幽门螺杆菌尿素酶B亚单位（UreB）一个新的B细胞抗原表位的鉴定
发布时间：2018-08-31 11:59
【摘要】： 目的 幽门螺杆菌(Helicobacter pylori,Hp)是一种定植于胃内的革兰氏染色阴性、螺杆状、微需氧菌,目前全球近50%的人口感染Hp,在发展中国家,其感染率可高达70%以上。Hp的抗感染治疗存在较大问题,使用单剂药物根除率低,而使用多联药物费用昂贵,且不能有效阻止Hp的再感染和复发。此外,耐药菌株的增加也使抗Hp治疗面临越来越复杂的难题。种种因素使Hp疫苗有望成为治疗Hp感染最有效的途径。Hp自然感染时,机体存在免疫耐受状态,激发的免疫应答不能起到保护作用,因而必须在抗原选择以及表位水平对抗原进行改造,激发更有效的免疫应答。表位疫苗是近年来发展起来的一种独特的疫苗设计思路,是目前研制某些感染性疾病、恶性肿瘤疫苗以及自身免疫性疾病等疫苗设计的新方向。基于表位的疫苗设计可以特异性的增强保护性免疫反应的强度而排除非保护性甚至是抑制性免疫反应的影响。因此,寻找鉴定Hp有效的抗原表位对发展Hp疫苗具有重要的理论指导意义。 幽门螺杆菌可以产生大量的尿素酶,该尿素酶对细菌在体内的定植及致病发挥着重要作用。尿素酶主要由A (UreA)和B (UreB)两个亚单位组成,分子量分别约为30kD和63kD。业已证实尿素酶B亚单位(UreB)是幽门螺杆菌的一个重要保护性抗原,近年已有很多学者对其展开了多方面研究。本实验室前期通过基因工程方法构建了重组幽门螺杆菌尿素酶B亚单位,制备了针对此重组UreB抗原的单克隆抗体,并在研究中发现其中一株单克隆抗体6E6具有抗Hp定植的作用。本研究拟通过体外尿素酶活性中和试验进一步鉴定Hp尿素酶B亚单位单克隆抗体6E6的中和作用,同时通过生物信息学方法和截短法分段表达UreB片段,并在此基础上采用表位作图的方法筛选出单克隆抗体所对应的B细胞抗原表位,分析其免疫学功能,为Hp新型表位疫苗的研制提供理论和实验依据。 方法 1.通过体外尿素酶活性中和试验对Hp尿素酶B亚单位单克隆抗体6E6的功能进行分析,根据不同剂量和同一剂量不同时间的依赖关系,鉴定单克隆抗体6E6的中和Hp尿素酶活性的作用。 2.通过生物信息学方法对尿素酶B亚单位蛋白的亲水性、抗原性和表面可及性等进行分析,结合尿素酶B亚单位蛋白的三级结构分析图,依据尿素酶B亚单位蛋白功能、结构稳定性,选择性的将UreB蛋白分成5段:U12、U13、U47、U15和U16,进行克隆表达。 3.采用PCR方法从Hp NCTC11637基因组中扩增U12、U13、U47、U15和U16目的基因片段,并构建到原核表达载体pET-11c,通过酶切、测序鉴定阳性重组子。将阳性重组子转化宿主菌大肠杆菌BL21 (DE3)中,IPTG诱导表达,Tris-Tricine PAGE电泳确定目的蛋白表达,通过免疫印迹方法初步确定单克隆抗体6E6识别B细胞抗原表位的区域。 4.根据免疫印迹方法初步确定的B细胞抗原表位的区域,采用步移合成表位多肽的方法合成4条15肽,通过ELISA和Dot blot方法双重鉴定,精确定位单克隆抗体6E6所对应的B细胞抗原表位。 5.将B细胞抗原表位与载体蛋白BSA应用戊二醛进行交联,然后将交联物皮下免疫BALB/c小鼠,同时做单一表位组和PBS组对照。初免加弗氏完全佐剂,次免加弗氏不完全佐剂,分别于0、7、14、28天免疫接种,剂量为100μg/只/次。末次免疫 7天后,ELISA检测免疫后抗血清,测定抗体水平。并通过免疫印迹方法检测抗血清能否识别不同天然Hp菌株的尿素酶B亚单位蛋白。 6.通过B细胞抗原表位与单克隆抗体6E6竞争抑制尿素酶活性的试验以及B细胞表位对临床感染Hp患者的血清学分析试验鉴定此表位的作用。 结果 1.体外尿素酶活性中和试验表明,单克隆抗体6E6能够中和尿素酶的活性,其抑制率随剂量的增加而增强,并且随时间的延长,其抑制率降低。 2.通过生物信息学方法分析,选择了UreB蛋白中5’端的第200、230、250、260、300和390位氨基酸分别作为构建UreB部分抗原的分割点,克隆U12(1～300aa)、U13(1～200aa)、U47(250～390aa)、U15(1～230aa)和U16(1～260aa) 5个目的片段。 3.成功构建了包含目的片段U12(1～300aa)、U13(1～200aa)、U47(250～390aa)、U15(1～230aa)和U16(1～260aa)的5个表达载体pET11c-U12、pET11c-U13、pET11c-U47、pET11c-U15和pET11c-U16,Tris-Tricine PAGE电泳确定目的蛋白表达,目的片段分子量分别约为33.0kD、22.0kD、15.4kD、25.3kD和28.6kD。免疫印迹方法初步确定单克隆抗体6E6所对应的抗原表位位于UreB蛋白5’端的第200～230位氨基酸内。 4.步移合成表位多肽U201-215、U206-220、U211-225和U216-230,经ELISA和Dot blot方法双重鉴定,单克隆抗体6E6与合成肽U211-225发生了特异性抗原抗体反应,说明单抗6E6识别的B细胞抗原表位即为U211-225。 5. B细胞抗原表位U211-225与载体蛋白BSA交联免疫动物后,产生了较高效价的抗血清,其效价高达1:32000。免疫印迹试验证实载体蛋白BSA和B细胞表位U211-225交联物产生的抗血清能够与4株Hp天然菌株的尿素酶B亚单位发生特异性抗原抗体反应。 6. B细胞表位U211-225与抗幽门螺杆菌尿素酶B亚单位单克隆抗体6E6的竞争抑制尿素酶活性试验表明,此B细胞表位U211-225能够拮抗单克隆抗体6E6中和尿素酶的活性。血清学分析试验显示,57份幽门螺杆菌感染患者血清中,有37份能够识别此B细胞表位U211-225。 结论 1.单克隆抗体6E6能够抑制尿素酶的活性,是针对Hp的一种中和性抗体。 2.单克隆抗体6E6所针对的B细胞抗原表位位于UreB蛋白5′端第211～225位氨基酸内。 3. B细胞抗原表位U211-225是Hp的一个优势(显性)表位,可以作为幽门螺杆菌的表位疫苗成分和诊断靶分子。
[Abstract]:objective
Helicobacter pylori (Hp) is a gram-negative, screw-like, micro-aerobic bacteria colonized in the stomach. At present, nearly 50% of the world's population is infected with Hp. In developing countries, the infection rate can be as high as 70%. The anti-infective treatment of Hp has great problems, the eradication rate of single drug is low, and the cost of using multi-drug is high. In addition, the increase of drug-resistant strains also makes anti-Hp treatment more and more complex. Various factors make Hp vaccine hopefully the most effective way to treat Hp infection. Epitope vaccine is a unique vaccine design idea developed in recent years. It is a new direction of vaccine design for some infectious diseases, malignant tumor vaccines and autoimmune diseases. Epitope-based vaccine design It can specifically enhance the intensity of protective immune response and exclude the influence of non-protective or even inhibitory immune response. Therefore, it is of great theoretical significance to identify the effective epitopes of Hp for the development of Hp vaccine.
Helicobacter pylori can produce a large number of urease, urease plays an important role in the colonization and pathogenesis of bacteria in vivo. Urease mainly consists of two subunits A (UreA) and B (UreB), the molecular weight of which is about 30 kD and 63 kD respectively. It has been confirmed that urease B subunit (UreB) is an important protective antigen of Helicobacter pylori. Many scholars have studied it in many aspects in recent years. In our laboratory, we constructed a recombinant urease B subunit of Helicobacter pylori by genetic engineering and prepared monoclonal antibodies against the recombinant UreB antigen. In our study, we found that one of the monoclonal antibodies, 6E6, had the effect of anti-HP colonization. The neutralization effect of monoclonal antibody 6E6 against Hp urease B subunit was further identified by urease activity neutralization test. The UreB fragment was segmented and expressed by Bioinformatics Method and truncation method. On this basis, the B cell epitopes corresponding to the monoclonal antibody were screened by epitope mapping, and its immunological function was analyzed. The development of epitope vaccine provides theoretical and experimental basis.
Method
1. The function of monoclonal antibody 6E6 to Hp urease B subunit was analyzed by urease activity neutralization test in vitro. The neutralization effect of monoclonal antibody 6E6 on Hp urease activity was identified according to the dependence of different doses and different time at the same dose.
2. The hydrophilicity, antigenicity and surface accessibility of urease B subunit protein were analyzed by bioinformatics method. According to the function and structural stability of urease B subunit protein, UreB protein was selectively divided into five segments: U12, U13, U47, U15 and U16.
3. Purpose gene fragments of U12, U13, U47, U15 and U16 were amplified from Hp NCTC11637 genome by PCR and constructed into prokaryotic expression vector pET-11c. The positive recombinant was identified by enzyme digestion and sequencing. Immunoblotting method preliminarily identified the region of B cell epitopes recognized by monoclonal antibody 6E6.
4. Four 15 peptides were synthesized by step-by-step synthesis of epitope polypeptides according to the region of B cell antigen epitopes preliminarily determined by Western blot. The B cell epitopes corresponding to monoclonal antibody 6E6 were accurately located by ELISA and Dot blot.
5. The B cell antigen epitope was cross-linked with the carrier protein BSA by glutaraldehyde, and then subcutaneously immunized BALB/c mice with the cross-linking substance. At the same time, the BALB/c mice were immunized with single epitope group and PBS group as control group.
After 7 days, ELISA was used to detect the anti-serum and the antibody level, and the immunoblotting method was used to detect whether the anti-serum could recognize the urease B subunit protein of different natural Hp strains.
6. The effect of B-cell epitope on urease activity was identified by competitive inhibition of B-cell epitope with monoclonal antibody 6E6 and serological analysis of clinical infected patients with HP.
Result
1. In vitro neutralization test of urease activity showed that monoclonal antibody 6E6 could neutralize the activity of urease, and its inhibition rate increased with the increase of dosage, and decreased with the increase of time.
2. By bioinformatics analysis, the amino acids at positions 200, 230, 250, 260, 300 and 390 of the 5'end of UreB protein were selected as the splitting points for constructing partial antigens of UreB. Five target fragments of U12 (1-300aa), U13 (1-200aa), U47 (250-390aa), U15 (1-230aa) and U16 (1-260aa) were cloned.
3. Five expression vectors including target fragments U12 (1-300aa), U13 (1-200aa), U47 (250-390aa), U15 (1-230aa) and U16 (1-260aa) were successfully constructed. The molecular weights of the target fragments were 33.0kD, 22.0kD, 15.4kD, 25.3kD, respectively. The antigen epitope of monoclonal antibody 6E6 was initially located in the 200-230 amino acids of UreB protein 5'.
4. The epitope peptides U201-215, U206-220, U211-225 and U216-230 were synthesized by step-by-step method. The monoclonal antibody 6E6 reacted with the synthetic peptide U211-225 by ELISA and Dot blot, indicating that the B cell epitope recognized by the monoclonal antibody 6E6 was U211-225.
5. The antiserum produced by the cross-linking of the B cell epitope U211-225 with the carrier protein BSA produced a high titer of 1:32000. The immunoblotting test showed that the antiserum produced by the cross-linking of the carrier protein BSA and the B cell epitope U211-225 could react with the urease B subunit of four natural strains of Hp.
6. Competitive inhibition of urease activity by B cell epitope U211-225 and monoclonal antibody 6E6 against urease B subunit of Helicobacter pylori showed that the B cell epitope U211-225 could antagonize the activity of urease neutralized by monoclonal antibody 6E6. Serological analysis showed that 37 of 57 sera from Helicobacter pylori infected patients could recognize the B. Cell epitopes U211-225.
conclusion
1. monoclonal antibody 6E6 can inhibit the activity of urease and is a neutralizing antibody against Hp.
2. The B cell epitope of monoclonal antibody 6E6 is located in the 211-225 amino acids of UreB protein 5'.
3. B cell epitope U211-225 is a dominant (dominant) epitope of Hp and can be used as an epitope vaccine component and diagnostic target molecule of Helicobacter pylori.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392
[Abstract]:objective
Helicobacter pylori (Hp) is a gram-negative, screw-like, micro-aerobic bacteria colonized in the stomach. At present, nearly 50% of the world's population is infected with Hp. In developing countries, the infection rate can be as high as 70%. The anti-infective treatment of Hp has great problems, the eradication rate of single drug is low, and the cost of using multi-drug is high. In addition, the increase of drug-resistant strains also makes anti-Hp treatment more and more complex. Various factors make Hp vaccine hopefully the most effective way to treat Hp infection. Epitope vaccine is a unique vaccine design idea developed in recent years. It is a new direction of vaccine design for some infectious diseases, malignant tumor vaccines and autoimmune diseases. Epitope-based vaccine design It can specifically enhance the intensity of protective immune response and exclude the influence of non-protective or even inhibitory immune response. Therefore, it is of great theoretical significance to identify the effective epitopes of Hp for the development of Hp vaccine.
Helicobacter pylori can produce a large number of urease, urease plays an important role in the colonization and pathogenesis of bacteria in vivo. Urease mainly consists of two subunits A (UreA) and B (UreB), the molecular weight of which is about 30 kD and 63 kD respectively. It has been confirmed that urease B subunit (UreB) is an important protective antigen of Helicobacter pylori. Many scholars have studied it in many aspects in recent years. In our laboratory, we constructed a recombinant urease B subunit of Helicobacter pylori by genetic engineering and prepared monoclonal antibodies against the recombinant UreB antigen. In our study, we found that one of the monoclonal antibodies, 6E6, had the effect of anti-HP colonization. The neutralization effect of monoclonal antibody 6E6 against Hp urease B subunit was further identified by urease activity neutralization test. The UreB fragment was segmented and expressed by Bioinformatics Method and truncation method. On this basis, the B cell epitopes corresponding to the monoclonal antibody were screened by epitope mapping, and its immunological function was analyzed. The development of epitope vaccine provides theoretical and experimental basis.
Method
1. The function of monoclonal antibody 6E6 to Hp urease B subunit was analyzed by urease activity neutralization test in vitro. The neutralization effect of monoclonal antibody 6E6 on Hp urease activity was identified according to the dependence of different doses and different time at the same dose.
2. The hydrophilicity, antigenicity and surface accessibility of urease B subunit protein were analyzed by bioinformatics method. According to the function and structural stability of urease B subunit protein, UreB protein was selectively divided into five segments: U12, U13, U47, U15 and U16.
3. Purpose gene fragments of U12, U13, U47, U15 and U16 were amplified from Hp NCTC11637 genome by PCR and constructed into prokaryotic expression vector pET-11c. The positive recombinant was identified by enzyme digestion and sequencing. Immunoblotting method preliminarily identified the region of B cell epitopes recognized by monoclonal antibody 6E6.
4. Four 15 peptides were synthesized by step-by-step synthesis of epitope polypeptides according to the region of B cell antigen epitopes preliminarily determined by Western blot. The B cell epitopes corresponding to monoclonal antibody 6E6 were accurately located by ELISA and Dot blot.
5. The B cell antigen epitope was cross-linked with the carrier protein BSA by glutaraldehyde, and then subcutaneously immunized BALB/c mice with the cross-linking substance. At the same time, the BALB/c mice were immunized with single epitope group and PBS group as control group.
After 7 days, ELISA was used to detect the anti-serum and the antibody level, and the immunoblotting method was used to detect whether the anti-serum could recognize the urease B subunit protein of different natural Hp strains.
6. The effect of B-cell epitope on urease activity was identified by competitive inhibition of B-cell epitope with monoclonal antibody 6E6 and serological analysis of clinical infected patients with HP.
Result
1. In vitro neutralization test of urease activity showed that monoclonal antibody 6E6 could neutralize the activity of urease, and its inhibition rate increased with the increase of dosage, and decreased with the increase of time.
2. By bioinformatics analysis, the amino acids at positions 200, 230, 250, 260, 300 and 390 of the 5'end of UreB protein were selected as the splitting points for constructing partial antigens of UreB. Five target fragments of U12 (1-300aa), U13 (1-200aa), U47 (250-390aa), U15 (1-230aa) and U16 (1-260aa) were cloned.
3. Five expression vectors including target fragments U12 (1-300aa), U13 (1-200aa), U47 (250-390aa), U15 (1-230aa) and U16 (1-260aa) were successfully constructed. The molecular weights of the target fragments were 33.0kD, 22.0kD, 15.4kD, 25.3kD, respectively. The antigen epitope of monoclonal antibody 6E6 was initially located in the 200-230 amino acids of UreB protein 5'.
4. The epitope peptides U201-215, U206-220, U211-225 and U216-230 were synthesized by step-by-step method. The monoclonal antibody 6E6 reacted with the synthetic peptide U211-225 by ELISA and Dot blot, indicating that the B cell epitope recognized by the monoclonal antibody 6E6 was U211-225.
5. The antiserum produced by the cross-linking of the B cell epitope U211-225 with the carrier protein BSA produced a high titer of 1:32000. The immunoblotting test showed that the antiserum produced by the cross-linking of the carrier protein BSA and the B cell epitope U211-225 could react with the urease B subunit of four natural strains of Hp.
6. Competitive inhibition of urease activity by B cell epitope U211-225 and monoclonal antibody 6E6 against urease B subunit of Helicobacter pylori showed that the B cell epitope U211-225 could antagonize the activity of urease neutralized by monoclonal antibody 6E6. Serological analysis showed that 37 of 57 sera from Helicobacter pylori infected patients could recognize the B. Cell epitopes U211-225.
conclusion
1. monoclonal antibody 6E6 can inhibit the activity of urease and is a neutralizing antibody against Hp.
2. The B cell epitope of monoclonal antibody 6E6 is located in the 211-225 amino acids of UreB protein 5'.
3. B cell epitope U211-225 is a dominant (dominant) epitope of Hp and can be used as an epitope vaccine component and diagnostic target molecule of Helicobacter pylori.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392
【参考文献】
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