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。     

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.     

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.     

幽门螺杆菌中性粒细胞激活蛋白DNA疫苗的构建及其免疫保护作用

目的:构建携带幽门螺杆菌中性粒细胞激活蛋白(Hp neutrophil-activating protein,Hp-NAP)基因(napA)活减毒鼠伤寒沙门菌重组DNA疫苗,初步观察其对慢性Hp感染的免疫保护作用.方法:应用基因工程技术扩增全长napA,测序并经同源性分析后,将其亚克隆入真核表达载体pIRES,鉴定正确后将重组质粒转化活减毒鼠伤寒沙门菌构建Hp-NAP口服DNA疫苗.口服Hp-SS1建立SS1长期感染小鼠模型,30周后随机均分为3组,每组各5只.治疗组予109cfu/0.4 ml疫苗菌灌胃,1次/周×3周;2个对照组分别予等体积生理盐水或空白质粒.末次免疫4周后行快速尿素酶检测,ELISA测定血清抗体效价.结果:重组真核表达质粒pIRES-napA成功转化活减毒鼠伤寒沙门菌SL7207;所克隆napA与GenBank中SS1-na-pA核苷酸和蛋白质的同源性均＞98%.免疫后4周治疗组75%(3/4)小鼠快速尿素酶检测阴性,对照组均阳性,差异显著(P＜0.05);治疗组血清抗Hp-NAP抗体效价明显升高.结论:成功构建了具有较好免疫保护作用的Hp-NAP口服重组DNA疫苗,为进一步研制多价抗Hp核酸疫苗奠定了基础.     

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。     

两种表达鼠精子受精素β蛋白的减毒沙门氏菌疫苗株的稳定性分析

目的 了解表达鼠精子抗原受精素β亚单位(Fertilin βsubumit,fβ)的两种重组减毒沙门氏菌疫苗株X4632(pFEC)和X4550(pFEC)的生长特性和体内外携带重组质粒的稳定性。方法 将两种重组沙门氏菌疫苗株X4632(pFEC)和X4550(pFEC)体外传代培养，观察比较其生长特性和所含重组质粒的稳定性；将该两种重组菌口服免疫BAIB／c雄性小鼠，观察重组菌侵入小鼠Peyer结、肠系膜淋巴结和脾脏等组织内繁殖生长情况和所回收重组菌携带质粒的阳性率。结果 该两种无抗药性的重组菌株在体内、外营养选择压力下均可较稳定携带重组质粒传代繁殖，X4550(pCFL)在体内可较稳定地定植于Peyer结、肠系膜淋巴结和脾脏；X4632(pCFL)可较稳定地定植于Peyer结，均可稳定表达被Fβ单抗识别的重组Fβ蛋日。结论 X4632(pFEC)、X4550(pFEC)疫苗株可作为递呈精子抗原的活菌载体。     

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.     

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。     

EV71-VP1抗原在双歧杆菌中表达的免疫效果检测

目的 检测表达肠道病毒71型（enterovirus 71,EV71）VP1基因的重组双歧杆菌免疫后小鼠IgG、IgA表达变化,并对免疫的孕鼠产下的新生小鼠注射EV71病毒,以检测免疫保护作用可否从母代传给子代。方法 ELISA法检测免疫小鼠血清IgG、IgA抗体效价;用EV71毒株对免疫孕鼠所产下的新生小鼠攻毒。结果 两种抗体效价都有明显升高（P<0.01）;实验组受病毒攻击新生小鼠存活率明显高于对照组。结论 口服表达VP1蛋白的双歧杆菌能够激活体内免疫系统产生特异性抗体,重组双歧杆菌可通过免疫孕鼠而使新生幼鼠获得保护性抗体。为研制EV71亚单位口服活疫苗奠定了研究基础。     

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     

幽门螺杆菌中性粒细胞激活蛋白DNA疫苗的构建及鉴定

目的 构建幽门螺杆菌(Hp)中性粒细胞激活蛋白(HP-NAP)DNA疫苗。方法 扩增全长napA(编码HP-NAP)，将其与克隆载体pBT连接，经测序及同源性分析后，将相应片段亚克隆入真核表达载体pIRES，经双酶切及PCR鉴定。结果 PCR扩增-435bp产物，核苷酸序列测定及BLAST分析表明，所克隆序列与基因库中Hp悉尼株(SS1)napA核苷酸及蛋白质的同源性均为98％。ANTHEPROT软件预测其蛋白质具有良好的疏水性和抗原性。经PCR及双酶切鉴定，证实成功构建携带，napA的重组真核表达质粒plRES-napA。结论 成功构建并鉴定了HP-NAP重组DNA疫苗，为进一步研究其免疫原性及研制口服DNA疫苗奠定了基础。     

口服轮状病毒减毒活疫苗与麻疹－风疹联合减毒活疫苗联合接种的免疫原性和安全性研究

目的:探讨口服轮状病毒减毒活疫苗(oral live attenuated rotavirus vaccine,ORV)与麻疹-风疹联合减毒活疫苗(measles and rubella combined attenuated live vaccine,MR)联合接种的可行性,为制定免疫规划程序提供可靠的科学依据。方法:设两种疫苗同时接种组和单苗接种组,观察疑似预防接种异常反应(AEFI)发生情况,接种前后、联合接种与单独接种组之间抗体几何平均滴度/浓度(GMT/GMC)、抗体阳性率和阳转率的差异。结果:各接种组临床反应都轻微,实验组与各对照组免前免后同种抗体GMT/GMC、阳性率,以及免后抗体阳转率,其差异均无显著意义(P>0.05)。结论:ORV与MR同时接种未发现严重异常反应和相互干扰免疫应答,可以同时接种。     

新型冠状病毒疫苗免疫BALB/c小鼠后的T细胞表位鉴定

目的 鉴定携带新型冠状病毒S、N和M基因的疫苗在BALB/c小鼠上的T细胞表位。方法 用携带新型冠状病毒S、N和M基因的DNA疫苗和痘病毒载体疫苗免疫6~8周龄雌性BALB/c小鼠,末次免疫1个月内取小鼠脾细胞,用ELISPOT法检测其对新型冠状病毒特异性多肽的T细胞免疫反应,筛选出具有阳性反应的多肽片段,并对其表位进行预测分析。结果 S、N、M基因分别鉴定出12、2、17条阳性多肽。S基因中多肽S49、S50、S100、S101、S102、S103阳性反应率为100%,且免疫刺激指数最高;覆盖M蛋白全长的41条多肽中有7条能刺激50%以上的小鼠产生阳性反应。预测分析结果显示,S、N、M基因分别有10、1、16个MHC-1 H-2限制性表位,主要位于S基因的RBD区、N基因RBD区以及M基因的跨膜区。这些表位在SARS-CoV-2及其变异株、SARS-CoV中高度保守。结论 新型冠状病毒DNA疫苗和痘病毒载体疫苗诱导了针对多个基因、多个表位的特异性T细胞应答,未来通用型疫苗设计应包含能诱导细胞免疫的多个抗原,特别是包含保守表位的结构抗原。     

艾滋病疫苗的研究进展

HIV疫苗研制的困难在于HIV病毒的高度遗传变异.根据已发现的10种亚型HIV-1型病毒研制的能激发CTL的新疫苗,已证实在临床上有了实际的应用效果.目前的疫苗研制策略包括质粒DNA载体,活重组载体和联合疫苗等.本文着重介绍这几种艾滋病疫苗的研究现状和存在的问题.     

儿童甲肝、风疹减毒活疫苗同时免疫效果观察

目的了解甲肝与风疹减毒活疫苗同时免疫的效果。方法对荆州市妇幼保健院预防接种门诊的356名1岁3个月至2岁的儿童进行免疫监测,并按同时免疫组与单独免疫组进行效果分析。结果同时免疫与单独免疫应答率差异无统计学意义(P>0.05),但同时免疫组两种疫苗的抗体几何平均滴度(GMT)明显高于单独组。结论采用同时免疫方法可增加疫苗的抗体几何平均滴度。     

基因重组卡介苗研究进展

卡介苗是用来预防结核病的牛型结核分枝杆菌减毒活疫苗。以卡介苗为载体的基因重组卡介苗因其具有集疫苗与佐剂于一体的特点,近年来成为研究的热点。基因重组卡介苗在免疫功能的研究、传染病和肿瘤的防治中均发挥了重要作用。本文就近年来这方面的研究作一简要概述。     

犬用口服狂犬病减毒活疫苗安全实验研究

目的:探讨犬用口服狂犬病减毒活疫苗(CTN-22毒株)的安全性。方法:依照WHO 专家会议关于犬和野生食肉动物口服狂犬病疫苗接种现场试验要求和标准的报告,以及美国动物与动物制品法规—兽医生物制品标准,进行本疫苗的安全性试验。结果:口服免疫的5只猫和口服、注射免疫的各5只黄毛鼠,观察21d,无任何症状发生,均健康存活;用本疫苗10个现场使用量的1ml,各对5只家犬肌肉和大隐神经及其周围组织浸润注射后,观察3～35d,无狂犬病症状,并按方法中所规定时间淘汰犬,剖取颈淋巴结、脑、唾液腺等组织分离病毒,均为阴性;3月龄10只犬以10个现场剂量口服免疫后,1、2、3d连续3次取唾液进行病毒分离亦均阴性,免疫后观察6个月未显示狂犬病任何征象,均健康存活。结论:证实CTN-22毒株制备的犬用口服狂犬病减毒活疫苗是安全的。     

幽门螺杆菌热休克蛋白60脂质体疫苗的制备及其免疫预防作用

目的 探讨制备脂质体包裹重组幽门螺杆菌(Hp)热休克蛋白60(Hsp60)口服疫苗的方法,并用Hp感染的小鼠模型评价其在预防Hp感染中的作用.方法 将PET-22(+)/Hsp60在BL21(DE3)大肠杆菌表达,Ni-NTA琼脂糖树脂纯化Hsp60重组蛋白,用薄膜分散法制备以卵磷脂和胆固醇为膜组分包裹的Hsp60重组蛋白口服疫苗,并用透射电镜测定其粒径.75只BALB/C小鼠分为5组,分别通过灌胃方法给予PBS、空白脂质体、Hsp60重组蛋白+霍乱霉素(CT)、脂质体包裹Hsp60重组蛋白、脂质体包裹Hsp60重组蛋白+CT,每周1次共次,末次攻击2周再用活Hp攻击3次,3周后处死小鼠,行胃组织快速尿素酶试验、Hp的定植半定量、炎症程度及其炎症活动度的评分.结果 可溶性表达产物占全菌总蛋白的27%,经纯化获得纯度为95%的重组蛋白,制备的脂质体粒径为(0.7±0.)μm.PBS组和空白脂质体组保护率均为0,而Hsp60重组蛋白+CT组、脂质体包裹Hsp60重组蛋白组、脂质体包裹Hsp60重组蛋白+CT组的保护率分别为73.3%、66.7%和86.7%,且均能使免疫小鼠胃粘膜Hp感染数目明显减少,炎症反应减轻.结论 口服脂质体能分地代替免疫佐剂,作为Hp疫苗的免疫佐剂,将具有广泛的应用前景.     

表达幽门螺杆菌过氧化氢酶的无抗性减毒鼠伤寒沙门氏菌株的构建

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.     

Anti-angiogenesis Effect on Glioma of Attenuated Salmonella Typhimurium Vaccine Strain with flk-1 Gene

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.