治疗性幽门螺杆菌表位疫苗的实验研究

发布时间：2018-03-31 01:24

本文选题：幽门螺杆菌　切入点：表位疫苗　出处：《第三军医大学》2007年博士论文

【摘要】： 幽门螺杆菌(Helicobacter pylori, H. pylori)是慢性胃炎、胃十二指肠溃疡的主要病原菌,与胃癌、胃粘膜相关性淋巴组织淋巴瘤密切相关,已被世界卫生组织列为Ⅰ类致癌因子。目前临床治疗H. pylori感染主要方法为联合应用质子泵抑制剂和抗生素的疗法,虽然在一定程度上能清除H. pylori,但患者对药物的依从性差、抗生素耐药菌株不断出现、再感染的发生及药物的不良反应等,这都使临床药物治疗受到限制。有效的预防性和治疗性H. pylori疫苗可以克服上述药物治疗H.pylori的不足,被认为是控制H. pylori感染的最有前景的方法,是根除H. pylori感染的希望。基于表位疫苗设计(Epitope-based Vaccine Design,EBVD)的策略被认为是第四代疫苗,其着眼点是免疫应答的功能单位——表位。表位疫苗明显的优势是设计灵活、针对性强,选择优势表位进行合理的组合设计,能够诱导高效、安全、特异的免疫应答。近年来,表位疫苗已经成为研制感染性疾病、恶性肿瘤以及自身免疫性疾病等疫苗设计的新方向,并取得显著进展。H.pylori自然感染可通过免疫偏差、免疫颠覆和免疫缺陷等机制逃避机体的清除,进而引起机体对H.pylori自身天然蛋白成分的免疫耐受,研究表明仅仅选用H.pylori天然抗原进行免疫治疗是很难打破机体免疫耐受,激发有效的免疫应答,因此,必须对天然抗原分子进行设计、修饰和改造。蛋白抗原发挥其功能主要通过表位来体现特异性,这些表位不仅包括B细胞表位、Th细胞表位、CTL表位等与免疫识别相关的结构,而且还同时存在毒性或抑制性表位等不利于免疫保护的因素。为了提高蛋白抗原的保护性,就必须在表位水平上作出选择和设计以得到理想的疫苗分子。借鉴其它疾病表位疫苗的设计经验,选择H.pylori优势抗原的保护性、特异性、免疫原性强的表位进行设计,辅以粘膜佐剂来激发更强、更特异的、不同于自然感染时的粘膜免疫应答,有可能打破免疫耐受,达到预防或治疗H.pylori感染的目的。 H.pylori感染免疫治疗后的保护机制较为复杂,所设计的不同类型H.pylori治疗疫苗的免疫应答机制也存在差异。在细胞免疫方面,已有很好的证据证实:CD4~+T细胞对于H.pylori的免疫保护是必需的,并且,随着对H.pylori疫苗治疗效果及机制研究的深入,逐渐认识到机体抵抗H.pylori感染的保护性机制可能为一种Th1/Th2同时存在的平衡应答模式;在体液免疫方面,不能完全排除特异性体液免疫应答的作用,基于对H.pylori感染免疫机制的进一步认识和本室先前H.pylori感染治疗的动物试验结果,H.pylori治疗性疫苗的设计可能遵循以CD4+ T细胞介导的Th1/Th2平衡应答为主,兼顾特异性体液免疫的基本策略。在H.pylori的已知蛋白抗原中,尿素酶B亚单位(UreB)和粘附素(HpaA)是已经证实具有很强的抗原性和免疫保护力的抗原。本课题在本室成功筛选和鉴定UreB多个Th细胞表位的基础上,选用UreB三个优势性Th细胞表位(两个Th2,一个Th1表位)和文献报道UreB的一个B细胞表位、HpaA的一个B细胞表位以及分子内佐剂LTB,通过计算机辅助设计和分析对天然抗原表位进行分子设计、重组、优化、计算机分子结构模建等获得理论上最佳组合方式,按此组合方式构建了表达H.pylori五个表位多肽基因(HUepi)与ltB基因的表位疫苗融合蛋白,经抗原特性及各表位功能分析后,进行口服免疫治疗小鼠H.pylori感染的试验,观察H.pylori清除效果及相应的免疫应答过程,为H.pylori治疗性疫苗的设计与完善奠定基础。本课题的主要研究方法、结果与结论如下: 1.治疗性幽门螺杆菌表位疫苗的设计选用UreB三个优势性Th细胞表位(两个Th2,一个Th1表位)和文献报道UreB的一个B细胞表位、HpaA的一个B细胞表位以及分子内佐剂LTB,利用RANKPEP软件、DNAstar软件进行表位组合顺序分析和串联表位与分子内佐剂LTB之间连接linker的设计,获得理论上最佳组合方式,采用Robetta、Modeller和SPDB viewer软件进行表位融合蛋白三级结构的模建,获得理论上最接近天然状态下的三级结构。 2.治疗性幽门螺杆菌表位疫苗融合蛋白抗原的构建、表达、纯化按照第一部分的设计,在本室成功构建表位融合蛋白Uepi的基础上,分别克隆了表位多肽基因HUepi和ltB基因,采用重叠延伸PCR的方法将两段基因按照预先设计的方向通过linker连接起来,获得表位多肽与ltB的融合基因(HUepi-ltB),通过限制性内切酶NcoⅠ、XhoⅠ分别构建原核表达载体pET22b-HUepi-ltB、pET22b-HUepi和原核表达工程菌株pET22b-HUepi-ltB/BL21、pET22b-HUepi/BL21,经IPTG诱导高效表达分子量分别为20.7 kDa(HUepi-LTB)和8.3 kDa(HUepi)的表位融合蛋白,分别采用不同的纯化方案对重组表位融合蛋白HUepi-LTB和HUepi进行纯化与鉴定,纯化后HUepi-LTB和HUepi的蛋白纯度分别为95.2%和97.6%。 3.治疗性幽门螺杆菌表位疫苗融合蛋白抗原特性及各表位功能分析表位疫苗融合蛋白分别免疫家兔制备兔抗HUepi-LTB和HUepi抗血清,采用Western blot和ELISA检测表位疫苗融合蛋白特异性抗体与rUreB和rHpaA的免疫反应性及LTB组分GM1神经节苷脂的结合活性;通过尿素酶抑制实验、H.pylori与胃上皮细胞(GES-1)的粘附与粘附抑制实验,血凝与血凝抑制实验检测B细胞表位的作用;表位融合蛋白注射免疫小鼠,通过特异性淋巴细胞增殖实验检测Th细胞表位作用。结果表明:表位疫苗融合蛋白产生的特异性抗体能够与rUreB、rHpaA、rLTB反应,且融合蛋白中LTB组分保持了与GM1神经节苷脂结合活性;抗表位融合蛋白的特异性抗体在一定程度上能够抑制H.pylori尿素酶的活性,并可阻止H.pylori与胃上皮细胞的粘附和抑制H.pylori与红细胞的凝集反应;淋巴细胞增殖实验证实三个Th表位肽能刺激表位疫苗免疫小鼠的淋巴细胞发生特异性增殖(SI2),并且表位疫苗致敏的淋巴细胞在rUreB的刺激下可以增殖。实验证明表位融合蛋白具有良好的免疫原性、免疫反应性并且所包含的各表位发挥了各自的免疫效应。 4.治疗性幽门螺杆菌表位疫苗免疫治疗BALB/c小鼠H.pylori感染效果评价及机制探讨 1)表位疫苗治疗BALB/c小鼠H.pylori感染效果评价:采用H.pylori动物适应菌株SS1感染BALB/c小鼠,成功建立H.pylori感染动物模型,同时建立H.pylori定量培养方法,根据H.pylori 16S rRNA编码基因设计引物建立H.pylori感染的PCR检测方法。以表位融合蛋白HUepi-LTB和HUepi+rLTB每间隔7～10d,连续4次对感染H.pylori的BALB/c小鼠进行口服免疫治疗,于治疗38d后进行H. pylori定量培养检测、尿素酶实验、PCR检测评价表位疫苗的治疗效果。结果显示表位融合蛋白能够显著降低小鼠胃粘膜H.pylori的定植数量,清除率分别为64.2%和61.5%。 2)表位疫苗治疗BALB/c小鼠H.pylori感染保护机制探讨: H.pylori感染小鼠予以表位疫苗口服免疫治疗38d后,流式细胞术分析脾淋巴细胞亚群变化,淋巴细胞增殖实验检测脾和肠粘膜淋巴细胞的特异性增殖,ELISA检测脾和粘膜淋巴细胞细胞因子的分泌,RT-PCR检测脾和肠粘膜淋巴细胞IL-4和IFN-γmRNA表达水平,ELISA检测特异性抗体IgG、IgA的产生。结果显示:H.pylori超声上清可显著刺激表位疫苗免疫治疗小鼠脾淋巴细胞和肠粘膜淋巴细胞特异性增殖,与PBS组、佐剂组和单纯表位蛋白组差异显著(P0.01);流式细胞术分析脾淋巴细胞亚群显示CD4~+ T淋巴细胞增殖明显;疫苗作用下脾淋巴细胞和肠粘膜淋巴细胞分泌IFN-γ、IL-4水平较对照组显著升高(P0.01),RT-PCR结果显示脾和肠粘膜淋巴细胞IL-4和IFN-γmRNA表达水平明显增高;体液免疫应答检测结果显示H.pylori表位疫苗可刺激机体产生血清特异性IgG抗体,刺激肠液、唾液及粪便中特异性sIgA水平显著升高(P0.01)。因此,H.pylori表位疫苗口服免疫后可诱导产生局部粘膜和全身性体液免疫应答和Th1/Th2混合型细胞免疫应答,起到清除H.pylori感染的作用。
[Abstract]:Helicobacter pylori ( H . pylori ) is the main pathogenic bacterium of chronic gastritis and gastroduodenal ulcer . It is closely related to gastric cancer and gastric mucosa associated lymphoid tissue lymphoma . It has been classified as type I oncogenic factor by the World Health Organization .

In recent years , it is very difficult to design , modify and modify the epitope of H . pylori . The epitope vaccine has become a new direction for the development of vaccine designs such as infectious diseases , malignant tumors and autoimmune diseases .

The protective mechanism of H . pylori ( H . pylori ) infection is complicated . There is also a difference in the immune response mechanism of different types of H . pylori . In the aspect of cellular immunity , it has been proved that the protective mechanism of CD4 ~ + T cells is necessary for the immune protection of H.pylori .

In the known protein antigens of H.pylori , the urease B subunit ( UreB ) and adhesin ( HpaA ) have been proved to have strong antigenicity and immune protection .

1 . Design of therapeutic Helicobacter pylori epitope vaccine

Three dominant Th cell epitopes of UreB ( two Th2 , one Th1 epitope ) and one B cell epitopes of UreB , one B cell epitope of HpaA and internal adjuvant LTB were reported . Using RANKPEP software , DNAstar software was used to analyze the epitope combined sequence analysis and the linkage linker between tandem epitope and internal adjuvant LTB . The best combination was obtained . The three - stage structure of epitope fusion protein was obtained by using Robetta , Modeller and SPDB viewer software .

2 . Construction , expression and purification of therapeutic Helicobacter pylori epitope vaccine fusion protein antigen

The huepi - LTB and HUepi - Huepi - ltB / BL21 , pET22b - HUepi - ltB / BL21 , pET22b - HUepi - ltB / BL21 , pET22b - HUepi - ltB / BL21 , pET22b - HUepi - ltB / BL21 , pET22b - HUepi - ltB / BL21 , pET22b - HUepi - LTB and HUepi were respectively constructed . The purity of HUepi - LTB and HUepi were 95.2 % and 97.6 % respectively .

3 . Analysis of the antigenic properties of the epitope vaccine fusion protein and the functional analysis of each epitope

The anti - HUepi - LTB and HUepi antiserum were prepared in rabbits immunized with epitope vaccine . Western blot and ELISA were used to detect the binding activity of the specific antibody of epitope vaccine fusion protein with rUreB and rHpaA .

Evaluation and mechanism of Helicobacter pylori epitope vaccine in BALB / c mice infected with H . pylori infection

1 ) The effect of epitope vaccine on H . pylori infection in BALB / c mice was evaluated : H.pylori infected BALB / c mice were infected with H . pylori ( H . pylori ) , and H . pylori quantitative culture was established . The results showed that the epitope fusion protein could significantly reduce the number of H.pylori colonization in mice . The results showed that the epitope fusion protein could significantly reduce the number of H.pylori colonization in mice . The clearance rate was 64.2 % and 61.5 % , respectively .

2 ) The protective mechanism of epitope vaccine in the treatment of H . pylori infection in BALB / c mice :

After oral immunization with the epitope vaccine of H . pylori infected mice , the lymphocyte subsets were analyzed by flow cytometry , the specific proliferation of lymphocytes of spleen and intestinal mucosa was detected by lymphocyte proliferation assay , the secretion of cytokines in spleen and mucosal lymphocytes was detected by ELISA , the levels of IL - 4 and IFN - 纬 mRNA were detected by RT - PCR , and the production of IgG and IgA was detected by ELISA .

The results showed that H . pylori could significantly stimulate the specific proliferation of lymphocytes and lymphocytes in spleen lymphocytes and intestinal mucosa of mice immunized with epitope vaccine . The results showed that the levels of IL - 4 and IFN - 纬 mRNA in lymphocytes of spleen and intestinal mucosa were significantly higher than those in control group ( P0.01 ) . The results of humoral immune response showed that the levels of IL - 4 and IFN - 纬 mRNA in lymphocytes and intestinal mucosa increased significantly ( P0.01 ) .

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R392

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