鼠源性抗EHEC O157:H7 Stx2噬菌体Fab抗体库的构建及筛选

目的构建鼠源性抗EHEC O157∶H7 Stx2噬菌体Fab抗体库,并从中筛选特异性的抗体。方法用EHEC O157∶H7 Stx2类毒素免疫BALB/c小鼠,取脾分离淋巴细胞,提取总RNA,RT-PCR分别扩增抗体轻、重链(к和Fd)基因,经双酶切依次克隆入噬粒载体pComb3X中,电转化大肠杆菌XL1-Blue,以辅助噬菌体M13K07进行超感染,构建抗EHEC O157∶H7 Stx2的Fab噬菌体抗体库。以纯化的Stx2为抗原进行筛选,获得抗EHECO157∶H7Stx2的特异性Fab抗体,Western blot法检测噬菌体抗体与毒素抗原的结合活性,并对所得阳性克隆进行基因序列分析。结果构建了一个库容为1.56×107的Fab抗体库,筛选出3株特异性较强的阳性克隆,其中2个可与Stx2A1亚单位抗原反应,1个可与Stx2B亚单位抗原反应。基因序列分析显示,轻、重链可变区氨基酸序列与GenBank中已注册的鼠免疫球蛋白可变区氨基酸序列同源性分别为98.5%和99.6%。结论已成功构建了鼠源性抗EHEC O157∶H7 Stx2噬菌体Fab抗体库,为进一步制备抗EHECO157∶H7Stx2的治疗性人源化抗体奠定了基础。     

霍乱毒素B亚单位植物细胞表达载体的构建及其在人参细胞中的表达

目的构建霍乱毒素B亚单位(CTB)植物细胞表达载体,并在人参细胞中进行表达。方法根据人参偏爱密码子,采用引物延伸PCR法合成CTB基因,连入pBI121质粒,构建植物细胞表达载体,转化人参细胞后,采用PCR、RT-PCR和Western blot进行鉴定。结果测序结果表明,PCR法合成的目的基因序列与设计完全一致,构建的植物细胞表达载体经双酶切鉴定显示,所含基因片段大小与预期相符。提取转基因人参细胞基因组DNA和mRNA,分别进行PCR和RT-PCR鉴定,均可见约400 bp的特异性片段。Western blot分析可见相对分子质量约12 000的特异性条带。结论已成功构建了含霍乱毒素B亚单位的植物细胞表达载体,并在人参细胞中获得表达。     

大肠杆菌不耐热肠毒素B亚单位的原核表达及活性鉴定

目的克隆大肠杆菌不耐热肠毒素B亚单位(ltB)基因,构建其原核表达质粒,并对表达产物进行活性鉴定。方法采用PCR技术从产毒大肠杆菌129株基因组DNA中扩增ltB基因,克隆入载体pET-32a(+)中,构建原核表达质粒pET-32a(+)-ltB,转化E.coli BL21(DE3),IPTG诱导表达,Ni2+-NTA树脂层析柱进行纯化,纯化产物采用ELISA法鉴定其与牛GM1的结合活性。结果重组表达质粒经菌落PCR鉴定证明构建正确,所克隆的ItB基因与GenBank中报道的相应核苷酸序列的同源性为99.9%。重组菌诱导4h,目的蛋白表达量最高,约占菌体总蛋白的25%。纯化的重组LTB蛋白纯度约为96%,具有与牛GM1特异性结合的生物学活性。结论已成功克隆了ltB基因,并构建了其原核表达质粒,表达的重组蛋白具有一定的生物学活性。     

EspA-Stx2A1融合蛋白的表达、纯化及其免疫学活性

目的在原核表达系统中表达肠出血性大肠杆菌O157∶H7(EHECO157∶H7)Ⅲ型分泌蛋白EspA与Stx2毒素A1亚单位(Stx2A1)的融合蛋白,并对表达蛋白进行纯化及免疫学活性检测。方法PCR扩增espA和stx2A1全长基因,利用重叠延伸PCR技术获得espA-stx2A1融合基因,T-A克隆后插入表达载体pET-28a(+),构建原核表达质粒pET-28a(+)-espA-stx2A1,转化大肠杆菌BL21(DE3),分别在37℃和25℃用IPTG诱导表达。以EspA单克隆抗体亲和层析柱纯化目的蛋白,免疫小鼠,检测其免疫原性及抗血清的反应原性,并以天然Stx2毒素攻击,观察保护效果。通过细胞毒试验检测免疫小鼠抗血清的体外中和作用。结果重叠延伸PCR方法扩增出1319bp的融合基因片段,重组表达质粒构建正确;目的蛋白在25℃时的表达量明显高于37℃,约占菌体总蛋白的40%。两种温度下,目的蛋白均主要以包涵体形式存在;纯化后蛋白纯度可达90%。Western blot结果证实,融合蛋白与EspA单克隆抗体和Stx2A1单克隆抗体均发生特异性反应。融合蛋白免疫小鼠制备的抗血清能分别与O157∶H7的EspA、Stx2A发生特异性免疫反应。融合蛋白免疫小鼠能够抵御致死剂量天然Stx2毒素的攻击,保护率达95%。免疫小鼠血清可以中和天然Stx2毒素对HeLa细胞的毒性作用。结论已成功表达了EspA-Stx2A1融合蛋白,纯化的蛋白显示出较好的免疫保护效果,为研制EHECO157∶H7基因工程多亚单位疫苗奠定了基础。     

以霍乱毒素B亚单位为载体的甲型副伤寒多糖结合疫苗理化及生物学特性

目的制备甲型副伤寒沙门菌(Salmonella paratyphi A,SPA)O特异多糖(O-specific polysaccharide,OSP)-霍乱毒素B亚单位(cholera toxin B subunit,CTB)结合疫苗,并初步探讨其理化及生物学特性。方法 SPA OSP多糖原液(简称OSP多糖)经溴化氰活化,己二酰肼(ADH)衍化,在碳二亚胺(EDAC)作用下与CTB偶联,结合物经Sepharose4FF柱纯化,获得多糖-蛋白结合物,对其理化指标、血清特异性、免疫原性及血清体外杀菌力进行检测。结果制备的SPA OSP-CTB结合物(简称OSP-CTB结合物)衍化度为(2. 63±0. 13)%、多糖回收率为(74±0. 2)%;鉴别试验显示OSP-CTB结合物与SPA O诊断血清、CTB抗体均有阳性沉淀线产生;免疫小鼠血清中OSP-CTB结合物抗体滴度显著增长,阳转率达100%,血清体外杀菌抗体滴度为1∶64。结论用溴化氰活化多糖制备的SPA OSP-CTB结合物具有良好免疫原性,可进一步进行疫苗研发。     

幽门螺杆菌尿素酶B亚单位基因的克隆及其高效表达

目的克隆幽门螺杆菌尿素酶B亚单位(UreB)基因,构建原核表达载体,并进行高效表达。方法以幽门螺杆菌基因组DNA为模板,PCR扩增UreB基因,双酶切后,与质粒pET-22b(+)连接,构建表达载体pET-22b(+)/UreB,分别转化E.coliBL21(DE3)、Origam(iDE3)和Rossetta(DE3),经IPTG诱导后,进行SDS-PAGE和Western blot分析。结果经酶切及测序,证明幽门螺杆菌UreB基因的原核表达载体构建正确。3种重组菌的诱导表达产物经SDS-PAGE分析,均可见相对分子质量为64000的目的蛋白条带,Rossetta(DE3)重组菌目的蛋白表达量最高,约占菌体蛋白的35%。Western blot结果表明,表达的目的蛋白具有良好的反应原性。结论已成功克隆了幽门螺杆菌UreB基因,并在大肠杆菌Rossetta(DE3)中获得了高效表达。     

重组大肠杆菌不耐热肠毒素B亚单位表达条件的优化及其佐剂作用

目的优化重组大肠杆菌不耐热肠毒素B亚单位(LTB)的表达条件,并检测其黏膜佐剂作用。方法通过向LB培养基中添加葡萄糖(终浓度为0.5%)、低温培养诱导等方法诱导LTB的表达;采用阳离子交换法对目的蛋白进行纯化;以LTB作为佐剂进行免疫试验。结果可溶性重组大肠杆菌不耐热肠毒素(LTB)的表达量明显提高,纯化后蛋白含量达95%以上,经纯化的LTB作为三价流感裂解疫苗或gD抗原的佐剂免疫BALB/c小鼠,可提高小鼠血清及黏膜的抗体水平,且其效果与铝化剂及福氏佐剂相当或更高。结论优化了LTB的表达条件,且表达的LTB具有较强的黏膜免疫佐剂作用。     

百日咳丝状血凝素与志贺毒素B亚单位的融合表达及其免疫保护效果评价

目的 融合表达百日咳丝状血凝素(filamentous haemagglutinin,FHA)片段FHA1877-2250与志贺毒素B亚单位(Shiga toxin B subunit,StxB)蛋白,并对融合蛋白StxB-Fha1877-2250的免疫保护效果进行评价.方法 设计引物将StxB片段连接至FHA1877...     

食品能力验证中大肠埃希氏菌O157:H7的分离鉴定及质量控制

目的:从能力验证样品中分离、鉴定大肠埃希氏菌O157:H7,提高食源性致病菌的检测能力。方法:本文参照GB4789.36-2016《食品微生物学检验大肠埃希氏菌O157:H7/NM检验》对2份样品进行检测,对符合VITEK 2 Compact生化鉴定结果的进行血清学鉴定,同时使用荧光定量PCR方法进行联合检验分析。结果:2份能力验证样品均检出大肠埃希氏菌O157:H7,结果均为满意。结论:国标法与荧光定量PCR方法结合进行检测,相互验证,结果可靠性更高,准确性更强。同时在检验过程中应注意培养基、试剂的质量控制,灵活应用目标菌特异性提高检出率等。     

百日咳毒素S1亚单位截短片段的表达、纯化及初步应用

目的表达并纯化百日咳毒素(PT)S1亚单位截短片段S146,以其制备抗体,初步建立检测PT的双抗体夹心ELISA法。方法PCR扩增S146基因,亚克隆至载体pUC18,鉴定正确后,插入表达载体pET16b,转化大肠杆菌BL21(DE3),IPTG诱导表达。表达产物经SDS-PAGE和Western blot鉴定后,用镍离子亲和层析柱纯化,复性后的蛋白免疫家兔,制备抗血清,纯化后作为包被抗体,建立检测PT的双抗体夹心ELISA法,并检测其特异性。结果表达的重组蛋白相对分子质量约为19000,主要存在于破菌沉淀中,表达量占菌体总蛋白的44%;纯化后蛋白纯度为94.5%;家兔抗血清可特异性识别天然PTSI;建立的双抗体夹心ELISA法特异性良好。结论已成功表达、纯化了PTS1亚单位截短片段S146,并以纯化的抗S146抗体作为包被抗体,初步建立了检测PT的双抗体夹心ELISA法,为研制特异性检测试剂和提供一种疫苗的质量控制方法奠定了基础。     

Characterization and Genomic Analysis of Escherichia coli O157:H7 Phage UAE_MI-01 Isolated from Birds

Verotoxin-producing Escherichia coli O157:H7 is responsible for the majority of foodborne outbreaks worldwide and may lead to death. Bacteriophages are natural killers of bacteria. All previously reported E. coli O157:H7 phages were isolated from ruminants or swine. Here, we report for the first time a phage isolated from bird feces in the United Arab Emirates (UAE), designated as UAE_MI-01, indicating birds as a good source of phages. Thus, phages could be a tool for predicting the presence of the host bacteria in an animal or the environment. UAE_MI-01 was found to be a lytic phage that was stable at wide ranges of pH, temperature, and chemical disinfectants, and with a burst size of almost 100 plaque-forming units per host cell after a latent period of 20 min and an adsorption rate constant (K) of 1.25 × 10⁻⁷ mL min⁻¹. The phage genome was found to be 44,281 bp long with an average GC content of 54.7%. The presence of the phage indicates the presence of the host cell E. coli O157:H7 in wild birds. Therefore, other birds, mainly poultry, could be also investigated for the presence of this pathogenic bacterium. To the best of our knowledge, this is the first report of an E. coli O157:H7 bacteriophage isolated from a bird.     

益生菌对大肠埃希菌O157:H7的抑制作用

以11%脱脂牛奶和MRS肉汤作为培养基,在适宜大肠埃希菌O157:H7生长的条件(37℃,有氧)下,考察益生菌混合物对大肠埃希菌O157:H7生存生长的影响。前期实验获得了5株益生菌和大肠埃希菌O157:H7各自的生长曲线,确认了酸性条件(培养基pH值为3.8)对大肠埃希菌O157:H7的抑制作用。经过对酸性环境的适应并不能提高大肠埃希菌O157:H7菌株对益生菌的抵抗能力。大肠埃希菌O157:H7(37℃,有氧状态培养8 h)不能在经过益生菌有氧状态下培养后的培养基澄清液中存活,但将该澄清液的pH值调到6.5(新鲜MRS肉汤培养基的pH值),大肠埃希菌O157:H7又可以在其中生长。结果表明,高浓度的益生菌对大肠埃希菌O157:H7菌株有抑制作用,这一方面是由于益生菌降低了培养环境的pH值,另一方面也是由于益生菌本身在有氧状态下产生了抑制大肠埃希菌O157:H7的代谢产物。     

幽门螺杆菌尿素酶表位疫苗的构建、表达及鉴定

目的构建和表达幽门螺杆菌(Helicobacter pylori,Hp)尿素酶B亚单位(UreB)表位串联体(Uepi)与大肠杆菌不耐热肠毒素B亚单位(LTB)融合蛋白表位疫苗,并对其生物学及免疫学特性进行鉴定。方法设计引物,采用PCR法分别扩增UreB表位多肽编码基因Uepi和LTB编码基因,重叠延伸PCR法将两段基因拼接,T-A克隆后,构建融合基因表达质粒pET-22b(+)-Uepi-LTB,经酶切鉴定后转化E.coliBL21(DE3),IPTG诱导表达,并对表达产物进行鉴定。结果PCR扩增出206bp和336bp的目的片段,重叠延伸PCR扩增出524bp的融合目的基因片段。原核表达质粒pET-22b(+)-Uepi-LTB经酶切及测序鉴定,与设计序列一致。重组工程菌pET-22b(+)-Uepi-LTB/BL21经IPTG诱导,目的蛋白表达率约25%,SDS-PAGE分析相对分子质量约20000,目的蛋白以包涵体形式表达,纯化后蛋白纯度达96%,Westernblot鉴定该融合蛋白与兔抗LTB多抗血清可发生特异性结合。结论HpUreB表位串联体与LTB融合蛋白的表位疫苗经基因克隆,可获得高效表达,并显示出较好的免疫活性,为新一代Hp疫苗的研制奠定基础。     

A sandwich-type assay based on quantum dot/aptamer bioconjugates for analysis of E. Coli O157:H7 in microtiter plate format

An optical assay based on CdSe/ZnS quantum dots (QD)/NH₂-Apt bioconjugates for the pathogen detection was presented. QDs with carboxyl functional groups and 72-mer aptamer (against E. coli outer membrane proteins) were used as probes and sensing element. E. coli O157:H7 was selected as a model pathogen and 96-well plate assay in the sandwich hybridization format was constructed. Poly-L-lysine-coated 96-well plate surfaces were used as support material where thiol functionalized aptamers were immobilized by 4-(N-Maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide (sulfo-SMCC). After incubation with the bacteria, CdSe/ZnS QDs/aptamer bioconjugates were added. The fluorescence signals were followed before and after addition of bioconjugates. Probe concentrations, incubation time with E. coli O157:H7 were also optimized. The bioassay could detect the pathogen down to 10² CFU/mL with high selectivity. The detection system was successfully employed in samples, in the presence of interfering compounds.     

E. coli O157:H7 Desorption by Rhamnolipid Biosurfactant in Water-Unsaturated Porous Media

Animal waste is a valuable resource, which can add organic matter, nitrogen, phosphorous and potassium as well as other nutrients needed for plant growth when it is used in the agricultural field. When applied to an agricultural field, infectious agents or pathogenic organisms in the animal waste are usually retained in the soil. At the same time, surfactants are popularly utilized in pesticide applications to aid pesticide solubility and mobility. Consequently, the retained pathogenic strains might be flushed out of the soil matrices with a possibility of contaminating the groundwater. In this research, we investigated desorption of E. coli O157:H7 pre-deposited in silica sand and its subsequent release and transport in the presence of rhamnolipid biosurfactant in unsaturated laboratory columns. Based on the experimental observations, it was discovered that retained E. coli O157:H7 was released during flushing with rhamnolipid biosurfactant solutions. E. coli O157:H7 release rate coefficient increased both with the increase of rhamnolipid biosurfactant concentration and the increase of water saturation. With the increase of rhamnolipid biosurfactant concentration, the air–water surface tension decreased. With the increase of water saturation, the air–water interfacial area decreased. Both the decreased air–water surface tension and air–water interfacial area favored E. coli O157:H7 release. Increase of E. coli O157:H7 release rate coefficient with the decrease of air–water surface tension was more pronounced for low air–water interfacial area (i.e., high water saturation). Similarly, increase of E. coli O157:H7 release rate coefficient with the decrease of air–water interfacial area was more pronounced for low air–water surface tension (i.e., high rhamnolipid biosurfactant concentration). E. coli O157:H7 retention in unsaturated systems was found to be controlled by the capillary force, which was greatly impacted by water saturation and air–water surface tension. Increase of water saturation and rhamnolipid biosurfactant concentration both contributed to the decrease of the capillary force, resulting in increased E. coli O157:H7 release rate.     

RV-G/LTB双基因融合真核表达质粒的构建及其在Vero细胞中的表达

目的 构建RV-G/LTB双基因融合真核表达质粒,并在Vero细胞中表达.方法 应用重叠延伸PCR方法,采用一定的linker序列(Gly4Ser)2将已克隆的狂犬病病毒糖蛋白(Rabies virus glycoprotein,RV-G)基因片段和大肠杆菌不耐热肠毒素B亚单位(E.coli heat-labile e...