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1.
沙眼衣原体重组口服活疫苗的构建及其口服免疫的特性   总被引:1,自引:1,他引:0  
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  相似文献   

2.
目的:探讨双启动子DNA疫苗载体在减毒沙门菌中的稳定性和重组减毒沙门菌在小鼠体内的定植及动态变化。方法:将人乳头瘤病毒双启动子DNA疫苗载体pCN-16L1E7转化减毒沙门菌S-SL3261,经口服、鼻饲和皮肤途径免疫小鼠,在免疫后的第3、4、5、6周取小鼠粘膜相关淋巴组织,电镜观察细菌在组织中的定植;组织匀浆,细菌培养计数确定细菌的增殖;从细菌中提取质粒酶切鉴定以确定质粒的稳定性。结果:重组减毒沙门菌S-SL3261能在小鼠脾脏、肝脏和粘膜相关淋巴组织中定植,电镜可检测到多个细菌定植灶;重组减毒沙门菌可有效进入机体细胞并有限增殖达6周,在第4~5周菌落数量达到高峰,然后逐渐减少以至最后被清除。pCN-16L1E7可在减毒沙门菌中稳定存在,载体的产量和酶切图谱没有明显变化。结论:双启动子DNA疫苗载体pCN-16L1E7与减毒沙门菌有较好的相容性,能够在小鼠体内定植及有限增殖。口服、鼻饲和皮肤粘膜接种途径均可有效地将减毒沙门菌携带的DNA疫苗载体导入粘膜相关淋巴组织。  相似文献   

3.
目的 构建表达人受精素 β去整合素结构域的重组沙门菌疫苗株。 方法 应用PCR方法获得人受精素 β去整合素结构域的cDNA片断 ,将其插入高拷贝表达载体pYA3 3 3 9中 ,构建重组质粒pYA3 3 3 9 hf2 79,将该重组质粒转入鼠沙门菌疫苗株X45 5 0 ,获得重组菌株X45 5 0 (pYA3 3 3 9 hf2 79)。结果 该重组疫苗株可稳定表达能被抗人受精素 β多克隆抗体识别的重组蛋白HF93 ,其分子量约为 16× 10 3。结论 该疫苗株的构建为进一步研究以人受精素 β为靶抗原的免疫避孕疫苗打下了基础  相似文献   

4.
[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.  相似文献   

5.
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   

6.
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   

7.
目的:构建携带NK4基因和IL-2基因真核表达载体的减毒沙门氏菌株.方法:用基因工程方法将NK4基因和IL-2基因克隆入真核表达载体pcDNA4,并进行基因测序.重组质粒经鉴定后再电转入减毒沙门氏菌Ty21a中,通过PCR和酶切鉴定,并对构建的重组减毒沙门氏菌进行体外稳定性观察.结果:经PCR和酶切证实,构建了分别含NK4基因和IL-2基因的重组真核表达质粒pcDNA4-NK4和peDNA4-IL-2,并将他们成功导入减毒沙门氏菌Ty21a中.稳定性实验结果表明该重组菌株在体外能稳定地繁殖、生长和传代.结论:成功构建携带NK4基因和IL-2基因真核表达载体的减毒沙门氏菌株,为探索制备携带NK4和IL-2基因的肿瘤减毒活疫苗奠定了基础.  相似文献   

8.
目的考察Pbr R大肠埃希菌外膜展示株在不同条件下的体内外生长特性,为其体内定植驱铅能力的研究奠定基础。方法 15%SDS-PAGE分析不同浓度诱导剂L-阿拉伯糖诱导展示菌p BAD-loa-pbrr/DH10B的重组蛋白表达情况;铅平板敏感实验对比重组菌诱导及非诱导条件下在含铅平板上的菌落形成能力;体外培养实验考察诱导剂的加入对重组菌生长能力及耐铅能力的影响;通过混合共培养实验对比诱导条件下重组菌与空宿主菌的竞争生长能力;考察重组菌6 h内的胃酸耐受性;考察诱导剂及染菌方式对重组菌小鼠体内定植的影响。结果低至0.002%的L-阿拉伯糖即可诱导重组蛋白的高效表达;Pbr R展示菌对铅表现出了一定的耐受力;诱导剂引入后重组菌生长能力降低;重组菌对胃酸有较好的抗性,经口染菌方式重组菌可定植于小鼠肠道,但摄入L-阿拉伯糖水后,粪便中重组菌快速丢失,通过连续染菌方式可维持其定植水平。结论重组菌体内外培养实验结果表明,基于商品化的诱导型表达载体构建的展示菌难以表达重组蛋白的同时实现小鼠体内的稳定定植。  相似文献   

9.
目的 基因重组构建以单增李斯特菌、绵羊李斯特菌为载体的新型结核疫苗候选株,并进行蛋白表达验证,为结核疫苗研究和结核防控提供新思路和新方法。方法 以体外基因连接、质粒转化等技术,将本实验室已有的分别编码结核抗原Ag85C、ESAT-6蛋白的两种基因盒,插入携带单增李斯特菌、绵羊李斯特菌同源序列的打靶质粒中。将打靶质粒电转化单增李斯特菌、绵羊李斯特菌,在42 ℃和30 ℃连续传代,利用同源重组杂交原理和基因打靶技术,将打靶质粒携带的两种编码结核抗原的抗原基因盒,分别整合至致病基因(actA和plcB)被敲除的单增李斯特菌、绵羊李斯特菌减毒株及未经减毒的绵羊李斯特菌基因组中。重组菌经蓝白斑、抗生素抗性及PCR筛选,再经Western blot检测其菌体蛋白及分泌蛋白的表达情况。结果 构建了携带抗原基因盒的重组菌株5株,其重组基因序列PCR验证结果均符合预期,且测序验证成功;重组菌不携带红霉素抗性基因,表型特征符合预期;其中,重组菌Li-Rv0129c、Li-ΔactAplcB-Rv0129c按预期表达了结核杆菌Ag85C抗原蛋白,Li-ΔactAplcB-Rv3875按预期表达了结核杆菌ESAT-6抗原蛋白,Lm重组菌未表达相应结核抗原蛋白。结论 成功构建并筛选得到3株以绵羊李斯特菌为载体的、携带结核杆菌Rv0129c(编码Ag85C)抗原基因盒或Rv3875(编码ESAT-6)抗原基因盒,且表达相应抗原蛋白的新型结核疫苗候选株。  相似文献   

10.
目的:观察构建含有Tip30与人IFN-γ基因的真核表达及共表达质粒的重组减毒沙门菌对腺样囊性癌荷瘤裸鼠肿瘤血管生成的抑制作用.方法:4周龄BALB/c nu nu雄鼠25只,随机分成5组,对照组(PBS组)、单纯减毒沙门菌组(SL7207组)、携带人IFN-γ基因减毒沙门菌治疗组(SL7207/pCI-IFN组)、携带Tip30基因减毒沙门菌治疗组(SL7207/pCI-Tip30组)、携带Tip30与人IFN-γ基因的真核共表达质粒的重组减毒沙门菌治疗组(SL7207/pCI-Tip30/IFN组),均于颌下接种腺样囊性癌(ACC-2)细胞.10 d后对照组裸鼠口服PBS,其余各治疗组口服相应的减毒沙门菌,隔周1次,共3次.然后检测肿瘤生长体积、带瘤存活期、裸鼠体内肿瘤的微血管密度(MVD).结果:各治疗组比较对照组、携带基因治疗组比较SL7207组、联合基因治疗组比较单基因治疗组肿瘤生长慢、体积小,带瘤存活期长(P<0.05);携带Tip30基因治疗组MVD较其他3组低,联合基因组较携带Tip30单基因组MVD低(P<0.05).结论:减毒沙门菌载体分别介导Tip30、IFN-γ基因有抑制肿瘤生长作用,联合基因较单基因治疗效果好,有协同作用.  相似文献   

11.
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   

12.
目的 构建能稳定表达pCDR1 Th表位和CTLA4Ig融合基因的真核表达载体,探讨其在小鼠体内的表达并对表达产物进行鉴定.方法 用Touchdown PCR法扩增CTLA4Ig基因,同时引入pCDR1 Th表位.将PCR产物连接真核表达载体pcDNA3.1( ),构建pcDNA3.1( )-CTLA41g-pCDR1.将构建表达CTLA4Ig-pCDR1减毒鼠伤寒沙门菌SL7207喂饲BALB/c小鼠,取脾脏进行免疫组化鉴定重组蛋白在动物体内的表达.结果 酶切鉴定和基因序列测定显示重组质粒构建成功.重组蛋白在BALB/c小鼠脾脏免疫细胞胞浆中有阳性表达.结论 成功构建了能稳定表达pCDR1 Th表位和CTLA4Ig融合基因的真核表达载体.  相似文献   

13.
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.  相似文献   

14.
板蓝根多糖对宿主抵抗鼠伤寒沙门菌感染能力的影响   总被引:1,自引:0,他引:1  
目的:观察板蓝根多糖注射鼠伤寒沙门菌感染的小鼠后,对小鼠抵抗鼠伤寒杆菌能力的影响。方法:水煮法提取板蓝根多糖,配成悬液,腹腔注射感染鼠伤寒的小鼠,观察小鼠的生存时间,脾脏的重量指数,脏器菌落计数,检测小鼠血清中抗体IgG水平,用ELISA法测定小鼠脾脏释放的细胞因子。结果:注射板蓝根多糖的小鼠,生存时间延长,脾脏的重量指数,菌落计数均比对照组明显降低,血清中抗体IgG水平升高,脾脏细胞分泌细胞因子IFN-γ水平上升。结论:板蓝根多糖能够通过增强抗体的产生,调节细胞因子的释放,增强宿主的体液免疫和细胞免疫功能,保护宿主抵抗胞内菌的感染。  相似文献   

15.
目的:探讨鼠伤寒沙门菌的L型变异对噬菌体敏感性的影响。方法:用12株鼠伤寒沙门菌分型噬菌体对3株体外诱导的鼠伤寒沙门菌L型及其原菌做噬菌体敏感性检测。结果:裂解原菌的噬菌体大多不能裂解其L型菌,只有少数噬菌体能同时裂解原菌及其L型菌。结论:鼠伤寒沙门菌的L型变异可引起噬菌体受体的丢失,从而导致对相应的噬菌体不再敏感。  相似文献   

16.
To investigate the anti-vasculature effects and the anti-glioma effects of attenuated Salmonella typhimurium vaccine strain expressing VEGFR2 (flk-1) gene, plasmid pcDNA3, 1-flk1 was constructed and electro-transfected into live attenuated Salmonella typhimurium strain SL7207. Mouse models of intracranial G1261 glioblastoma were treated with an orally administered attenuated Salmonella typhimurium expressing flk-1 gene. The survival period was recorded and vessel density was observed by immunofluorescence. CTLs activity was measured by MTT assay. Our results showed that attenuated Salmonella typhimurium vaccine strain expressing flk-1 gene could significantly inhibit glioblastoma growth, reduce vessel density, prolong the survival period and improve the survival rate in these mice. The flk-1 specific CTLs activity was increased obviously after the vaccination. Our study showed that attenuated Salmonella typhlmurium vaccine strain expressing flk-1 gene could break peripheral immune tolerance a in glioma gainst this self-antigen and kill endothelial cells by the orally administered vaccine and can be used for both prophylactic and therapeutic purposes.  相似文献   

17.
Malariaisoneofthemajortropica1diseasesaffectinghumantoday,contributinggreatlytothemortalityrateinthedevelopingworld-Sincenoef-fectivetreatmentcouldsavethelifeofsomanypeople,thereisanurgentnecessitytodevelopaneffectivemalariavaccine.Nowresearcheshavebeentoncentratedonthestage-specificantigensofsporozoites,merozoites,exoerythrocyticandery-throcyticstagesandgametocytes.Monovalentvac-cineshavebeenprovedtohavelowprotectiveim-munity['],sopolyvalentvaccines,whichincludeseveralepitopesofdifferentantig…  相似文献   

18.
目的观察重组减毒鼠伤寒沙门菌作为基因载体在人涎腺腺样囊性癌裸鼠移植瘤中的富集情况和对外源基因的呈递能力。方法以绿色荧光蛋白基因(GFP)为报告基因,以减毒鼠伤寒沙门菌SL7207为转基因载体,分别构建原核启动GFP表达的重组减毒鼠伤寒沙门菌SL7207-pUC-GFP和真核启动GFP表达的重组减毒鼠伤寒沙门菌SL7207-pEG-FP-N1。原核菌SL7207-pUC-GFP在体外连续传代,观察GFP基因表达的稳定性;同时,对荷人涎腺腺样囊性癌皮下移植瘤裸鼠模型口服给予原核菌SL7207-pUC-GFP(0.1mL,1×109cfu/mL),在口服后24h、48h、5d、10d、20d、30d处死荷瘤裸鼠,获取肝、脾及肿瘤组织并制成匀浆液进行重组菌培养及GFP表达检测,观察重组菌在瘤体细胞内富集情况。对荷瘤裸鼠模型口服给予真核菌SL7207-pEGFP-N1,5d后取肝、脾及肿瘤组织进行冰冻组织切片,荧光显微镜下观察GFP的表达,了解重组菌携带外源基因在肿瘤细胞内的表达。结果携带GFP原核表达的重组减毒鼠伤寒沙门菌SL7207-pUC-GFP在体外连续传代10次未见表达减少或缺失。荷瘤裸鼠口服原核表达GFP基因重组菌SL7207-pUC-GFP菌液实验表明,重组菌SL7207-pUC-GFP在肝、脾及肿瘤组织中能长期存活,以肿瘤组织中聚集明显(P<0.05)且维持时间较长。荷瘤裸鼠口服真核表达GFP基因重组菌SL7207-pEGFP-N1菌液实验表明,相对肝脏和脾脏组织,外源基因在肿瘤细胞内表达量最高。结论重组减毒鼠伤寒沙门菌可以在瘤体细胞内富集存活,并且携带的外源基因可以释放到肿瘤细胞内表达,具有作为基因治疗载体的双重优势。  相似文献   

19.
了解鼠伤寒沙门菌L型变异对细菌质粒和外膜蛋白的影响。方法:在鼠伤寒沙门菌L型的诱导 传代过程中,取传不同代的L型细菌提取质粒,观察质粒图谱的变化。提取鼠伤寒沙门菌稳定L型的外膜蛋白,通 过聚丙烯酰胺凝胶电泳观察外膜蛋白的变化。结果:鼠伤寒沙门菌L型在诱导传代过程中原菌的质粒逐渐以致全 部丢失,大多数外膜蛋白亦丢失。结论:鼠伤寒沙门菌发生L型变异后,原菌的质粒不能稳定遗传,且有外膜蛋白 的丢失。  相似文献   

20.
Twenty colostrum-fed piglets from three sows were separated from the sows 24 hours after birth and were randomly divided into five groups of four piglets each. Every piglet in each of four test groups was orally inoculated with about 10(10) colony forming units of Salmonella typhimurium, Salmonella choleraesuis var Kunzendorf or one of two isolates of Klebsiella pneumoniae. One group served as uninoculated controls. Piglets infected with K. pneumoniae developed severe diarrhea beginning about 12 hours after inoculation. They became dehydrated and weak but continued to drink. There were no morphological alterations in intestinal mucosa when piglets were killed and necropsied 48 or 72 hours after inoculation. Klebseilla pneumoniae was isolated from intestine and feces but not from liver or spleen. Piglets inoculated with S. choleraesuis became lethargic and disinterested in food by 24 hours after inoculation. Diarrhea developed by 48 hours after inoculation. Lesions at necropsy 60 or 72 hours postinoculation were subcutaneous edema, mesenteric lymphadenitis, diffuse intestinal superficial mucosal necrosis with villous atrophy, and focal deep ulceration in the ileum. Salmonella choleraesuis was isolated from all segments of intestine and from feces, liver and spleen. Piglets inoculated with S. typhimurium developed a relatively mild diarrheal disease with lesions similar to those with S. choleraesuis infection but less severe. The inoculated organism was recovered from all areas of intestine and from feces, liver and spleen. Serum from infected and control piglets had high (greater than 1:256) agglutinating titres against S. typhimurium but low titres (0 to 1:8) against S. choleraesuis. The agglutinins were assumed to originate from colostral antibodies.  相似文献   

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