chronic and persistent, and even leads to infectionfor life. It is indicated that the human body exists immune tolerance and the immune response can not pay a role. So the protective antigens must be chosen and reshapedto induce effective immune response in the design of Hp vaccine. With thedevelopment of genome, proteome, immunome and vaccinome of Hp, manyimportant protective antigens in Hp infection were discovered, which lay foundationfor the understanding of Hp invading the human, causing infection, immunemechanism and the design of a new generation vaccine. Previous studiesdemonstrated that VacA, Lpp20, HspA, CagA, UreaseA, UreaseB, Catalase, NapA,adhesion et. al antigens were alone used as antigens in the animal experiment and hadcertain immune protective effect. But the protective effect had the limitation, in whichthe weak immunity did not induce effective immune response and the individual didnot response to the re-infection of Hp. Telford et al demonstrated that the protectiveratio of alone Hp antigen was under 80% and two or more Hp combination antigenshad the ideal protective effect. Though whole molecular antigens remarkably improvevaccine protection, it does not been avoided that the side-effects harm to the humanbody. To further improve the security and validity of vaccine, epitope vaccines basedon major protective antigens of Hp become a new study aspect. The immune mechanism of Hp infection demonstrated that the host producesspecific and non-specific immune response against the infection. In the specificimmune response, antigens inducing antibodies may play an important role in the Hpimmune protection because Hp infection is mainly extracelluar. So the Identificationof B epitopes within major protective antigens is extremely important. The presentstudy determined B epitopes of major protective antigens of Hp by phage randompeptide library technology. According to corresponding reference, Lpp20, HspA, UreaseA, CagA, UreaseBand Catalase antigens were chosen as our study target. At first, specific PCR primersof six genes are designed according to the sequence of Hp standard strain 26695 on Genbank. The genes encoding Lpp20, HspA, UreaseA, CagA, UreaseB and Catalaseof helicobacter pylori (Hp) were amplified from Hp NCTC11639 chromosomal DNAby PCR .They were first operated T-A clone and sequenced. The amplified Lpp20、HspA、UreaseA、CagA、UreaseB fragments were composed of 528bp, 351bp,675bp,855bp, 1704bp,1515bp and accessed on GenBank(accession No DQ106902,DQ141574, DQ141577, DQ141575, DQ141576 and DQ333889).The nucleotide andamino acid sequences were high homology compared with other Hp strains onGenBank. Then the genes of Lpp20, HspA, UreaseA, CagA, UreaseB and Catalasecloned into pGEX-4T-1 fusion expression vector were expressed in E.coIi Top10 with48000Da, 41000Da, 52000Da, 60000Da, and 91000Da and 85000Da fusion proteinrespectively. Expression products were ultrasonicated and the target proteins weresoluble in the supernatants, which were purified by Glutathione Sepharose 4B affinitychromatography. Western blotting proved that six recombinant proteins werespecifically recognized by the serum of Hp -infected patients. This proved that sixrecombinant proteins preserved original antigenicity. To prepare monoclonal antibodies (mAbs) against Hp, the BALB/c mice wereimmunized with supernatant and precipitation of culture combination of Hp standardand clinical strains after ultra sonication. Thirty-four mAbs were obtained byhybridoma technology and the subtype of identified mAbs were all IgG1 (κ). The titerof culture supernatant was 1:32～1:128 and the titer of ascites was 1:32000～1:128000 with affinity constants (K_(aff)) from 1×10~8 L/M to 1×10~(10)L/M. Recombinantexpression Lpp20、HspA、UreaseA、CagA、UreaseB、Catalase proteins were usedto identify mAbs by ELISA and Western-blotting and specific mAbs were obtainedrespectively against these six antigens. We labeled mAbs with HRP and identifiedepitopes by competitive ELISA. Three mAbs against Lpp20 were the same epitope,two mAbs against HspA were the same epiotpe, four mAbs against UreaseA were thesame epiotpe, six mAbs against UreaseB were two epitopes and two mAbs againstCatalase were the same epitope. The preparation and determination of these mAbs laid foundation for the screening and identification of antigenic determinant by phagerandom display library. In order to map the epitope of UreaseB antigen, the Ph.D.-12 library wasscreened with specific anti- UreaseB mAb U001 as selective molecular. After threerounds of biopanning, the phages bound to mAb were well enriched and phage cloneswere randomly selected to test for their immunoreactivity with mAb L001. Thesequences of the DNA of 15 positive clones were determined and the deduced aminoacid sequences of the corresponding peptides were analyzed. The most positive phageclones presented the sequence of. EHWSHDMFSPGD. Compared with the originalamino acid sequence of UreaseB protein, E, H, D, M of this 12 peptides sequence in1,5,6,7 positions had high homology with amino acid residues (347-353) of UreaseBprotein. E, H, D, M may determine the property of antigenic epitope and may be keyamino acids to form a motif of UreaseB protein. This notion was further confirmed bycompetitive ELISA that the positive phage clone inhibited UreaseB antigen binding tomAb. To identify the immunigenity of this peptide, the positive phage clone was usedto immunize BALB/c mice and ELISA and Western-blotting proved that the UreaseBantigen was recognized by antiserum. The epitope postion was adjacent to aneutralizing epitope of UreaseB at 321-339 aa reported by a foreign researcherpreviously. It suggested that there might be predominant antigenic epitopes onUreaseB protein. Still, more investigation is needed. In order to map the epitope of Lpp20 antigen, the Ph.D.-12 library was screenedwith specific anti- Lpp20 mAb L001 as selective molecular. After three rounds ofbiopanning, the phages bound to mAb were well enriched and phage clones wererandomly selected to test for their immunoreactivity with mAb L001. The sequencesof the DNA of 15 positive clones were determined and the deduced amino acidsequences of the corresponding peptides were analyzed. The most positive phageclones presented the sequence of SWPLYSDASGLG. Compared with the original amino acid sequence of Lpp20, D, A, S, G of this 12 peptide sequence had the samehomology with Lpp20 amino acid residues (114-117). D, A, S, G may determine theproperty of antigenic epitope and may be key amino acids to form a motif of Lpp20protein. It is speculated that this epitope may be liner. This notion was furtherconfirmed by competitive ELISA that the positive phage clone inhibited Lpp20antigen binding to mAb. To identify the immunigenity of this peptide, the positivephage clone was used to immunize BALB/c mice and ELISA and Western-blottingproved that the Lpp20 antigen was recognized by antiserum. In order to map the epitope of Catalase antigen, the Ph.D.-12 library wasscreened with specific anti- Catalase mAb C001 as selective molecular. After threerounds of biopanning, the phages bound to mAb were well enriched and phage cloneswere randomly selected to test for their immunoreactivity with mAb C001. Thesequences of the DNA of 15 positive clones were determined and the deduced aminoacid sequences of the corresponding peptides were analyzed. The most positive phageclones presented the sequence of SVSLPYANLAHI. Compared with the originalamino acid sequence of Catalase, S, P, N, L, A of this 12 peptide sequence in 1, 5,8,9,10 positions had high homology with amino acid residues (394-405) of Catalaseprotein. S, P, N, L, A may determine the property of antigenic epitope and may be keyamino acids to form a motif of Catalase. This notion was further confirmed bycompetitive ELISA that the positive phage clone inhibited Catalase antigen binding tomAb. To identify the immunigenity of this peptide, the positive phage clone was usedto immunize BALB/c mice and ELISA and Western-blotting analysis proved that theCatalase antigen was recognized by antiserum. In conclusion, the present study screened and identified B cell epitopes ofUreaseB, Lpp20, Catalase of Hp major protective antigens by phage random peptidelibrary technology .From the above results, the study platform of B cell epitope was set up and laid theoretic foundation for the development vaccine for Hp vaccine. Atthe same time, the results also indicated the potential of using phagotopes asalternative vaccine components. The comparison of mimotopes selected from apeptide library with mAb and the corresponding antigen sequence may lead to abetter understanding of the epitope of Hp from molecular level and to the design ofspecific peptides for vaccines.
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