幽门螺杆菌尿素酶减毒鼠伤寒杆菌疫苗诱导小鼠粘膜免疫应答的研究

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。     

表达幽门螺杆菌UreB减毒鼠伤寒沙门氏菌对小鼠的免疫保护作用

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。     

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.     

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.     

表达幽门螺杆菌UreB减毒鼠伤寒沙门氏菌对小鼠的免疫保护作用

[目的]构建表达幽门螺杆菌(Helicobacterpylori,Hplori)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SL261.用SDS聚丙烯酰胺凝胶电泳、Westernblot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SL261.重组菌SL3261(pTrc99A-ureB)表达了约66ku的UreB.与对照组比,重组菌免疫组H.pylori定植水平明显下降(P＜0.05).[结论]构建了表达H.pyloriUreB的重组减毒鼠伤寒沙门氏疫苗菌,该菌株对C57BL/6有免疫保护作用.     

携带flk-1的减毒沙门疫苗菌抗胶质瘤血管生成的研究

目的观察表达鼠血管内皮生长因子受体2（VEGFR2，flk-1）的重组减毒鼠伤寒沙门疫苗菌对胶质瘤荷瘤小鼠的抗肿瘤血管及肿瘤生长抑制作用。方法构建真核表达载体pcDNA3、1-flk-1通过电转化法将pcDNA3．1-flk-1导入减毒鼠伤寒沙门菌SL7207中，经由胃管饲于C57BL／6J小鼠，对胶质瘤荷瘤小鼠进行免疫预防及治疗。通过观察免疫动物的生存期，免疫荧光法检测肿瘤血管密度，评价重组疫苗菌的抗血管及肿瘤生长抑制作用。分离免疫小鼠的脾细胞，分析重组疫苗菌免疫后小鼠体内的特异性细胞毒性T细胞（CTL）应答。结果重组疫苗菌免疫能够明显减少肿瘤血管密度，延缓胶质瘤的生长，延长小鼠生存期，获得明显的抗肿瘤效果。重组疫苗菌免疫后可诱导小鼠脾淋巴细胞产生针对flk-1的CTL活性。结论表达鼠flk-1的重组减毒鼠伤寒沙门疫苗菌经口服免疫，可打破小鼠对于自身抗原flk-1的免疫耐受，诱导小鼠产生抗flk-1的特异性免疫反应，特异性杀伤肿瘤血管内皮细胞，预防和治疗小鼠胶质瘤。     

减毒伤寒杆菌为载体的甲肝疫苗候选株的构建和免疫应答

目的：构建以减毒鼠伤寒杆菌为载体的甲肝疫苗候选株,并证明其免疫源性。方法：将甲型肝炎病毒编码区cDNA插入高效表达质粒pBV221。转化大肠杆菌DH5α。用核酸探针和限制性内切酶筛选和鉴定阳性克隆。将阳性重组质粒用电转移法转化减毒鼠伤寒杆菌BRD509。用抗生素平板筛选法筛选出转化成功的伤寒杆菌,用温度诱导法诱导目的基因在伤寒杆菌中表达。结果：蛋白质印迹法（Western blotting）和酶联免疫吸附测定（ELISA）检测表明,表达蛋白可与人抗甲型肝炎IgG发生特异性反应。用此伤寒杆菌口服或腹腔注射免疫小鼠,小鼠血清检测到抗甲型肝炎病毒的抗体。 结论：以减毒鼠伤寒杆菌为载体的甲肝疫苗构建成功,并可在体内外产生免疫应答。     

携带flk-l的减毒沙门疫苗菌抗胶质瘤血管生成的研究

目的观察表达鼠血管内皮生长因子受体2(VEGFR2,flk-1)的重组减毒鼠伤寒沙门疫苗菌对胶质瘤荷瘤小鼠的抗肿瘤血管及肿瘤生长抑制作用。方法构建真核表达载体pcDNA3 1.-flk-l,通过电转化法将pcDNA3 1.-flk-1导入减毒鼠伤寒沙门菌SL7207中,经由胃管饲于C57BL/6J小鼠,对胶质瘤荷瘤小鼠进行免疫预防及治疗。通过观察免疫动物的生存期,免疫荧光法检测肿瘤血管密度,评价重组疫苗菌的抗血管及肿瘤生长抑制作用。分离免疫小鼠的脾细胞,分析重组疫苗菌免疫后小鼠体内的特异性细胞毒性T细胞(CTL)应答。结果重组疫苗菌免疫能够明显减少肿瘤血管密度,延缓胶质瘤的生长,延长小鼠生存期,获得明显的抗肿瘤效果。重组疫苗菌免疫后可诱导小鼠脾淋巴细胞产生针对flk-l的CTL活性。结论表达鼠flk-l的重组减毒鼠伤寒沙门疫苗菌经口服免疫,可打破小鼠对于自身抗原flk-1的免疫耐受,诱导小鼠产生抗flk-1的特异性免疫反应,特异性杀伤肿瘤血管内皮细胞,预防和治疗小鼠胶质瘤。     

沙眼衣原体重组口服活疫苗的构建及其口服免疫的特性

目的 观察所构宾沙眼衣原体（Ｃｔ）重组疫苗株对小鼠的免疫特性。方法 以减毒鼠伤寒杆菌致死性平衡系统为载体构建Ｃｔ重组疫苗株。将重组疫苗株用ＬＢ培养基连续传代５０次，比较传代前后携带质料、生化反应性及蛋白表达情况，以鉴定重组子的稳定性。并以不同浓度的重组菌活菌液口服免疫ＢＡＬＢ／ｃ小鼠，检测外源基因的插入和表达对减毒株毒性的影响，口服免疫后不同时间检测小鼠小肠Ｐｅｙｅｒ氏结、肠系膜淋巴结、脾脏及子宫     

表达幽门螺杆菌过氧化氢酶的减毒沙门氏菌疫苗株的构建及其免疫保护作用的观察

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。