产肠毒素大肠杆菌基因工程疫苗在小鼠中的免疫原性

目的观察肠毒素大肠杆菌(ETEC)基因工程疫苗的抗原性效果,筛选有效的防治ETEC腹泻病基因工程疫苗株。方法将表达ETEC抗原成分的重组质粒pZCF16和pZHY22分别转化入载体痢疾福氏2a菌株FWL01中,构成含不同ETEC抗原成分的工程疫苗株FE3和FE16。分别用FE3与FE16活菌和灭活菌制成的疫苗以同等剂量梯度,通过口服和鼻饲途径免疫小白鼠,经过一定时间检测2组小白鼠的免疫效果。结果在免疫后的小白鼠血液中均产生了特异性抗IgG抗体,在活菌免疫组肠道内也产生了特异性分泌型抗体IgA;抗体IgG 1周后开始升高,且在8周后达到高峰。抗体IgA的产生规律与IgG相似。结论肠毒素大肠杆菌载体菌苗FE3和FE16有较强的免疫原性,作为多价疫苗的构建是有效的,从而为ETEC腹泻的特异性免疫防治提供了可能。     

一株具有痢疾志贺菌抗原与基因相关性的侵袭性大肠埃希菌O29

目的对造成婴儿腹泻的粪便标本中所分离出的一株菌株进行鉴定。方法使用VITEK 2和志贺菌血清以及大肠埃希菌血清对此株菌进行生化和血清学鉴定。毒力基因和药敏试验以及对菌株进行多位点序列分析找出它与大肠杆菌和志贺菌之间的基因相关性。结果生化鉴定为大肠埃希菌,此菌对侵袭性大肠埃希菌O29以及痢疾志贺菌1型血清都凝集。检测出ipa H基因。多位点序列分析显示属于ST38这一簇。结论此菌株是侵袭性大肠埃希菌。     

聚集性大肠埃希菌生物膜形成及毒力基因型研究

目的:通过肠道门诊标本检出的聚集性大肠埃希菌,研究其相关致病因子的毒力基因。方法:使用生物膜形成试验筛检聚集性大肠埃希菌,获得菌株后,进行转译激活因子基因,CVD432质粒基因,聚集性大肠埃希菌Ⅰ型鞭毛基因、Ⅱ型鞭毛基因、Ⅲ型鞭毛基因、Ⅳ菌毛基因,质粒编码毒素基因,聚集性大肠埃希菌耐热性肠毒素基因等各种基因检测,研究不同基因的分布和生物膜实验的相关性。结果:可以通过生物膜形成试验筛检聚集性大肠埃希菌,并根据毒力基因分型。结论:生物膜形成实验作为利用生物学特性筛检聚集性大肠埃希菌有一定的应用价值,聚集性大肠埃希菌的生物膜的形成与其各类毒力基因有不同程度的相关性。     

食品中致泻性大肠埃希菌PCR确认试验检测的探讨

目的 建议修订GB 4789.6—2016《食品安全国家标准食品微生物学致泻大肠埃希氏菌检验》PCR确认试验中，大肠埃希菌ATCC25922不宜作为检测肠道集聚性大肠埃希菌(EAEC)毒力基因的阴性对照菌株。方法 参照GB 4789.6—2016,采用市面在售的2种不同品牌规格致泻性大肠埃希菌核酸检测试剂盒进行PCR确认试验。结果 阴性对照菌株大肠埃希菌ATCC25922在2种不同品牌规格致泻性大肠埃希菌核酸检测试剂中均检测出pic基因，存在PCR确认实验过程中阴性对照失控现象。结论 PCR确认试验中大肠埃希菌ATCC25922作为阴性对照菌参与检测EAEC毒力基因，会影响试验结果的判读，建议采用等效标准菌株(如ATCC8739)作为阴性对照菌株参与试验。     

辽宁省生活饮用水中大肠埃希菌的检测及毒力和血清分析

目的 了解辽宁省部分地区生活饮用水被大肠埃希菌污染的状况，并为水源性疾病的防控和生活饮用水的卫生安全监测和预警提供参考依据。方法 采用多管发酵法对2010—2021年辽宁省各类生活饮用水中的大肠埃希菌微生物指标进行检测，同时采用聚合酶链反应（polymerase chain reaction,PCR）方法对分离出的大肠埃希菌进行致病性毒力基因和O抗原血清分型鉴定并分析。结果 共检出大肠埃希菌78株，总样本检出率为11.84%。水样的处理方式对水样中大肠埃希菌指标检出有明显差异，未消毒样品的合格率明显低于消毒处理的水样。其中检出16株致泻大肠埃希菌，总检出率为0.024%，水源水的致泻大肠埃希菌检出率明显高于其他类样品。16株致泻大肠埃希菌中检出4株肠道致病性大肠埃希菌、5株产肠毒素大肠埃希菌和7株肠道集聚性大肠埃希菌，除5株不能分型外，其余11株分为8种血清型。毒力基因和O抗原均具有优势分型。结论 辽宁省各类生活饮用水中水源水的大肠埃希菌检出率最高，生活饮用水消毒处理对降低大肠埃希菌的检出率至关重要；水源水、分散式供水、集中式供水中均检出致泻大肠埃希菌，其存在优势毒力基因，且热源稳定性...     

婴幼儿致病性大肠埃希菌的毒力基因检测研究

目的通过对婴幼儿腹泻病例粪便标本进行致病性大肠埃希菌的分离鉴定及毒力基因检测,从分子生物学方面研究致病性大肠埃希菌引起婴幼儿腹泻的分布,以降低婴幼儿致病性大肠埃希菌的感染率。方法收集江西省儿童医院2011-2012年婴幼儿腹泻病例357份粪便标本,按照致病性大肠埃希菌分离鉴定程序用EC肉汤增菌过夜,增菌后用麦康凯琼脂培养基分离培养,可疑菌用API 20E生化鉴定出大肠埃希菌,然后用聚合酶链反应(PCR)检测致病性大肠埃希菌相应的毒力基因。结果 357份腹泻患者粪便标本中,检出37份携带毒力基因的致病性大肠埃希菌,检出率为10.36%,携带毒力基因的肠聚集性大肠埃希菌(EAggEC)、肠道致病性大肠埃希菌(EPEC)、肠道侵入性大肠埃希菌(EIEC)、肠道出血性大肠埃希菌(EHEC)检出率分别为8.4%、0.56%、1.12%、0.28%,产肠毒素大肠埃希菌(ETEC)未检出。结论婴幼儿腹泻病例中致病性大肠埃希菌主要由EaggEC引起,其次为EIEC,进一步证实对致泻大肠埃希菌进行PCR毒力基因检测具有重要临床意义。     

腹泻症候群致泻性大肠埃希氏菌血清型及毒力基因检测

目的：通过对腹泻症候群中致泻大肠埃希菌的血清型和多重PCR检测,了解本地区致泻大肠埃希菌的血清群、型分布规律,所携带毒力基因及相互之间的相关性,为腹泻症候群中致泻大肠埃希菌的检测、鉴定提供科学依据,以提高阳性株的检出率。方法：对本地区2012-2013年腹泻症候群监测医院门诊未使用过抗生素腹泻患者的稀便、水样便、黏脓便或脓血便标本通过増菌、各种选择性培养基筛选出病原菌,后经生化试验、多重PCR试验和血清型分型试验进行鉴定。结果：本次检测共检出65株血清凝集致泻大肠埃希菌,分属EPEC、EIEC、ETEC,以EPEC为主,占83.08%,共8个血清型,其中血清型O55：H59、O128：H67占58.47%;共检出4种相关毒力基因,其中escV 49株（75.38%）。未分血清型菌株27株,检出相关毒力基因共5种,分属EPEC、EIEC、ETEC、EAEC,其中escV 15株（55.56%）。结论：本地区腹泻症候群中血清学阳性致泻大肠埃希菌以EPEC为主,常见血清型为O55：H59、O128：H67,毒力基因为escV、escV＋bfpB、invE、elt。未分血清型致泻大肠埃希菌毒力基因为escV、escV＋bfpB、invE、elt、astA,与已分型菌株携带基因基本一致。腹泻患者的大肠埃希菌检测中,在传统的细菌分离培养、生化试验、血清学鉴定的基础上,有必要进行大肠埃希菌的毒力基因检测,以提高阳性检出率,避免漏检,提高致泻性大肠埃希菌的诊断。     

致腹泻大肠埃希菌引起急性肠道感染调查分析

目的为检测急性肠道感染性腹泻中大肠埃希菌并了解其在人群中的分布状况。方法于2004年5～10月收集了我中心肠道门诊急性感染性腹泻病人543份粪便标本进行分离培养、生化鉴定和血清凝集实验以检测致腹泻大肠埃希菌，并用10种抗生素检测该菌的药物敏感性。结果致腹泻大肠埃希菌是急性肠道感染的主要病原之一。结论重视大肠埃希菌的检测，有利于早期作出明确的病原学诊断。     

与志贺菌和大肠埃希菌O157诊断血清交叉凝集反应细菌的分离和鉴定

目的 了解食品中能与志贺菌、大肠埃希菌O157发生交叉凝集的细菌,为防止误诊提供参考.方法 采用染色形态鉴别、血清学及生化等方法进行检测.结果 对食品风险监测205件样品中检出2种(3株)肠道杆菌能与志贺菌、大肠埃希菌O157诊断血清交叉凝集.结论 肠杆菌科、弧菌科某些种属细菌可携带与志贺菌、大肠埃希菌O157相同或相关抗原,故在鉴定这些杆菌时除依血清学外,还应加用生化试验等检测作出最后判定.     

两种菌与沙门菌菌体抗原交叉凝集报告

大肠埃希菌与枸橼酸杆菌属、沙门菌属等,在O抗原上存在很多交叉反应[1]。但两种细菌恰好与沙门菌菌体抗原凝集却很少有人报道。在今年从业人员健康体检大便培养中检出一株大肠埃希菌和一株弗劳地枸橼酸杆菌,它们分别与沙门菌O15、O3,19抗原诊断血清发生凝集。现将试验结果报道如下。     

浙江省舟山市269例海岛渔民病毒性肝炎型别分析

为探讨海岛渔民病毒性肝炎的病毒种类及其与临床的关系，对269例海岛渔民肝炎患者的型别进行了分析。研究对象为浙江省舟山市人民医院2001年1月至2004年12月住院的269例渔民病毒性肝炎患者，均为男性，年龄18～65岁，平均32．5岁。诊断标准根据2000年9月中华医学会传染病与寄生虫病学分会、肝病学分会联合修订的病毒性肝炎防治方案。患者人院后常规进行甲、乙、丙、丁、戊、庚型肝炎血清标志物检测，采用酶联免疫吸附法，HCV—RNA，HBV—DNA（采用PCR）。     

Recombinant M. smegmatis vaccine targeted delivering IL-12/GLS into macrophages can induce specific cellular immunity against M. tuberculosis in BALB/c mice

In the present study, we constructed a viable therapeutic vaccine of recombinant M. smegmatis mediated IL-12/GLS (granulysin) gene transfer into murine macrophages to exert the immunotherapy effects on the Mycobacterium tuberculosis infection. We tested this recombinant therapeutic vaccine in an in vivo study to determine its capability of stimulating host specific immune responses against M. tuberculosis. BALB/c mice intranasally immunized with the therapeutic vaccine developed an efficient Th1 protective immune response against M. tuberculosis which was equal to that of the BCG strain. Inoculation intranasally with this viable vaccine induced high level of serum IFN-gamma, IL-12 and IgG2a. The viable vaccine was capable of inducing purified protein derivative (PPD) antigen-specific splenocytes proliferation and IFN-gamma production from T cells in spleens of the immunized mice. In addition, intranasally inoculation with the viable vaccine can induce PPD antigen-specific sIgA production in the broncho-alveolar lavage fluid (BALF) of the immunized mice. No change of IL-4 level was found in all groups. The therapeutic mechanism of this viable vaccine against M. tuberculosis infection observed here appeared to be a result of the specific Th1 immune response activated by mycobacterium antigen from M. smegmatis and the expression of sIL-12/GLS in alveolar macrophages via the M. smegmatis-mediated gene transfer method. This research demonstrates that the therapeutic gene can be introduced into a host by viable mycobacteria works to induce the host specific immune response against M. tuberculosis infection in vivo. Since this therapeutic vaccine can strongly induce specific Th1 responses against M. tuberculosis in BALB/c mice and has no obviously harmfulness to the host simultaneously, the recombinant vaccine might be a potential candidate therapeutic vaccine against tuberculosis.     

Intranasal immunization of Balb/c mice against prion protein attenuates orally acquired transmissible spongiform encephalopathy

To test whether prion protein (PrP) specific secretory immunoglobulin A (sIgA) can be induced and protect against oral transmission of spongiform encephalopathy (SE) we immunized Balb/c mice either intragastrically or intranasally (i.n.) with a recombinant PrP-fragment (PrP90-231) and cholera toxin (CT) adjuvant. Since PrP90-231 was rapidly digested in intestinal lavage, aprotinin was added to some vaccine formulations. While an anti-CT response was elicited via both routes, solely i.n. immunization without aprotinin induced PrP-specific sIgA. They recognize predominantly PrP-oligomers as the antigen was aggregated in the vaccine formulations. Challenge experiments showed that the immune response induced by our protocol could not prevent disease, but increases the median survival of the animals. We conclude that PrP-specific sIgA reduce the infectivity of the inoculum and that complete protection against transmission of SE should be achievable by optimized immunization regimens.     

Enhanced mucosal immunogenicity of prion protein following fusion with B subunit of Escherichia coli heat-labile enterotoxin

Mucosal vaccine against prion protein (PrP), a major component of prions, is urgently awaited since the oral transmission of prions from cattle to humans is highly suspected. In the present study, we produced recombinant bovine and mouse PrPs fused with or without the B subunit of Escherichia coli heat-labile enterotoxin (LTB) and intranasally immunized mice with these fused proteins. Fusion with LTB markedly enhanced the mucosal immunogenicity of bovine PrP, producing a marked increase in specific IgG and IgA titer in serum. Mouse PrP also showed slightly increased immunogenicity following fusion with LTB. These results demonstrate that LTB-fused PrPs might be potential candidates for protective mucosal prion vaccines.     

The first experimental study on a candidate combined vaccine against hepatitis A and hepatitis E

To test the possibility of developing a combined vaccine against hepatitis A and E, groups of mice were immunized with different formulations containing different dosages of a commercially inactivated hepatitis A vaccine and a candidate recombinant hepatitis E vaccine. Monovalent vaccine components were used as controls. The experimental results showed that the combined vaccine could induce neutralizing antibodies against both hepatitis A virus (HAV) and hepatitis E virus (HEV) effectively in mice. Moreover, the inactivated hepatitis A vaccine could increase the immunogenicity of the recombinant HEV protein, and the recombinant HEV protein had no adverse effects on the immunogenicity of the inactivated HAV vaccine. Thus, the present study demonstrates an important first step for the further development of a combined hepatitis A and E vaccine.     

A comparison of immunogenicity and in vivo distribution of Salmonella enterica serovar Typhi and Typhimurium live vector vaccines delivered by mucosal routes in the murine model

We evaluated the immune responses elicited by attenuated Salmonella enterica serovar Typhi vaccine strain CVD 908-htrA and serovar Typhimurium strain SL3261 alone or as live vectors carrying a plasmid encoding fragment C of tetanus toxin (pTETnir15) in mice immunized intranasally and orogastrically, as well as the in vivo distribution of vaccine organisms following immunization. Higher serologic and proliferative responses against both vector and the foreign antigen were elicited when vaccines were delivered by intranasal route. Whereas both Salmonella strains were detected in the nasal tissue, lungs, and Peyer's patches following intranasal and orogastric immunization, larger numbers of vaccine organisms were recovered from these tissues when the vaccines were delivered intranasally.     

空肠弯曲菌peb1A基因的克隆表达及免疫性鉴定

目的:克隆空肠弯曲菌peb1A基因,构建其原核表达载体;鉴定重组表达产物的免疫原性。方法:PCR扩增空肠弯曲菌peb1A基因,构建pET-28a(+)-peb1A表达载体。转化E.coliBL21(DE3)后诱导表达,用SDS-PAGE鉴定表达产物。采用Ni-NTA亲和层析法收集表达的目的重组蛋白PEB1。采用CJ全菌抗体的Western blot鉴定rPEB1免疫反应性,双向免疫扩散试验鉴定rPEB1免疫家兔的抗血清效价。结果:所克隆的基因与报道的核苷酸序列同源性为99.9%,转化E.coliBL21(DE3)后,表达产物最高表达量占全菌总蛋白的33%左右,纯化后获得纯度为96%的重组蛋白。Western blot证实rPEB1能与CJ全菌抗体发生特异性免疫结合反应。兔抗rPEB1血清的双向免疫扩散效价为1:16。结论:成功地构建了高效表达载体pET-28a(+)-peb1A,并获得rPEB1,所表达的rPEB1具有良好的免疫原性和免疫反应性,为基因工程疫苗的研制奠定了基础。     

Evaluation of different promoter sequences and antigen sorting signals on the immunogenicity of Bacillus subtilis vaccine vehicles

Recombinant Bacillus subtilis strains, either in the form of spores or vegetative cells, may be employed as safe and low-cost vaccine vehicles. In this study, we studied the role of promoter sequences and antigen-sorting signals on the immunogenicity based on previously constructed B. subtilis episomal expression systems. Mice orally immunized with spores or cells encoding the B subunit of the heat labile toxin (LTB), originally expressed by some enterotoxigenic Escherichia coli (ETEC) strains, under control of the stress-inducible gsiB promoter developed higher anti-LTB serum IgG and fecal IgA responses with regard to vaccine strains transformed with plasmids encoding the antigen under control of IPTG-inducible (Pspac) or constitutive (PlepA) promoters. Moreover, surface expression of the vaccine antigen under the control of the PgsiB promoter enhanced the immunogenicity of vegetative cells, while intracellular accumulation of LTB led to higher antibody responses in mice orally immunized with recombinant B. subtilis spores. Specific anti-LTB antibodies raised in vaccinated mice recognized and neutralized in vitro the native toxin produced by ETEC strains. Nonetheless, only mice orally immunized with recombinant B. subtilis strains, either as vegetative cells or spores, expressing intracellular LTB under the control of the gsiB promoter conferred partial protection to lethal challenges with purified LT. The present report further demonstrates that B. subtilis plasmid-based heterologous protein expression systems are adequate for antigen delivery via the oral route.     

Clinical trial to evaluate safety and immunogenicity of an oral inactivated enterotoxigenic Escherichia coli prototype vaccine containing CFA/I overexpressing bacteria and recombinantly produced LTB/CTB hybrid protein

We have developed a new oral vaccine against enterotoxigenic Escherichia coli (ETEC) diarrhea containing killed recombinant E. coli bacteria expressing increased levels of ETEC colonization factors (CFs) and a recombinant protein (LCTBA), i.e. a hybrid between the binding subunits of E. coli heat labile toxin (LTB) and cholera toxin (CTB). We describe a randomized, comparator controlled, double-blind phase I trial in 60 adult Swedish volunteers of a prototype of this vaccine. The safety and immunogenicity of the prototype vaccine, containing LCTBA and an E. coli strain overexpressing the colonization factor CFA/I, was compared to a previously developed oral ETEC vaccine, consisting of CTB and inactivated wild type ETEC bacteria expressing CFA/I (reference vaccine). Groups of volunteers were given two oral doses of either the prototype or the reference vaccine; the prototype vaccine was administered at the same or a fourfold higher dosage than the reference vaccine.