表达幽门螺杆菌粘附素保守区的减毒鼠伤寒沙门菌的构建及其免疫治疗作用的研究
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摘要
背景／目的：幽门螺杆菌(Helicobacter pylori,Hp)感染是慢性活动性胃炎和消化性溃疡的主要病因，与胃腺癌、胃黏膜相关淋巴样组织(MALT)淋巴瘤的发生亦密切相关。临床实践证明，消化性溃疡愈合后Hp仍(+)者一年复发率达50％以上；根除Hp后不仅溃疡可以愈合，而且一年复发率可降至10％以下。此外，根除Hp后可以治愈MALT淋巴瘤。然而目前根除Hp的主要手段是抗菌疗法，尽管有较高的根除率，但存在诸如费用较高、耐药菌株的不断增加致根除率逐年降低、药物的副作用以及病人的依从性差等问题，并非理想的治疗方法。随着Doige等在1994年发现Hp疫苗的治疗作用后，免疫防治Hp成为治疗Hp最有希望的方法之一。鉴于Hp已证实的四种粘附素保守区(AB)是外膜蛋白(OMP)和膜孔素(porin)样成分，而外膜蛋白和膜孔素样成分是优秀的疫苗候选抗原。因此本研究的目的是：选择暴露于Hp表面的AB为抗原，首先通过基因工程技术将保守区基因克隆入原核表达载体并在大肠杆菌中表达，表达产物经过纯化后在体外探讨其安全性、免疫原性和生物活性；进一步将保守区基因克隆入减毒鼠伤寒沙门菌平衡-致死系统，在已感染Hp的小鼠模型中研究其免疫治疗效果，以及可能的免疫治疗机制，以确定AB及表达AB的减毒鼠伤寒沙门菌在Hp疫苗研制中的应用价值。
方法：(1)运用PCR技术从Hp总DNA中扩增出AB结构基因，装入pET-22b(+)载体进行序列及生物信息学分析，并在大肠杆菌BL21(DE3)中表达，采用金属螯合亲和层析(MCAC)的方法纯化表达产物AB，免疫印迹法检测其抗原性；(2)二苯胺(DPA)法和流式细胞术检测AB致人外周血淋巴细胞(PBL)凋亡的作用，体外评价AB的安全性；光镜计数法研究AB抗体对Hp与胃癌细胞粘附的影响；ELISA法测定Hp感染者血清中抗AB抗体，MTT法测定PBL对AB的增殖反应，体外评价AB对体液和细胞免疫的影响；(3)采用缺失腺苷酸环化酶(Δcya)、环腺苷酸受体蛋白基因(Δcrp)以及天冬氨酸β-半醛脱氢酶(Δasd)的鼠伤寒沙门菌
比4072）作为宿主，将编码 AB的基因插入 ASd”的组成型表达载体
PYA248，通过两次转化引入宿主菌，构建了表达AB基因平衡致死的减毒
鼠伤寒沙门重组菌X4072（PYA248－AB），采用桥联法ELISA测定X4072
（pYA248－AB）培养上清液和裂解上清液中 AB的抗原性，参照 Meacock
叙述的方法及重组菌的生长曲线的测定来确定重组菌株的稳定性，通过
C57BL巧小鼠口服测定半致死量来确定重组菌的安全性；④建立 Hp感
染小鼠模型，在该模型中评价 X4072（PYA248－AB）治疗 HP感染的效果，
运用细菌培养法观察HP根除率及定植量的改变，流式细胞术分析免疫后
小鼠脾细胞亚型，ILZ和 IL4细胞依赖株的细胞测活法检测细胞因子，
ELISA 法检测小鼠血清及肠液中抗AB抗体产生情况，探讨X4072
（PYA248－AB）可能的免疫治疗机制。
结果：＊）成功构建了AB的重组质粒pET22b卜＊／AB，序列分析显示
获得了一段588hP的基因，完整的包含了粘附素C－端的7个保守区。
Parker法和Welling法分析显示AB具有良好的抗原性和疏水性。进一
步应用 INTERNET EXPASY、NNPREDICT、ISREC等生物信息软件分析，发
现其为膜孔素样结构，是跨膜区膜外区域的重要表位组成部分。在
Genebank中BLAST分析了836767个蛋白序列，与其具有同源性者均为
HP外膜蛋白序列。蛋白电泳分析和凝胶扫描表明获得了高效表达AB的
克隆株，其分子量为22．shD，占菌体总蛋白的29．6儿 其中可溶性表达
占上清的21．9％，纯化后获得纯度达卯％的重组蛋白AB，免疫印迹显示
该蛋白可被AIPA兔抗血清和HP感染患者的血清抗体识别；狸）体外安全
性实验表明AB无明显调节PBL细胞表达FasL和致PBL细胞凋亡的作用；
ELISA法共检测了55份血清，同时以RUT作为平行对照，两法的评价判
断一致性（agreement）程度的指标卡帕系数（KaPPa值）为0．76，同
时，低剂量 AB即可刺激 HPPBL的增殖；AB抗血清能部分阻断 Hp与胃癌
细胞系的粘附，在光镜下表现为经抗AB兔血清预处理后，每细胞周围粘
附的细菌数较免疫前兔血清预处理组显著减少…m．05＼ ①成功构建
了表达AB 的减毒鼠伤寒沙门菌重组菌株S．tyPhimurium
X4072（pYA248－AB），桥联法ELISA测定表明重组菌X4072（PYA248－AB）
培养上清中AB的含量高于菌体裂解液，重组菌PYA248｛B在没有选择压
－2－
力的情况下培养100代，随机挑选的重组菌全部都能生长，且在ELISA
测定妞抗原时均显阳性。重组菌的生长曲线测定表明，X4072叩YA248）
和X4072（wAZ抡－AB）的生长状态基本一致；口服 重组菌株
X4072（PYA248－AB）1．OX 10“CfU．30天后，C57BL／6存活率仍为 100％。（4）
免疫治疗后疫苗治疗组根除率为53．3兄 显著高于其它两组…州．of人
未根除HP的小鼠，疫苗治疗组伽的定植密度明显低于其它两组
h州．of人应用流式细胞仪分析免疫小鼠脾CD4＊CDS＊淋巴细胞的比值
Backgroud/Objectives: Helicobacter pylori(H.pylori) infection is the major etiological factor of chronic active gastritis and most peptic ulcer disease, and is closely associated with gastric cancers tumors such as adenocarcinoma and MALT lymphoma. Clinic practice proved the peptic ulcer recurrence rate was above 50% one year after cure in patients infected with Hp, Eradication of Hp cured peptic ulcer and reduced the recurrence rate of one year to below 10%. In addition, Eradication of Hp was able to cure MALT lymphoma. Current therapies for eradicating H. pylori depend on the use of
combined antibiotics, although having relatively high eradication rate, the high cost, low patient compliance, side effect, and increasing of resistant strains make these therapies imperfect. In 1994, Doige et al discovered the treatment of Hp vaccination. Scince then, Immunization against Hp infection has been one of the most prospective treaments. Since the Hp adhesin conservation region(AB) is outer membrane protein and porin type component, while these two proteins are the excellent immunogen candidates of vaccination, we prepared AB as a target to obtain the recombinant AB by genetic engineering and evaluated
its safety, biological activity and immunogenicity in vitro, Furthmore, we constructed the attenuated Salmonella typhimurium strain expressing AB which was a balanced lethal recombinant and investigated its efficacy and the potential mechanism of treatment in mouse model infected with Hp. Finally the feasibility of AB and the attenuated Salmonella typhimurium strain expressing AB in H. pylori vaccination was determined.
Methods: (l)Gene ab which was amplified from Hp chromosomal
DNA by PCR technique, was sequenced and the biological information was analysised. Then it was cloned into the expression vector pET-22b ( + ) and expressed in E. coli BL2KDE3). The recombinant protein(rAB) was purified with affinity chromatography. Antigenicity of rAB was identified by Western blot. (2) Flow cytometry was used to evaluate the increase in FasL expression on T cells under the stimulation of Hp AB,T cell apoptosis induced by AB was detected by DAP assay .The effect of anti-AB serum on Hp binding of human gastric carcinoma cell lines was determined by light microscopy. ELISA assay was used to measure AB-specific antibody in serum of Hp infected patients. The proliferation of peripheral blood lymphocytes(PBL) in response to rAB was examined by MTT test. (3) The ab gene was amplified by PCR and inserted into the expression vector pYA248 containing asd gene and was introduced into the delta Cya,delta Crp,delta Asd attenuated Salmonella typhimurium strain that is a balanced lethal recombinant by twice transformations. ELISA assay was used to measure AB expressed in sonicate and culture supernatant. According to Meacock's way and growth curve, stability of the recombinant is evaluated. Semi-lethal capacity test was used to evaluate the safty of recombinant. (4) H. pylori infected mouse model was established and applied in oral vaccination. The density of bacterial colonization was determined by the semi-quantitative bacterial culture assay. The T cell subsets of spleenocytes were assayed by flow cytometry (FCM) after immunization. IL-2 and IL-4 were detected through MTT. Mice sera and intestinal fluid were separatly tested for AB-specific IgG and IgA by ELISA.
Results: (1) The recombinant plasmid pET-22b(+)/AB was succefully constructed.DNA sequencing showed one open reading frame with the length of 588 bp.lt encoded 7 conservative regions that showed good antigencity and hydrophobicity by Parker and Welling method. Furthmore, INTERNET ExPASY, NNPREDICT and ISREC predicted it was a porin like structure consisting of 3 -pleated sheets, that were embeded in the outer membrane. BLAST analysed 836767 protein sequences and found the similar sequences were all belong to Hp OMP sequences. SDS-PAGE and scan analysis showed
the molecular weight of AB was 22.5kD and recombinant protein amounted to 29% of the total bacterial protein
方法：(1)运用PCR技术从Hp总DNA中扩增出AB结构基因，装入pET-22b(+)载体进行序列及生物信息学分析，并在大肠杆菌BL21(DE3)中表达，采用金属螯合亲和层析(MCAC)的方法纯化表达产物AB，免疫印迹法检测其抗原性；(2)二苯胺(DPA)法和流式细胞术检测AB致人外周血淋巴细胞(PBL)凋亡的作用，体外评价AB的安全性；光镜计数法研究AB抗体对Hp与胃癌细胞粘附的影响；ELISA法测定Hp感染者血清中抗AB抗体，MTT法测定PBL对AB的增殖反应，体外评价AB对体液和细胞免疫的影响；(3)采用缺失腺苷酸环化酶(Δcya)、环腺苷酸受体蛋白基因(Δcrp)以及天冬氨酸β-半醛脱氢酶(Δasd)的鼠伤寒沙门菌
比4072）作为宿主，将编码 AB的基因插入 ASd”的组成型表达载体
PYA248，通过两次转化引入宿主菌，构建了表达AB基因平衡致死的减毒
鼠伤寒沙门重组菌X4072（PYA248－AB），采用桥联法ELISA测定X4072
（pYA248－AB）培养上清液和裂解上清液中 AB的抗原性，参照 Meacock
叙述的方法及重组菌的生长曲线的测定来确定重组菌株的稳定性，通过
C57BL巧小鼠口服测定半致死量来确定重组菌的安全性；④建立 Hp感
染小鼠模型，在该模型中评价 X4072（PYA248－AB）治疗 HP感染的效果，
运用细菌培养法观察HP根除率及定植量的改变，流式细胞术分析免疫后
小鼠脾细胞亚型，ILZ和 IL4细胞依赖株的细胞测活法检测细胞因子，
ELISA 法检测小鼠血清及肠液中抗AB抗体产生情况，探讨X4072
（PYA248－AB）可能的免疫治疗机制。
结果：＊）成功构建了AB的重组质粒pET22b卜＊／AB，序列分析显示
获得了一段588hP的基因，完整的包含了粘附素C－端的7个保守区。
Parker法和Welling法分析显示AB具有良好的抗原性和疏水性。进一
步应用 INTERNET EXPASY、NNPREDICT、ISREC等生物信息软件分析，发
现其为膜孔素样结构，是跨膜区膜外区域的重要表位组成部分。在
Genebank中BLAST分析了836767个蛋白序列，与其具有同源性者均为
HP外膜蛋白序列。蛋白电泳分析和凝胶扫描表明获得了高效表达AB的
克隆株，其分子量为22．shD，占菌体总蛋白的29．6儿 其中可溶性表达
占上清的21．9％，纯化后获得纯度达卯％的重组蛋白AB，免疫印迹显示
该蛋白可被AIPA兔抗血清和HP感染患者的血清抗体识别；狸）体外安全
性实验表明AB无明显调节PBL细胞表达FasL和致PBL细胞凋亡的作用；
ELISA法共检测了55份血清，同时以RUT作为平行对照，两法的评价判
断一致性（agreement）程度的指标卡帕系数（KaPPa值）为0．76，同
时，低剂量 AB即可刺激 HPPBL的增殖；AB抗血清能部分阻断 Hp与胃癌
细胞系的粘附，在光镜下表现为经抗AB兔血清预处理后，每细胞周围粘
附的细菌数较免疫前兔血清预处理组显著减少…m．05＼ ①成功构建
了表达AB 的减毒鼠伤寒沙门菌重组菌株S．tyPhimurium
X4072（pYA248－AB），桥联法ELISA测定表明重组菌X4072（PYA248－AB）
培养上清中AB的含量高于菌体裂解液，重组菌PYA248｛B在没有选择压
－2－
力的情况下培养100代，随机挑选的重组菌全部都能生长，且在ELISA
测定妞抗原时均显阳性。重组菌的生长曲线测定表明，X4072叩YA248）
和X4072（wAZ抡－AB）的生长状态基本一致；口服 重组菌株
X4072（PYA248－AB）1．OX 10“CfU．30天后，C57BL／6存活率仍为 100％。（4）
免疫治疗后疫苗治疗组根除率为53．3兄 显著高于其它两组…州．of人
未根除HP的小鼠，疫苗治疗组伽的定植密度明显低于其它两组
h州．of人应用流式细胞仪分析免疫小鼠脾CD4＊CDS＊淋巴细胞的比值
Backgroud/Objectives: Helicobacter pylori(H.pylori) infection is the major etiological factor of chronic active gastritis and most peptic ulcer disease, and is closely associated with gastric cancers tumors such as adenocarcinoma and MALT lymphoma. Clinic practice proved the peptic ulcer recurrence rate was above 50% one year after cure in patients infected with Hp, Eradication of Hp cured peptic ulcer and reduced the recurrence rate of one year to below 10%. In addition, Eradication of Hp was able to cure MALT lymphoma. Current therapies for eradicating H. pylori depend on the use of
combined antibiotics, although having relatively high eradication rate, the high cost, low patient compliance, side effect, and increasing of resistant strains make these therapies imperfect. In 1994, Doige et al discovered the treatment of Hp vaccination. Scince then, Immunization against Hp infection has been one of the most prospective treaments. Since the Hp adhesin conservation region(AB) is outer membrane protein and porin type component, while these two proteins are the excellent immunogen candidates of vaccination, we prepared AB as a target to obtain the recombinant AB by genetic engineering and evaluated
its safety, biological activity and immunogenicity in vitro, Furthmore, we constructed the attenuated Salmonella typhimurium strain expressing AB which was a balanced lethal recombinant and investigated its efficacy and the potential mechanism of treatment in mouse model infected with Hp. Finally the feasibility of AB and the attenuated Salmonella typhimurium strain expressing AB in H. pylori vaccination was determined.
Methods: (l)Gene ab which was amplified from Hp chromosomal
DNA by PCR technique, was sequenced and the biological information was analysised. Then it was cloned into the expression vector pET-22b ( + ) and expressed in E. coli BL2KDE3). The recombinant protein(rAB) was purified with affinity chromatography. Antigenicity of rAB was identified by Western blot. (2) Flow cytometry was used to evaluate the increase in FasL expression on T cells under the stimulation of Hp AB,T cell apoptosis induced by AB was detected by DAP assay .The effect of anti-AB serum on Hp binding of human gastric carcinoma cell lines was determined by light microscopy. ELISA assay was used to measure AB-specific antibody in serum of Hp infected patients. The proliferation of peripheral blood lymphocytes(PBL) in response to rAB was examined by MTT test. (3) The ab gene was amplified by PCR and inserted into the expression vector pYA248 containing asd gene and was introduced into the delta Cya,delta Crp,delta Asd attenuated Salmonella typhimurium strain that is a balanced lethal recombinant by twice transformations. ELISA assay was used to measure AB expressed in sonicate and culture supernatant. According to Meacock's way and growth curve, stability of the recombinant is evaluated. Semi-lethal capacity test was used to evaluate the safty of recombinant. (4) H. pylori infected mouse model was established and applied in oral vaccination. The density of bacterial colonization was determined by the semi-quantitative bacterial culture assay. The T cell subsets of spleenocytes were assayed by flow cytometry (FCM) after immunization. IL-2 and IL-4 were detected through MTT. Mice sera and intestinal fluid were separatly tested for AB-specific IgG and IgA by ELISA.
Results: (1) The recombinant plasmid pET-22b(+)/AB was succefully constructed.DNA sequencing showed one open reading frame with the length of 588 bp.lt encoded 7 conservative regions that showed good antigencity and hydrophobicity by Parker and Welling method. Furthmore, INTERNET ExPASY, NNPREDICT and ISREC predicted it was a porin like structure consisting of 3 -pleated sheets, that were embeded in the outer membrane. BLAST analysed 836767 protein sequences and found the similar sequences were all belong to Hp OMP sequences. SDS-PAGE and scan analysis showed
the molecular weight of AB was 22.5kD and recombinant protein amounted to 29% of the total bacterial protein
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