重组人幽门螺杆菌尿素酶B亚单位及其生物学性质

目的基因重组人幽门螺杆菌(Hp)尿素酶B亚单位(UreB)并分析重组蛋白的生物学性质。方法采用PCR从我国临床分离的Hp菌株中克隆UreB基因,并通过DNA重组技术构建非融合表达UreB的重组质粒pET-11C-UreB,转化至大肠杆菌BL21(DE3),经IPTG诱导表达。结果重组UreB在大肠杆菌中表达,相对分子质量约62 000,表达率26%,N端15个氨基酸序列为MKKISRKEYVSMYGP,肽图及氨基酸组成成分分析结果与理论预测值吻合。以重组UreB免疫BalB/c小鼠,能产生抗UreB抗体。ELISA及Western blotting分析显示,重组UreB具有良好的免疫原性和反应性,表现出与天然Hp UreB相似的生物学功能。结论为重组UreB作为Hp亚单位疫苗成分奠定了免疫学基础。     

重组人幽门螺杆菌尿素酶B亚单位的免疫学性质研究

目的 分析重组幽门螺杆菌尿素酶B亚单位的免疫学性质。方法 重组尿素酶B亚单位注射免疫BALB／c小鼠、家兔，双向琼脂扩散、ELISA、免疫印迹分析特异抗体的产生及性质。结果 重组尿素酶B亚单位能有效刺激机体的免疫应答，且产生的抗体能与天然幽门螺杆菌发生抗原抗体反应。结论 重组尿素酶B亚单位表现出天然幽门螺杆菌尿素酶B亚单位的免疫学性质，提示可作为幽门螺杆菌特异性抗体检测的包被抗原及幽门螺杆菌疫苗的有效成分。     

过氧化氢酶和尿素酶B亚单位二价疫苗预防小鼠幽门螺杆菌感染

目的 利用人类幽门螺杆菌(H.priori)感染的小鼠模型研究过氧化氢酶和尿素酶B亚单位二价疫苗预防H.pylori感染的作用.方法 把实验动物无特定致病菌C57BL/6小鼠分成7组,分别通过灌胃方法给予过氧化氢酶和尿素酶B亚单位(各100μg)加霍乱毒素(CT)(2 μg)、过氧化氢酶(100 μg)加CT(2 μg)、尿素酶B亚单位(100 μg)加CT(2 μg)、PBS、单纯过氧化氢酶(100μg)、单纯尿素酶B亚单位(100μg)、单纯CT(2μg),共4次.2周后再用活H.pylori灌胃,再4周后处死动物.取胃粘膜行半定量细菌培养检查H. pytori情况.结果 实验各组H.pylori完全保护率分别为:过氧化氢酶和尿素酶B亚单位加CT组83.3%(20/24)、过氧化氢酶加CT组41.7%(10/24)、尿素酶B亚单位加CT组54.2%(13/24);生理盐水组、单纯过氧化氢酶、单纯尿素酶B亚单位、单纯CT组根除率均为0%.未完全保护的小鼠,疫苗组H. pylori的定植密度明显低于其它4组(P<0.05).此外,二价疫苗组的H. pylori完全保护率及定植密度较单价疫苗组均有显著性差异(P<0.05).结论 由过氧化氢酶和尿素酶B亚单位加免疫佐剂组成的二价口服疫苗较单价口服疫苗有更好的免疫预防效果.     

幽门螺杆菌基因重组尿素酶亚单位免疫预防作用的实验研究

目的 评价幽门螺杆菌（Ｈｐ）基因重组尿素酶Ａ、Ｂ亚单位（ｒＵｒｅＡ、ｒＵｒｅＢ）的免疫预防作用。方法 将ＢＡＬＢ／Ｃ小鼠分为如下７组：阴性对照组（１０只）、霍乱毒素Ｂ亚单位（ＣＴＢ）对照组（１０只）、ｒＵｒｅＡ对照组（１０只）、ｒＵｒｅＢ对照组（１０只）、ｒＵｒｅＡ＋ＣＴＢ实验组（３０只）、ｒＵｒｅＢ＋ＣＴＢ实验组（３０只）、Ｈｐ超声粉碎物＋ＣＴＢ实验组（３０只）,胃内分别接种如下物质：１５０ｍｌ     

幽门螺杆菌尿素酶B亚单位优势Th表位的系统筛选与鉴定

目的 筛选幽门螺杆菌(Helicobacter pylori,Hp)尿素酶B亚单位(UreB)中CD4+细胞优势应答表位,并确定其MHC限制性.方法 以重组UreB蛋白(rUreB)与弗氏佐剂联合皮下多点注射免疫BALB/c小鼠,从小鼠脾脏中体外扩增UreB特异性T淋巴细胞,采用步移重叠合成肽法筛选CD4+T细胞优势应答表位,分析其MHC分子限制性.结果 18mer短肽筛选发现UreB403-420和UreB409-426可显著刺激UreB特异性CD4+T细胞产生IFN-γ.13mer短肽鉴定实验结果显示,UreB409-421可刺激产生与18mer短肽UreB403 420和UreB409-426等同的应答强度.同时抗体阻断实验表明,抗H-2d (I-A)单克隆抗体能有效阻断该表位的应答.结论 UreB403-420和UreB409-426为UreB抗原中的优势Th表位,其刺激免疫反应的核心位置为UreB409-421,且存在H-2d(I-A)限制性.     

BalB/c小鼠口服重组幽门螺杆菌尿素酶B亚单位的免疫应答

目的：评价重组尿酶B亚单位（rUreB）作为幽门螺杆菌（Hp)口服疫苗成份的效果、探讨Hp的免疫保护机制。方法：采用重组Hp保护性抗原UreB与粘膜免疫佐剂霍乱毒素B亚单位（CTB）共口服免疫BalB/c小鼠，ELISA分析血清、唾液和粪便、胃粘液中IgG、IgA抗体水平，体外培养脾淋巴细胞在Hp刺激下的增殖情况及IL－4、IFN－γ的变化。结果：BalB/c小鼠口服rHreB和CTB混合制剂能有效激发机体产生全身和局部特异性的体液免疫应答和细胞免疫应答。结论：rUreB可以作为Hp疫苗成分用于Hp感染的预防和治疗。     

幽门螺杆菌尿素酶B亚单位功能片段的纯化及活性研究

目的 建立一种有效的尿素酶功能片段的纯化方法.方法 应用已构建的尿素酶B功能片段表达载体,IPTG诱导表达,包涵体鉴定试验判断目的蛋白表达形式,AKTA100上分别采用亲和层析和离子层析等多级纯化方式优化纯化条件,HPLC检测纯化蛋白纯度,尿素酶活性阻断试验对纯化后功能片段进行活性鉴定.结果 IPTG诱导后目的蛋白高表达,包涵体鉴定试验证实目的蛋白以可溶形式存在宿主细胞中,优化后方案得到目的蛋白纯度＞90%且能被尿素酶B免疫的兔血清识别,纯化后蛋白免疫家兔后产生兔抗血清能够特异性中和Hp尿素酶活性.结论 成功建立了尿素酶功能片段的纯化方法.     

幽门螺杆菌尿素酶B亚单位功能片段构建及免疫原性研究

目的 构建表达免疫活性的尿素酶功能片段原核表达体系.方法 根据尿素酶B基因序列以及文献报道的尿素酶活性中心区域,primer5.0设计相应的引物,以Hp基因组为模版,PCR扩增拟表达的尿素酶活性片段,经测序证实后将片段亚克隆于原核表达载体pET28中,转入宿主茵BL21后IPTG诱导表达后,Tris-Tricine PAGE电泳及抗Hp特异性抗体western-tbollting鉴定目的蛋白表达.结果 PCR从Hp基因组中扩增出414bp目的片段,经酶切及测序证实构建了功能片段表达阳性重组子,命名为pU414,IPTG诱导后电泳分析可以发现相应分子量附近出现可疑目的蛋白,能够被特异性血清识别.结论 成功建立尿素酶活性功能片段蛋白表达载体,为下一步实验研究奠定基础.     

幽门螺杆菌尿素酶B亚单位基因在减毒鼠伤寒杆菌中的表达

目的：制备能表达幽门螺杆菌（Ｈｐ）尿素酶Ｂ亚单位（ＵｒｅＢ）的减毒鼠伤寒杆菌,初步确定是其否可用作抗ＨＰ的口服疫苗。方法应用ＰＣＲ扩增ＨＰＵｒｅＢ基因片段,测序后克隆入原核表达载体ＰＴｃ０１,转化ＬＢ５０００修饰后再转化ＳＬ３２６１,得到重组的减毒鼠伤寒杆菌ＳＬ３２６１／ＰＴｃ０１－ＵｒｅＢ。应用抗ＨＰ菌体蛋白兔血清行Ｗｅｓｔｅｒｎ－ｂｌｏｔ检测ＵｒｅＢ在ＳＬ３２６１中的表达,ＳＬ３２６１／ＰＴｃ     

幽门螺杆菌尿素酶B基因的克隆、表达及其抗原性的鉴定

目的构建含人幽门螺杆菌(Helicobacterpylori,Hp)尿素酶B(UreaseB,UreB)编码基因的重组质粒,测定、分析其核苷酸序列,并在E.coliTop10中表达,研究其抗原性。方法应用PCR技术从HpDNA染色体中扩增UreB编码基因片段,将其T-A克隆和测序,并与GenBank公布的其它Hp菌株基因序列比较,再将目的基因克隆至表达载体pGEX-4T-1中进行表达,用GST亲和层析对其进行纯化,纯化产物用于对29株小鼠抗Hp-全菌单克隆抗体(mAb)的鉴定及与Hp感染患者血清进行Westernblot。结果扩增的UreB基因全长1704bp,并在GenBank上登录(No.DQ141576),与GenBank公布的其它Hp菌株的核酸同源性为97%～99%,表达的UreB融合蛋白的相对分子量为91000。29株小鼠抗Hp-全菌mAb中有6株是针对UreB抗原的,纯化产物可被病人血清和抗UreB的鼠单克隆抗体识别。结论重组UreB具有较好的抗原性,为Hp检测诊断和疫苗的研究奠定了基础。     

幽门螺杆菌UreB核酸疫苗的构建

目的:构建含幽门螺杆菌(Helicobacter pylori,Hp)尿素酶B亚单位(UreB)基因的核酸疫苗.方法:抽提Hp标准菌株CCUG17874基因组DNA,应用PCR技术从基因组DNA扩增UreB基因,克隆入PUCmT载体,检测UreB基因序列.经过一系列酶切、连接反应将其克隆入真核表达载体pIRES,转入感受态大肠杆菌DH5α,筛选阳性克隆,通过PCR和酶切反应进行鉴定.通过脂质体法将构建好的重组载体pIRES-UreB转染COS-7细胞,Western印迹分析检测pIRES-UreB表达UreB蛋白的免疫原性.结果:扩增出长约1 700 bp的UreB基因,与基因库Hp UreB序列一致,PCR和酶切鉴定结果证实成功构建了含UreB基因的Hp核酸疫苗pIRES-UreB,并且Western 印迹分析检测到特异性的蛋白条带.结论:构建了具有免疫反应性UreB基因的Hp核酸疫苗,为进一步探索其免疫作用奠定了基础.     

Oral immunization of mice with attenuated Salmonella typhimurium expressing Helicobacter pylori urease B subunit

Objective To establish attenuated Salmonella typhimurium producing Helicobacter pylori (H. pylori) urease subunit B （UreB） and determine whether it could be used as an oral vaccine against H. pylori. Methods H. pylori (SS1 strain) UreB gene fragment amplified by PCR was cloned into the prokaryotic expression vector pTC01 after sequencing, and then transformed into attenuated Salmonella typhimurium SL3261 to acquire SL3261/pTC01- UreB. The expression of H. pylori UreB in SL3261 was detected by Western blot. Twelve weeks after oral immunization of mice,antibody responses were evaluated using serum and intestinal fluid by ELISA assay. Interferon- γ （IFN- γ） and interleukin- 10 （IL- 10） in the supernatant of spleen cells culture were also assessed by ELISA. In vitro stability of pTC01- UreB plasmid in SL3261 was confirmed by growing in Luria Broth (LB) medium to 80 generations.Results The UreB gene fragment amplified by PCR was consistent with the sequence of the H. pylori UreB as evidenced by sequence analysis. Enzyme digestion revealed that the correct pTC01- UreB was obtained. Western blot showed that a 61kDa protein was expressed in SL3261/pTC01- UreB, which could be recognized by anti- H. pylori UreB antiserum. After 80 generations of continuous culture, the recombinant plasmid pTC01- UreB was stable in SL3261 and had no obvious toxicity. Multiple oral immunizations with SL3261/pTC01- UreB could significantly induce H. pylori- specific mucosal IgA response as well as serum IgG response. Moreover, there were significant increases of IFN- γand IL- 10 in the SL3261/pTC01- UreB group. Finally, no obvious side effects for mice and no change in gastric inflammation were observed.Conclusion Attenuated Salmonella typhimurium expressing H. pylori UreB may be used as oral vaccine against H. pylori infection.     

Construction and identification of recombinant attenuated Salmonella typhimurium vaccine strain expressing Helicobacter pylori urease subunit B

Objective To construct a recombinant live attenuated Salmonella typhimurium vaccine strain expressing Helicobacter pylori urease subunit B (ureB).Methods ureB gene was amplified by PCR and cloned into a prokaryotic expression plasmid pTrc99a, and the identified recombinant plasmid was then used to transform an attenuated Salmonella typhimurium vaccine strain SL3261. The ureB expressed in the recombinant vaccine strain was analyzed by SDS-PAGE and optical density scanning. Two and 10 days after recombinant strain intragastric immunization, the C57BL/6 mice were sacrificed, and the spleens and terminal ileums were cultured.Results The ureB gene could be amplified from the recombinant prokaryotic expression plasmid pTrc99A-ureB and the plasmids extracted from transformed SL3261 strain. SDS-PAGE and optical density scanning indicated that ureB was expressed in the recombinant vaccine strain SL3261 (pTrc99A-ureB) as a protein with 66*!kD of molecular weight. Recombinant strain was found in both spleen an terminal ileum of each mouse two and ten days after intragastric immunization.Conclusions A recombinant liver attenuated Salmonella typhimurium vaccine strain expressing Helicobacter pylori ureB was constructed and identified, and this study will help to develop an oral recombinant live vaccine against Helicobacter pylori infection.     

携带幽门螺杆菌尿素酶B亚单位的重组减毒鼠伤寒沙门菌核酸疫苗的构建

目的 构建含幽门螺杆菌(Hp)尿素酶B亚单位(ureB)基因重组减毒鼠伤寒沙门菌核酸疫苗。方法 抽提Hp标准菌株CCUG17874基因组DNA,应用聚合酶链反应(PCR)技术从基因组DNA扩增ureB基因,克隆入pucmT载体,检测ureB基因序列,经过酶切、连接反应将其克隆入真核表达载体pIRES,转入感受态大肠杆菌DH5α,筛选阳性克隆,通过PCR和酶切反应进行鉴定。重组载体pIRES-ureB转入减毒鼠伤寒沙门菌LB5000,抽提质粒,再次转入SL7207,反复传代,鉴定重组核酸疫苗菌的稳定性。通过脂质体法将构建好的重组载体pIRES-ureB转染COS-7细胞,SDS-PAGE Western印迹法检测pIRES-ureB表达蛋白的免疫原性。结果 扩增出长约1700bp的ureB基因,测序结果表明扩增出的ureB基因与基因库Hp ureB序列一致,PCR和酶切鉴定结果证实ureB基因克隆人真核表达载体pIRES,并成功构建了Hp ureB基因的减毒鼠伤寒沙门菌核酸疫苗,重组核酸疫苗稳定,并且Westem印迹法检测到特异性的蛋白条带。结论 构建了具有免疫反应性的Hp UreB减毒鼠伤寒沙门菌核酸疫苗,为进一步探索其体内的免疫作用奠定了基础。     

Oral immunization of mice with vaccine of attenuated Salmonella typhimurium expressing Helicobacter pylori urease B subunit

Objective To prepare the live recombinant vaccine of attenuated Salmonella typhimurium SL3261 expressing Helicobacterpylori （H. pylori） B subunit （UreB） and to determine whether it could be used as an oral vaccine against H. pylori infection. Methods Using genomic DNA of H. pylori Sydney strain （SS1） as template, the H. pylori UreB gene fragment was amplified by PCR and subcloned into the expression vector pTC01. The recombinant plasmid pTC01-UreB was then transferred into LBS000 to obtain modified forms, and further conversed into the attenuated Salmonella typhimurium SL3261 to obtain recombinant SL3261/pCT01-UreB as an oral immunization reagent, which was then used to orally immunize Balb/c mice twice at a three-week interval. Twelve weeks later, anti-UreB IgA antibodies in intestinal fluid and IgG antibodies in sera were determined by ELISA. The relating data in control groups （including body weight, gastric inflammation, etc.） were also collected. Results The sequencing analysis showed that the UreB gene fragment amplified by PCR was consistent with the sequence of the H. pylori UreB gene. The restriction enzyme digestion revealed that the correct pTC01-UreB was obtained. SDS-PAGE and Western blot showed that a 61KD protein was expressed in SL3261/pTC01-UreB, which could be recognized by anti-H, pylori UreB antiserum and was absent in the control containing only Salmonella typhimurium SL3261 strain. The multiple oral immunization with SL3261/pTC01-UreB could significantly induce H. pylori specific mucosal IgA response as well as serum IgG responses. IFN-T and IL-10 levels were significantly increased in SL3261/pTC01-UreB group, and no obvious side effect and change in gastric inflammation were observed. Conclusion The attenuated vaccine of Salmonella typhimurium expressing H. pylori UreB can be used as an oral vaccine against H. pylori infection.     

幽门螺杆菌尿素酶减毒鼠伤寒杆菌疫苗诱导小鼠黏膜免疫应答的研究

目的研究表达幽门螺杆菌((H．pylori)尿素酶B亚单位(UreB)的减毒鼠伤寒杆菌活菌重组疫苗口服免疫Balb／c小鼠后的黏膜免疫应答状况。方法将已构建成功的表达UreB的重组减毒鼠伤寒杆菌SL3261／pTC01-UreB口服免疫Balb／c小鼠，12周后检测肠液和血清中的特异性抗体反应。结果疫苗组小鼠的肠液和血清中可分别检测到针对UreB的特异性抗体IgA和IgG，病理学检查显示疫苗组小鼠较对照组小鼠胃黏膜炎症程度的差异无统计学意义。结论表达H．pylori UreB的减毒鼠伤寒杆菌SL3261／pTC01-ureB能够诱导小鼠产生抗H．pylori的黏膜免疫，可用作抗H．pylori感染的口服疫苗。