EHECO157∶H7志贺样毒素Ⅱ结合亚单位Stx2B的基因克隆、表达与纯化

目的克隆表达肠出血性大肠杆菌(EHEC)O157∶H7志贺样毒素B亚单位(Shiga liketoxin2B,Stx2B),并对其初步纯化。方法设计引物采用PCR法自EHEC O157∶H7基因组扩增志贺样毒素B亚单位的编码基因stx2b,T-A克隆后构建原核表达质粒pET-22b(+)-stx2b,经测序鉴定后转化E.coliBL21(DE3),IPTG诱导表达,以Tricine-PAGE和Western blot分析目的蛋白的表达,并采用固相镍离子亲和层析纯化重组蛋白。结果扩增的stx2b基因约210bp;原核表达质粒pET-22b(+)-stx2b经酶切和测序鉴定与预期序列一致;并在E.coliBL21(DE3)中得到高效可溶性表达,蛋白表达率约30%;PAGE初步测定目的蛋白的相对分子质量(Mr)约7×103;经固相镍离子亲和层析纯化后,蛋白纯度可达90.1%。结论EHEC O157∶H7志贺样毒素B亚单位的高效表达与初步纯化,有利于进一步的生物学性质研究。     

EHEC O157:H7志贺样毒素Ⅱ(Stx2)的克隆表达与活性研究

目的克隆表达肠出血性大肠杆菌(EHEC)O157：H7志贺样毒素Ⅱ(Shiga like toxinⅡ,Stx2),并鉴定其生物学活性。方法采用PCR法自EHEC O157：H7基因组扩增志贺样毒素Ⅱ的编码基因stx2,T-A克隆后构建原核表达质粒pET-11c(+)-six2,经测序鉴定后转化E．coli BL21(DE3),IPTG诱导表达,以SDS-PAGE、Western blot分析目的蛋白的表达。以EHEC O157：H7野生菌株作对照,通过对HeLa细胞的细胞毒作用及对小鼠攻毒实验,鉴定重组蛋白的生物学活性。结果扩增的stx2基因约1230bp；原核表达质粒pET-11c(+)-stx2经酶切和测序鉴定与预期序列一致；并在E．coli BL21 (DE3)中得到表达,蛋白表达率约25％；PAGE初步测定目的蛋白的相对分子质量(M_r)约72×10~3；重组蛋白Stx2对HeLa细胞具有细胞毒作用,CD_(50)为35pg/ml；Stx2对小鼠致死剂量LD_(100)为10μg。结论成功克隆并表达了Stx2重组蛋白,为探讨O157：H7菌的感染机制及其防治研究奠定了一定的基础。     

EHEC O157:H7志贺毒素Ⅱ型A、B亚单位的表达及功能鉴定

目的 克隆、表达肠出血性大肠杆菌(enterohemorrhagic Escherichia coli,EHEC)O157:H7志贺毒素(Shiga toxin,Stx)Ⅱ型A、B亚单位,并鉴定其免疫学功能.方法 从EHECO157:H7基因组中克隆编码Stx Ⅱ型A、B亚单位的基因,经测序、酶切后,导入表达载体pGEX-4T-2,转化大肠杆菌Topl0F',经诱导、纯化得到谷胱甘肽S-转移酶(glutathione S-transferase,GST)-A和GST-B融合蛋白.分别用A、B亚单位融合蛋白免疫BALB/c小鼠获得高免血清,观察A、B亚单位血清对小鼠进行毒素攻击的保护作用.结果 Stx Ⅱ型A、B亚单位的GST融合蛋白获得高效表达并纯化;A、B亚单位的高免血清可以保护小鼠对致死量毒素的攻击.结论 Stx Ⅱ型A、B亚单位蛋白可以作为EHEC O157:H7疫苗的候选分子,同时针对A、B亚单位蛋白的中和单抗可以对易感人群起到紧急保护作用.     

黄连素和6种抗生素对大肠杆菌O157∶H7中国分离株产生志贺毒素的影响

目的：研究黄连素和6种常用抗生素对大肠杆菌O157：H7中国分离株产生志贺样毒素的影响。方法：利用志贺毒素对Vero细胞具有细胞毒性，并可使其释放乳酸脱氢酶（LDH）的原理，使用Vero细胞的细胞毒性检测试剂盒，检测培养基中的LDH的含量。结果：将大肠杆菌O157：H7在环丙沙星，氨苄西林，庆大霉素，链霉素，青霉素，红霉素的亚抑制浓度中培养后，可增加大肠杆菌O157：H7对Vero细胞的毒性，但不同菌株有差异，环丙沙星对大肠杆菌O157：H7的最小抑制浓度很低，但其对vero细胞毒性作用，随着药浓度的降低而增高，黄连素在试管内对大肠杆菌O157：H7没有抑制作用，和抗生素相比，黄连素对大肠杆菌O157：H7产生和释放志贺毒素比较小。结论：黄连素在试管内对大肠杆菌O157：H7的生长抑制作用不明显，其刺激大肠杆菌O157：H7产生和释放志贺毒素的作用较抗生素小。     

EHEC O157:H7志贺毒素ⅡA1亚单位蛋白的构建表达与免疫原性鉴定

目的 构建表达GST-STX2A1融合蛋白并鉴定其免疫原性.方法 通过PCR反应从EHEC O157:H7 EDL933标准株菌中扩增STX2A1基因,将此基因克隆到GST融合蛋白原核表达载体PGEX-6p-1,得到阳性克隆PGEX-6p-1/STX2A1,转化大肠杆菌BL21,IPTG诱导GST-STX2A1融合蛋白的表达,采用亲和层析纯化该蛋白,SDS-PAGE分析其表达量、表达形式和纯度;用纯化的融合蛋白免疫Balb/c小鼠,免疫双向扩散鉴定抗血清效价,Western blotting鉴定抗血清的特异性.结果 成功构建重组质粒PGEX-6p-1/STX2A1,经DNA测序证实插入序列与设计完全一致,并在大肠杆菌中表达出Mr约为5.3×104大小的可溶性目的 蛋白,经亲和层析纯化后纯度可达90%,免疫Balb/c小鼠产生的抗血清效价为1:16,Western blotting表明可与天然STx2A蛋白反应.结论 在大肠杆菌中成功表达了可溶性的GST-STX2A1融合蛋白,该蛋白具有较好的免疫原性,为O157:H7亚单位疫苗及制备抗STX2A1的单克隆抗体提供了必要的物质基础.     

肠出血性大肠杆菌O157:H7基因工程疫苗免疫小鼠的实验研究

目的 研究肠出血性大肠杆菌O157:H7基因工程疫苗免疫小鼠后对该菌感染的保护能力.方法 将60只BALB/c小鼠平均分为5组,分别为3种抗原单独免疫、三抗原联合免疫和PBS对照组,用注射方式免疫3次,抗原和佐剂均为每次100μg/只.采集免疫前和各次免疫后7d的小鼠血清检测抗体,末次免疫10d后以109CFU的0157全菌液经口感染小鼠.观察小鼠临床症状及死亡情况,检测粪便与肠道带菌情况,并作病理组织学检测.结果 各抗原免疫组小鼠特异抗体明显增加,感染后各组小鼠均有死亡,IntiminC300、Stx2B、HlyAN436和联合免疫组保护率分别为73%、64%、36%和91%.未病死小鼠粪便排菌情况:对照组22d,实验组为5～14d.肠道带菌情况:对照组阳性率为100%,实验组为25%～83%.病死小鼠肺脏、肝脏均有明显组织病理学改变.结论 Intim-inC300、Stx2B和HlyAN436疫苗对于O157感染具有一定的预防作用,而联合免疫效果又大于单独免疫.     

产志贺毒素大肠杆菌O157∶H7的亚碲酸钾抗性基因簇分布和抗性水平

目的 了解产志贺毒素大肠杆菌O157：H7的亚碲酸盐抗性基因簇的分布和抗性水平的关系。方法运用PCR和核酸杂交方法进行基因检测，使用平皿法检测亚碲酸钾抗性水平。结果在所检测的志贺菌、沙门菌、霍乱弧菌、耶尔森菌、泌尿道致病性大肠杆菌、肠产毒性大肠杆菌、肠致病性大肠杆菌、肠侵袭性大肠杆菌、肠黏附性大肠杆菌、肠出血性大肠杆菌中，只有大肠杆菌O157：H7具有ter(tellurite resistance，ter)基因簇。所检测的34株产志贺毒素的大肠杆菌O157：H7有33株具有ter基因簇，所检测的18株不产生志贺毒素的大肠杆菌O157，4株具有ter基因簇，但是没有该毒力岛的其它基因。ter基因阳性的33株菌中，亚碲酸钾的抗性水平一般较高，4株在750μg/ml以上，19株为500～750μg/ml，10株为250～500μg/ml；而4株ter基因簇阴性的菌株，也表现了高水平的耐药性，其抗性水平均为500～750μg/ml。其它ter基因簇阴性的菌株，20株耐药性为5～10μg/ml，1株10～20μg/ml，6株20～50μg/ml，2株50～100μg/ml，1株100～250μg/ml。结论ter基因簇在产志贺毒素的大肠杆菌O157：H7中高频率存在，而在其它病原菌中没有检测到。高水平的亚碲酸钾抗性可以作为选择性分离产志贺毒素大肠杆菌O157：H7的指标之一。     

肠出血性大肠杆菌O157∶H7志贺毒素I型A亚基的表达与鉴定

目的表达肠出血性大肠杆菌(EHEC)O157∶H7志贺毒素1型A亚基(Stx1A)重组蛋白并鉴定。方法从EHEC O157∶H7基因组中扩增编码Stx1A的基因,经测序无误后,克隆入表达载体pET-22b(+),转化大肠杆菌BL21(DE3),经诱导、纯化获得目的蛋白Stx1A,并对纯化的目的蛋白进行质谱鉴定。重组的Stx1A蛋白免疫BALB/c小鼠,观察小鼠抗血清与EHEC O157∶H7毒株特异性反应。结果 PCR扩增的Stx1A基因为945 bp,成功构建重组质粒pET22b(+)-Stx1a,重组蛋白在原核细胞中获得高效表达,通过AKTATM-His亲和层析柱获得纯化。质谱分析表明目的蛋白为Stx1A。Western blot显示鼠抗Stx1A血清可与EHEC O157∶H7毒株产生的天然毒素蛋白结合。结论成功克隆了EHEC O157∶H7 Stx1A基因,并获得重组表达,为后续研究奠定基础。     

大肠杆菌O157∶H7基因组分析的启示

大肠杆菌Ｏ15 7∶Ｈ7是 1982年新发现的一种病原菌 ,近年来在美国、日本、欧洲等地已经成为重大的公共卫生问题。 2 0 0 1年美国和日本先后发表了大肠杆菌Ｏ15 7∶Ｈ7ＥＤＬ933和ＳＡＫＡＩ菌株的基因组序列〔1,2〕。Ｐｅｒｎａ等报道〔2〕,与大肠杆菌Ｋ12ＭＧ16 5 5菌株的序列比较 ,ＥＤＬ933菌株有很多外源性基因的插入。因此 ,将大肠杆菌Ｋ12ＭＧ16 5 5菌株特异性的序列命名为“Ｋ”岛 ,将大肠杆菌Ｏ15 7∶Ｈ7ＥＤＬ933菌株特异性的序列命名为“Ｏ”岛 ,发现ＥＬＤ933菌株有分子量大于 5 0ｂｐ的 177个“Ｏ”岛和 2 34个“Ｋ”岛。“Ｏ”…     

应用酶联免疫吸附试验检测肠出血性大肠杆菌O157:H7

目的制备和纯化O157：H7抗原，免疫家兔和豚鼠，获得高效价的抗O157：H7免疫血清并进行纯化及酶标记。建立对O157：H7感染患者快速诊断方法，做到早期发现及时治疗，有效控制疫情的蔓延。方法ELISA双抗体夹心法检测O157：H7抗原。步骤：包被特异性抗体，加处理后的粪便等标本，然后加入抗O157：H7酶结合物，最后加入底物显色。结果本课题所研制的抗O157：H7酶结合物只对O157：H7呈阳性反应，而与其他相关细菌无交叉反应。结论应用酶联免疫吸附试验(即双抗体夹心法)对肠出血性大肠杆菌O157：H7抗原的检测较常规法实验程序简捷、快速、敏感。临床和现场验证结果表明，其方法具有灵敏度高，特异性强操作简单等特点，为肠出血性大肠杆菌O157：H7的鉴定及快速诊断提供了一种新的检测手段。     

肠出血性大肠杆菌O157∶H7紧密黏附素免疫保护性片段的基因克隆与表达

目的　克隆表达肠出血性大肠杆菌 (EHEC)O1 5 7∶H7紧密黏附素 (intimin)免疫保护性片段 (intiminC30 0 )。方法　设计引物采用PCR法自EHECO1 5 7∶H7基因组扩增紧密黏附素及其免疫保护性片段的编码基因eae与eaeC30 0 ,T A克隆后构建原核表达质粒pET 2 8a(+) eae及pET 2 8a(+) eaeC30 0 ,经测序鉴定后转化E .coliBL2 1 (DE3) ,IPTG诱导表达 ,PAGE检测。结果　PCR法自EHECO1 5 7∶H7基因组扩增出了约 2 80 0bp和 90 0bp的目的片段 ;原核表达质粒pET 2 8a(+) eae及pET 2 8a(+) eaeC30 0经酶切及测序鉴定与预期序列一致。转化E .coliBL2 1 (DE3)后IPTG诱导目的蛋白表达率约 2 5 %和 30 %;PAGE初步测定目的蛋白的相对分子质量 (Mr)约 96× 1 0 3和 35× 1 0 3,破菌后电泳证实目的蛋白均以包涵体形式表达。结论　EHECO1 5 7∶H7紧密黏附素免疫保护性片段经基因克隆后获得了较高的表达量 ,为进一步的生物学性质及免疫学特性研究奠定了基础。     

肠出血型大肠埃希菌O157:H7Tir的表达、纯化及不同免疫途径对其免疫效价的影响

目的 克隆、表达和纯化肠出血型大肠埃希菌(enterohaemorrhagic E.coli,EHEC)O157:H7转位紧密黏附素受体蛋白(Tir),观察不同免疫途径对其免疫效价的影响,为EHEC O157:H7亚单位疫苗的研究提供了实验资料.方法 扩增tir基因,克隆到pET-30a(+)载体上,转化至大肠埃希菌BL21/DE3,诱导表达目的 蛋白,通过Ni-IDA亲和层析进行纯化;将重组蛋白Tir免疫小鼠,检测血清和粪便提取物中抗体效价.结果 双酶切和测序鉴定结果均显示重组质粒pET-30a (+)-tir构建成功.SDS-PAGE结果表明,目的 蛋白Tir在大肠埃希菌BL21(DB3)中得到表达.皮下免疫和鼻腔免疫小鼠,血清中均能检测到高效价的IgG类抗体,鼻腔免疫组小鼠血清和粪便lgA类抗体效价明显高于皮下免疫组.结论 Tir蛋白具有一定的免疫原性.

Abstract：

Objective To clone and express translocation intimin receptor(Tir)of enterohemorrhagic Escherichia coli(EHEC)O157:H7,and to analyze the effect of different routes on the induction of immunity to the recombinant protein.Methods The tir eucoding genes were amplified from EHEC O157:H7 strain guangzhou 246 genome,and genes were cloned into the vector pET-30a(+).The pET-30a(+)tir recombinant was transformed into E.coli BL21.and expression was induced bv IPTG.The expressed product was analyzed by SDS-PAGE and purified by Ni-IDA affinity chromatography.The immunized mice sera and fecal against the recombinant protein was detected.Resuits The length of the tir is 1677 bp,with the initiation codon ATG and the termination codon TAA.Double enzyme digestion and DNA sequencing confirmed that the recombinant expression plasmid pET-30a(+)-tir was constructed.The recombinant protein was expressed in Escherichia coli expression system,and was purified by Ni-IDA affinity chromatography.The mice were able to produce a high serum IgG antibody titer after both subeutaneous and intranasal immunizations.Meanwhile,the intranasal immunization induced serum and fecal IgA antibody titer was significantly higher than that of the subcutaneous immunization group.Conclusion Tir molecule is potential vaccine candidate for preventing EHEC disease.     

Antimicrobial resistance patterns of Shiga toxin-producing Escherichia coli O157:H7 and O157:H7- from different origins

Shiga toxin-producing Escherichia coli (STEC) serotypes including O157:H7 (n = 129) from dairy cows, cull dairy cow feces, cider, salami, human feces, ground beef, bulk tank milk, bovine feces, and lettuce; and O157:H7- (n = 24) isolated from bovine dairy and bovine feedlot cows were evaluated for antimicrobial resistance against 26 antimicrobials and the presence of antimicrobial resistance genes (tetA, tetB, tetC, tetD, tetE, tetG, floR, cmlA, strA, strB, sulI, sulII, and ampC). All E. coli exhibited resistance to five or more antimicrobial agents, and the majority of isolates carried one or more target antimicrobial resistance gene(s) in different combinations. The majority of E. coli showed resistance to ampicillin, aztreonam, cefaclor, cephalothin, cinoxacin, and nalidixic acid, and all isolates were susceptible to chloramphenicol and florfenicol. Many STEC O157:H7 and O157:H7-isolates were susceptible to amikacin, carbenicillin, ceftriaxone, cefuroxime, ciprofloxacin, fosfomycin, moxalactam, norfloxacin, streptomycin, tobramycin, trimethoprim, and tetracycline. The majority of STEC O157:H7 (79.8%) and O157:H7- (91.7%) carried one or more antimicrobial resistance gene(s) regardless of whether phenotypically resistant or susceptible. Four tetracycline resistant STEC O157:H7 isolates carried both tetA and tetC. Other tetracycline resistance genes (tetB, tetD, tetE, and tetG) were not detected in any of the isolates. Among nine streptomycin resistant STEC O157:H7 isolates, eight carried strA-strB along with aadA, whereas the other isolate carried aadA alone. However, the majority of tetracycline and streptomycin susceptible STEC isolates also carried tetA and aadA genes, respectively. Most ampicillin resistant E. coli of both serotypes carried ampC genes. Among sulfonamide resistance genes, sulII was detected only in STEC O157:H7 (4 of 80 sulfonamide-resistant isolates) and sulI was detected in O157:H7- (1 of 16 sulfonamide resistant isolates). The emergence and dissemination of multidrug resistance in STEC can serve as a reservoir for different antimicrobial resistance genes. Dissemination of antimicrobial resistance genes to commensal and pathogenic bacteria could occur through any one of the horizontal gene transfer mechanisms adopted by the bacteria.     

Shiga toxin 2 overexpression in Escherichia coli O157:H7 strains associated with severe human disease

Variation in disease severity among Escherichia coli O157:H7 infections may result from differential expression of Shiga toxin 2 (Stx2). Eleven strains belonging to four prominent phylogenetic clades, including clade 8 strains representative of the 2006 U.S. spinach outbreak, were examined for stx2 expression by real-time PCR and western blot analysis. Clade 8 strains were shown to overexpress stx2 basally, and following induction with ciprofloxacin when compared to strains from clades 1–3. Differences in stx2 expression generally correlated with Stx2 protein levels. Single-nucleotide polymorphisms identified in regions upstream of stx2AB in clade 8 strains were largely absent in non-clade 8 strains. This study concludes that stx2 overexpression is common to strains from clade 8 associated with hemolytic uremic syndrome, and describes SNPs which may affect stx2 expression and which could be useful in the genetic differentiation of highly-virulent strains.     

Purification and some properties of Shiga-like toxin from Escherichia coli O157:H7 that is immunologically identical to Shiga toxin

A cytotoxin to Vero cells (Shiga-like toxin), which was neutralized by antibody against purified Shiga toxin produced by Shigella dysenteriae 1, was purified from Escherichia coli O157:H7, isolated from a patient with hemorrhagic colitis. The purification procedure consisted of ammonium sulfate fractionation, DEAE-cellulose column chromatography, chromatofocusing column chromatography and high performance liquid chromatography. About 200 micrograms of purified Shiga-like toxin was obtained from cell extracts of 14 liters of culture with a yield of about 15%. The purified Shiga-like toxin showed identical physicochemical, biological and immunological properties to those of Shiga toxin. Purified Shiga-like toxin and Shiga toxin also had the same mobilities on polyacrylamide disc gel electrophoresis and polyacrylamide gel isoelectrofocusing. On sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis, purified Shiga-like toxin migrated as two bands corresponding to the A and B subunits, and these migrated to the same positions as A and B subunits of Shiga toxin. The amino acid composition of the purified Shiga-like toxin was also similar to that of Shiga toxin. The purified Shiga-like toxin showed various biological activities: lethal toxicity to mice when injected intraperitoneally, the LD50 being 30 ng per mouse; cytotoxicity to Vero cells, killing about 50% of the cells at 6 pg; and fluid accumulation in rabbit ileal loops at concentrations of more than 1.25 micrograms/loop. These values are comparable with those obtained with Shiga toxin. In an Ouchterlony double gel diffusion test, the lines formed by the purified Shiga-like toxin and Shiga toxin fused, indicating that the two toxins were immunologically identical.     

Purification and some properties of a Vero toxin from Escherichia coli O157:H7 that is immunologically unrelated to Shiga toxin

A cytotoxin to Vero cells (Vero toxin) was purified from Escherichia coli O157:H7 isolated from a patient with hemorrhagic colitis by ammonium sulfate fractionation, DEAE-cellulose column chromatography, repeated chromatofocusing column chromatography and repeated high performance liquid chromatography. About 440 micrograms of purified Vero toxin was obtained from 12 liters of culture with a yield of about 22%. The purified Vero toxin showed similar cytotoxic activity to that of Shiga toxin to Vero cells and killed about 50% of the Vero cells at 1 pg. The activity was lost on heating the toxin at 80 degrees C for 10 minutes, but not at 60 degrees C for 10 minutes. The toxin also showed lethal toxicity to mice when injected intraperitoneally, the LD50 being 1 ng per mouse. The purified Vero toxin consisted of A and B subunits with molecular weights of about 35,000 and 10,700, respectively, which were slightly larger than those of Shiga toxin. On polyacrylamide gel disc electrophoresis, the mobility of the purified Vero toxin differed from that of Shiga toxin. The isoelectric point of the toxin was 4.1, which was also different from that of Shiga toxin (pI = 7.0). Furthermore, Vero toxin and Shiga toxin were found to be immunologically unrelated; anti-Vero toxin did not react with Shiga toxin, and similarly anti-Shiga toxin did not react with the Vero toxin in either the Ouchterlony double gel diffusion test or enzyme-linked immunosorbent assay. The Vero toxin purified in this work was found to be immunologically identical to VT2 and Shiga-like toxin II reported previously.     

Isogenic strain of Escherichia coli O157:H7 that has lost both Shiga toxin 1 and 2 genes.

An Escherichia coli O157:H7 strain isolated from a patient with hemorrhagic colitis was found to exhibit two slightly different colony morphology types on differential medium. Each morphological type, designated TT12A and TT12B, was isolated, and serological testing using various assays confirmed that both strains carried the O157 and the H7 antigens. Biochemical testing showed that the strains had identical profiles on AP120E analysis and, like typical O157:H7 strains, did not ferment sorbitol or exhibit beta-glucuronidase activity. Analysis with a multiplex PCR assay showed that TT12B did not carry the gene for either Shiga toxin 1 (Stx1) or Stx2, whereas these genes were present in TT12A and the toxins were produced. Apart from that, both strains carried the +93 gusA mutation, the cluster I ehxA gene for enterohemolysin, and the eae gene for gamma-intimin, which are all characteristics of the O157:H7 serotype. Phenotypic assays confirmed that both strains exhibited enterohemolysin activity and the attachment and effacing lesion on HeLa cells. Multilocus enzyme electrophoresis analysis showed that the strains are closely related genetically and belong in the same clonal group. Pulsed-field gel electrophoresis (PFGE) typing of XbaI-digested genomic DNA revealed that the two strains differed by two bands but shared 90% similarity and clustered in the same clade. All other non-Stx-producing O157:H7 strains examined clustered in a major clade that was distinct from that of Stx-producing O157:H7 strains. The findings that TT12B was identical to TT12A, except for Stx production, and its PFGE profile is also more closely related to that of Stx-producing O157:H7 strains suggest that TT12B was derived from TT12A by the loss of both stx genes.