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1.
目的:构建表达幽门螺杆菌(Helicobacter pylori,H.pylori)尿素酶B亚单位(UreB)减毒鼠伤寒沙门氏疫苗菌,研究其对小鼠抗H.pylori的免疫保护作用。方法:用PCR扩增ureB,将其克隆入高效原核表达质粒pTrc99A,进行基因测序,重组质粒鉴定后导入减毒鼠伤寒沙门氏菌SL3261。用SDS聚丙烯安凝胶电泳、Western blot和薄层扫描进行目的蛋白表达分析。C57BL/6小鼠用重组菌免疫,4周后用H.pyloriSS1攻击,再4周后处死小鼠,取胃做快速尿素酶试验和H.pylori定量培养,对照观察免疫效果。结果:构建了携带ureB的重组原核表达质粒pTrc99A-ureB,并将它成功转化了减毒鼠伤寒沙门氏菌SL3261。重组菌SL3261(pTrc99A-ureB)表达了约66ku的UreB。与对照组相比,重组菌免疫组H.lpylori定植水平明显下降(P<0.05)。结论:构建了表达H.pylori UreB的重组减毒鼠伤寒沙门疫苗菌,该菌株对C57BL/6有免疫保护作用。  相似文献   

2.
目的构建表达幽门螺杆菌(Hp)过氧化氢酶的无抗性减毒鼠伤寒沙门氏菌疫苗。方法采用缺失腺苷酸环化酶(△cya)、环腺苷酸受体蛋白(△crp)及天门冬氨酸β-半醛脱氢酶(△asd)的鼠伤寒沙门菌(X4072)作为宿主,将编码过氧化氢酶的基因CAT插入Asd 的组成型表达载体pYA248,通过两次转化引入宿主菌,构建表达过氧化氢酶基因平衡致死的减毒鼠伤寒沙门重组菌X4072(pYA248-CAT)。采用桥联法ELISA测定X4072(pYA248-CAT)培养上清液和裂解上清液中过氧化氢酶的抗原性,参照Meacock的方法及重组菌生长曲线的测定来确定重组菌株的稳定性,通过C57BL/6小鼠口服测定半致死量来确定重组菌的安全性。结果成功构建了表达过氧化氢酶的减毒鼠伤寒沙门菌重组菌株X4072(pYA248-CAT)。桥联法ELISA测定表明重组菌X4072(pYA248-CAT)培养上清中过氧化氢酶的含量高于菌体裂解液;重组菌pYA248-CAT在没有选择压力的情况下培养25代,随机挑选的重组菌全部都能生长,且在ELISA测定过氧化氢酶抗原时均显阳性;重组菌的生长曲线测定表明,X4072(pYA248)和X4072(pYA248-CAT)的生长状态基本一致。口服重组菌株X4072(pYA248-CAT) 1.0×1010 cfu,30 d后C57BL/6存活率仍为100%。结论成功构建了表达过氧化氢酶的无抗性的减毒鼠伤寒沙门菌疫苗X4072(pYA248-CA  相似文献   

3.
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。  相似文献   

4.
目的 探讨表达幽门螺杆菌(Hp)过氧化氢酶(KatA)的减毒鼠伤寒沙门氏菌疫苗株在防御Hp感染中的作用。方法 构建表达KatA的重组质粒,用IPTG进行诱导表达,再将其转入减毒鼠伤寒沙门氏菌SL3261株中构建成口服活疫苗株,经口服免疫C57BL/6小鼠,并以Hp翻尼株进行攻击,用快速尿素酶试验和Hp定量培养对胃粘膜中的Hp进行检测。结果 SDS-PAGE凝胶电泳上显示一条相对分子质量约79000的新生蛋白带,占细菌总蛋白的19%,并能与抗谷胱甘肽-s-转移酶(GST)抗体发生特异性反应,动物实验结果显示;免疫小鼠能有效防御Hp的感染,结论 表达KatA的减毒沙门氏菌疫苗株能诱导抗Hp保护性免疫反应,有望在Hp感染及其相关性疾病的防治中发挥积极作用。  相似文献   

5.
【目的】采用减毒鼠伤寒沙门菌S .typhimuriumSL32 6 1(pET 17b/A)防龋疫苗经不同途径免疫定菌大鼠 ,观察其对变形链球菌在大鼠牙面黏附、龋病发生的影响等免疫防龋效果。【方法】将 5 0只大鼠随机分组 ,经口服、皮下、黏膜下途径免疫 ,接种变形链球菌并给予致龋饲料。对各组大鼠口腔中变形链球菌的定居菌落进行计数、黏附率计算 ,收集大鼠颌骨标本 ,根据Keyes评分标准作龋齿计分统计。【结果】口服活菌苗 S typhimuriumSL32 6 1(pET 17b/A)组变形链球菌数为(3 4± 1 4)× 10 4 CFU ;磨牙龋齿Keyes计分总和为 30 7± 6 0 ,均明显低于其它组 (P <0 0 5 )。【结论】口服S typhimuriumSL32 6 1(pET 17b/A)活菌苗可抑制变形链球菌黏附于牙面 ,并能减少龋病发生 ,具有较好的防龋效果。  相似文献   

6.
OBJECTIVE: To construct a non-resistant attenuated Salmonella typhimurium (S.typhimurium) strain capable of expressing Helicobacter pylori (Hp) catalase. METHODS: After PCR amplification, the gene fragment encoding Hp catalase was inserted into the expression vector pYA248 containing asd gene, and the recombinant vector was then introduced into the host S.typhimurium strain X4072 depleted of genes encoding adenylate cyclase (delta cya), cyclic adenosine monophosphate receptor protein (delta crp) and aspartate-beta-semialdehyde dehydrogenase (delta asd). Bridged enzyme-linked immunosorbent assay (ELISA) was employed to measure the antigenicity of the catalase expressed in the sonicate and culture supernatant. According to Meacock's method and with the assistance of the growth curve, the stability of the recombinant strain was evaluated. A half lethal oral dose test was conducted to evaluate the safety of recombinant strain. RESULTS: S.typhimurium X4072 (pYA248-CAT) with expected capacity was successfully constructed, and bridged ELISA demonstrated higher catalase levels in the culture supernatant than in the sonicate of the recombinant strain X4072 (pYA248-CAT). After the strain was passaged for 100 generations without selection pressure, all the randomly selected colony of the recombinant strain grew well with positive catalase antigenicity as identified by ELISA. The growth curve of the recombinant strain showed comparable growth status of the 2 strains X4072 (pYA248) and X4072 (pYA248-CAT). The survival rate of C57BL/6 mice was 100% 30 d after oral administration of 1.0x10(10) cfu X4072 (pYA248-CAT). CONCLUSION: Non-resistant S. typhimurium vaccine X4072 (pYA248- CAT) is constructed successfully, which is stable in vitro and safe as confirmed by animal experiment. This vaccine provides a new candidate for viable oral vaccine against Hp infection.  相似文献   

7.
减毒鼠伤寒沙门菌防龋疫苗的免疫防龋效果   总被引:2,自引:1,他引:1  
《中山医科大学学报》2002,23(4):257-259
  相似文献   

8.
目的:了解内置重组大肠杆菌不耐热肠毒素B亚单位(rLTB)佐剂、幽门螺杆菌(Helicobacter pylori,Hp)尿素酶B亚单位(rUreB)和黏附素(rHpaA)双价基因工程疫苗对Hp SS1株感染BALB/c小鼠的保护作用及其机制.方法:采用NTA-Ni亲和层析法分别收集rUreB、rHpaA、rLTB-rUreB-rHpaA、重组大肠杆菌不耐热肠毒素A亚单位突变体(rLTKA63)、rLTB和霍乱肠毒素B亚单位(rCTB).用Western blot检测各重组蛋白抗原的免疫反应性.用GM1-ELISA检测rLTB、rCTB、rLTB-rUreB-rHpaA的佐剂活性.建立Hp SS1株感染BALB/c小鼠模型,检测不同组合抗原和佐剂的免疫保护效果.采用分离培养和镀银染色法检查感染小鼠胃窦组织中的Hp.建立ELISA,检测免疫小鼠特异性胃液S-IgA和血清IgA.结果:rUreB、rHpaA和rLTB-rUreB-rHpaA可被兔抗Hp全菌抗体识别,并与牛GM1结合.单用rUreB或rHpaA 免疫小鼠,其保护率低于70%;若与rLTB或rCTB合用,其保护率可增至75.0%~83.3%.在不同抗原及佐剂组合中,rLTB-rUreB-rHpaA保护率达100%,其次为rHpaA rUreB rCTB rLTKA63(91.7%)和rUreB rHpaA rLTB(90.9%).rLTB和rCTB均有明显的诱生免疫小鼠特异性血清IgA和胃液S-IgA作用,但前者诱生S-IgA的能力强于后者.特异性血清IgA,尤其是胃液S-IgA的阳性率与小鼠免疫保护率基本吻合,rLTB-rUreB-rHpaA免疫小鼠rUreB 和rHpaA 特异性S-IgA阳性率分别高达100%和91.7%.结论:rUreB和rHpaA有良好的免疫反应性和抗原性,rLTB和rCTB有黏膜免疫佐剂活性.内置佐剂UreB和HpaA双价重组疫苗对小鼠高保护率与使用高剂量rLTB、抗原分子量增大和局部高水平特异性S-IgA抗体有关.  相似文献   

9.
目的:观察口服减毒鼠伤寒杆菌活菌重组疫苗后小鼠的粘膜免疫应答状况。方法:将已构建成功的表达幽门螺杆菌(H.pylori)尿素酶B亚单位(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粘膜炎症程度差异无统计学意义。结论:表达H.pyloriUreB的减毒鼠伤寒杆菌SL3261/pTC01-UreB能够诱导小鼠产生抗H.pylori的粘膜免疫,可用作抗H.pylori感染的口服疫苗。  相似文献   

10.
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。  相似文献   

11.
目的:研究幽门螺杆菌超声粉碎抗原与霍乱毒素B亚单位组成的口服疫苗预防Hp感染的作用,探讨其保护性免疫应答的机制。方法:用Hp超声粉碎抗原2mg加CTB 10 μg作为免疫预防组,另设单纯Hp超声粉碎抗原组、单纯CTB组和PBS组为对照组。免疫4w后以活菌攻击,观察各组小鼠的免疫保护率,小鼠胃粘膜分泌性IgA抗体变化情况。结果:(1)免疫预防组小鼠免疫保护率为73.33%和对照组全部感染Hp(P <0 .0 0 1) ;(2 )免疫后4w各组小鼠未见明显的炎症反应;Hp攻击感染后4w免疫预防组小鼠可见明显的炎症反应,而各对照组相对较轻(P <0 .0 1) ;(3)Hp攻击前后免疫预防组小鼠局部胃粘膜IgA抗体显著升高(P <0 .0 1)。结论:Hp超声粉碎抗原加CTB可预防Hp感染。  相似文献   

12.
目的分析特发性血小板减少性紫癜(ITP)患者的幽门螺杆菌(Hp)感染情况,探讨抗Hp治疗对Hp感染阳性ITP患者的疗效。方法 ITP组45例,正常对照组32例,利用双抗夹心ELISA法检测两组Hp感染情况,对Hp感染阳性的ITP患者在治疗ITP的同时加用抗Hp治疗,与ITP组中Hp阴性病例治疗后的血小板数进行比较。结果 ITP组及正常对照组的Hp检出阳性率分别为66.7%与43.8%,ITP组Hp阳性率明显较高(P〈0.05);25例Hp阳性的ITP患者经抗Hp治疗后,转阴21例的血小板数明显高于ITP组中Hp阴性组(P〈0.05);Hp根除后的21例中,有12例血小板计数明显上升或恢复正常。结论 ITP患者Hp感染阳性率高于正常人;抗Hp治疗可以提高Hp感染的部分ITP患者血小板数量;Hp感染可能与ITP发病有关。  相似文献   

13.
为了建立幽门螺杆菌(Hp)感染BALB/c小鼠动物模型,在小鼠胃大弯窦部粘膜下埋上带腐蚀性药物的丝线致胃溃疡,或用2号手术针穿过小鼠胃大弯致假溃疡,随后灌服经微生物酶学和PCR鉴定为Hp的菌液。结果,在灌服Hp后的17,65,72天,取小鼠胃粘膜做Hp条件培养,见有乳白色菌落生长。表明通过这两种方法均可使Hp定植于BALB/c小鼠胃粘膜上。  相似文献   

14.
STAg和霍乱毒素不同程序滴鼻免疫小鼠诱导抗弓形虫作用   总被引:4,自引:0,他引:4  
目的观察弓形虫可溶性速殖子抗原(soluble tachyzoite antigen,STAg)和霍乱毒素(cholera toxin,CT)佐剂不同程序滴鼻免疫小鼠诱导的抗弓形虫感染能力,确定STAg和CT滴鼻免疫的最佳程序。方法BALB/c小鼠随机分为3组:1次、2次和3次免疫组,用20 μg STAg 1 μg CT/只分别滴鼻免疫1次,2次或3次,前2次间隔2周,末次间隔1周。末次免疫后第14天,用4×104个速殖子/只灌胃攻击所有小鼠,观察小鼠健康及死亡情况,攻击后第30天处死,ELISA法检测血清IgG和粪便IgA,计数肝、脑组织内弓形虫速殖子,分离并计数派伊尔结(Peyer′spatches,PP)和脾淋巴细胞数。结果2次和3次免疫组小鼠存活率明显高于1次免疫组(P<0.05),肝、脑组织内虫荷显著低于1次免疫组(P<0.001),血清IgG和粪便IgA高于1次免疫组,PP和脾淋巴细胞数无显著性变化。结论STAg和CT佐剂滴鼻免疫2次或3次能有效诱导小鼠抗弓形虫感染。  相似文献   

15.
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.  相似文献   

16.
目的研究重组日本血吸虫32kDa蛋白和铁蛋白联合免疫对感染小鼠的免疫保护作用。方法大量制备重组日本血吸虫32kDa蛋白(rSjGST-Sj32)和铁蛋白(GMCSF-SjFer)。48只昆明小鼠随机分为A,B,C,D4组,每组12只。B组和C组分别为rSjGST-Sj32和GMCSF-SjFer免疫组,每鼠注射50μg重组蛋白加弗氏完全佐剂(FCA);D组为联合免疫组,每鼠注射50μg重组32kDa蛋白和50μg铁蛋白的混合物加FCA;A组为FCA对照组。分别于0、2、6w共免疫3次。第3次免疫后2w,每鼠经腹部皮肤攻击感染40±1条尾蚴,感染后第45天剖杀所有小鼠,比较各组间减虫率和鼠肝卵减少率。结果联合免疫组与FCA对照组相比,获得了36.53%的减虫率和68.44%的每条雌虫肝组织产卵减少率,而单独rSjGST-Sj32免疫组的减虫率及每条雌虫肝组织产卵减少率分别为22.80%和43.12%,均显著低于联合免疫组(P<0.05及P<0.001);单独GMCSF-SjFer免疫组分别为21.05%和41.91%,均显著低于联合免疫组(P<0.05及P<0.001)。结论rSjGST-Sj32蛋白和GMCSF-SjFer蛋白联合免疫可显著地提高这两种蛋白单独应用时的免疫保护作用。  相似文献   

17.
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.  相似文献   

18.
[摘要]目的 初步探讨口腔扁平苔癣(OLP)与胃幽门螺杆菌(HP)的相关性.方法 应用聚合酶链反应(PCR)技术和14C尿素呼气试验相结合的方法,对65例确诊为OLP患者的口腔及胃分别测定HP的存在.结果 OLP患者口腔HP感染率均明显高于胃内HP感染率,2组比较差异有统计学意义(P<0.05); OLP伴有慢性胃病史的患者,胃和口腔中HP感染结果阳性为23例和27例;OLP不伴慢性胃病史的患者,胃和口腔中HP感染结果阳性为11例和20例.结论 口腔OLP可能与胃HP感染有相关性.  相似文献   

19.
目的探讨幽门螺杆菌在反流性食管炎发病中的作用.方法选择有典型反流症状的患者并经胃镜检查诊断为反流性食管炎53例作为病例组,无典型反流症状的患者并经胃镜检查诊断为慢性胃炎25例作为对照组,分别取食管下端黏膜及胃窦黏膜各3块,并分别行快速尿素酶试验、病理Hp检查和Hp培养,食管及胃窦黏膜Hp菌株应用PCR方法行cagA基因、vacA基因检测.结果病例组、对照组中食管黏膜的Hp阳性率分别为14/53(26.4%)、8/25(32%),两者差异无显著性(P>0.05);胃窦黏膜的Hp阳性率分别为20/53(37.8%)、16/25(64%),两者差异有显著性(P<0.05);病例组中胃窦黏膜Hp的cagA+检出率(6/20,30%)明显低于对照组(10/16,62.5%)(P<0.05),而vacA+检出率分别为6/20(30%)、7/16(43.8%),两者差异无显著性(P>0.05).不同程度食管炎中食管黏膜Hp阳性率差异无显著性(P>0.05),胃窦黏膜Hp阳性率差异有显著性(P<0.01),而cagA+及vacA+的Hp菌株的差异无显著性(P>0.05),但cagA+菌株多见于程度轻的食管炎.结论食管黏膜中检测到的Hp可能来源于胃窦黏膜的Hp,胃窦Hp感染、尤其是cagA+的Hp感染可能在RE的发生中有一定的保护作用.  相似文献   

20.
麻疹疫苗及加佐剂经滴鼻免疫小鼠的抗体应答   总被引:2,自引:0,他引:2  
目的:观察麻疹减毒活疫苗和灭活疫苗经滴鼻免疫小鼠后的抗体应答。方法:用活的或灭活麻疹疫苗及分别加明胶、植物血凝素(PHA)的疫苗滴鼻免疫小鼠,于末次免疫后10d取血清、唾液、呼吸道分泌物,用间接ELISA法检测特异性IgG,IgA抗体水平,并与皮下注射组及对照组进行比较。结果:各滴鼻组小鼠血清及呼吸道分泌物中均产生一定水平的特异性IgG及In抗体,皮下注射组小鼠呼吸道分泌物中未产生特异性IgA抗体;加明胶后的免疫效果优于加PHA的。结论:麻疹疫苗滴鼻免疫小鼠后可诱导全身及局部抗体应答,明胶可增强其免疫效果,而麻疹疫苗皮下注射免疫小鼠未能诱导局部抗体应答。  相似文献   

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