猪源大肠杆菌脂多糖的提取、纯化及活性分析

为了从猪源大肠杆菌中获得纯度高、产率高的脂多糖（lipopolysaccharides,LPS）,并评价其毒力,本试验采集仔猪腹泻粪便作为病料,进行细菌分离鉴定和16S rDNA鉴定,采用热酚水法提取细菌的LPS,通过DNaseⅠ、RNaseA、蛋白酶K和醇沉法对LPS进行纯化,通过苯酚硫酸法、Bradford法和紫外分光光度法测定LPS多糖、蛋白及核酸含量,鲎试剂定量法测定其活性,采用小鼠急性毒性试验方法测定小鼠半数致死量（LD₅₀）。结果显示,分离获得一株具有高致病性的猪源大肠杆菌,纯化的猪源大肠杆菌LPS平均产率为3.74%,其中多糖含量为13.40%,蛋白含量为0.48%,核酸含量为0.06%,主要条带集中在14~160 ku范围内,鲎试剂定量法测定其活性为1.21×10⁷ EU/mg,提取LPS对小鼠的LD₅₀为31.86 mg/kg。该猪场仔猪腹泻是由致病性大肠杆菌引起,获得其毒力因子LPS纯度高、产率高、毒力强,这将为临床防制猪大肠杆菌病及LPS提取纯化提供理论依据。     

铜绿假单胞菌混合蛋白疫苗制备研究

为了制备免疫力高、不良反应少的铜绿假单胞菌混合蛋白疫苗,试验采用间接ELISA方法测定内毒素蛋白、类毒素和外膜蛋白免疫后小鼠的血清抗体效价,鲎试剂凝胶半定量方法测定上述3种蛋白的内毒素含量,采用"高免疫力+低内毒素"作为配伍指标制备5组混合蛋白疫苗,分别编为1～5号,间接ELISA法测定混合蛋白疫苗的免疫力水平,鲎试剂凝胶半定量方法测定混合蛋白疫苗的内毒素含量,小鼠腹腔注射法测定混合蛋白疫苗的异常毒性。结果表明:三种蛋白免疫后,小鼠血清抗体效价均随免疫进程持续升高,内毒素蛋白抗体效价最高,达到1∶12 500;内毒素蛋白中内毒素含量(E_内)为8.00 EU/mL,类毒素中内毒素含量(E_类)为1.73 EU/mL,外膜蛋白中内毒素含量(E_外)为2.24 EU/mL;3号混合蛋白疫苗(内毒素蛋白∶类毒素∶外膜蛋白=60∶20∶20)血清抗体效价达到了1∶12 800,其内毒素含量(E_3)为5.66 EU/mL;3号混合蛋白疫苗异常毒性试验合格。说明3号混合蛋白疫苗免疫效果好,与内毒素蛋白持平,且内毒素含量低于内毒素蛋白。     

副猪嗜血杆菌脂多糖的提取、鉴定及初步应用

为建立一种有效提取高纯度副猪嗜血杆菌(Haemophilus parasuis,HPS)脂多糖(Lipopolysaccharides,LPS)的方法,进一步阐明HPS的致病机制,通过热酚水法提取LPS,浓缩后,采用酶解法和超离法纯化提取物,分别用硫酸-苯酚法、BCA法、紫外分光光度法,测定LPS提取物中多糖、蛋白及核酸的含量,通过SDSPAGE电泳银染方法鉴定提取物。将提取的LPS作用于猪肺泡巨噬细胞,通过荧光定量方法检测IL-1β的表达量差异。结果显示:纯化的LPS平均产率为2.56%;LPS提取物中不含还原性单糖,多糖含量为3.27%,蛋白含量为0.77%,核酸含量为0.90%;提取的LPS能够引起猪肺泡巨噬细胞IL-1β表达量显著上调。因此,通过该方法提取纯化的LPS可以用于HPS与宿主细胞间作用机制的研究。     

一株内毒素缺陷型大肠杆菌的构建与鉴定

为了降低大肠杆菌表达外源蛋白时产生的内毒素(LPS),本研究利用Red重组和Cre-Lox敲除系统相结合的方法,敲除大肠杆菌DH10B-2B中与LPS合成相关的基因kdsD、lpxL、lpxM、pagP、lpxP、eptA,并在其OmpT基因处插入弗朗西斯菌的磷酸酶基因lpxE去除LPS中类脂A的1-磷酸基团,获得改造的E.coli DH10B-2B-Δ6-lpxE。利用鲎试剂检测10~8cfu/mL～10~5cfu/mL DH10B-2B与DH10B-2B-Δ6-lpxE经超声破碎的裂解液中LPS含量;将9组小鼠每组5只分别腹腔注射PBS及浓度为10~(10)cfu/mL～10~7cfu/mL的两种裂解液,观察小鼠的临床症状并利用ELISA试剂盒检测小鼠脾脏中IL-6及TNF-a的表达水平。结果显示,经鲎试剂检测10~6cfu/mL DH10B-2B裂解液中的LPS结果为阳性,LPS含量≥10 000 U/mL,而10~8cfu/mL DH10B-2B-Δ6-lpxE裂解液LPS结果虽为阳性,但LPS含量≤100 U/mL。将DH10B-2B-Δ6-lpxE裂解液以10~9cfu/mL的剂量腹腔注射的小鼠与对照组小鼠均无任何异常,而注射10~9cfu/mL DH10B-2B裂解液的小鼠出现精神不振,反应迟钝,眼角有脓性分泌物以及腹泻等症状;IL-6及TNF-a检测结果显示,与DH10B-2B组小鼠相比,DH10B-2B-Δ6-lpxE组小鼠细胞因子的表达水平明显降低。本研究通过对E.coli LPS合成过程中关键基因的改造获得一株LPS缺陷的大肠杆菌DH10B-2B-Δ6-lpxE,其产生LPS的能力明显下降。该菌株既可以用作低LPS重组蛋白表达系统,也可以用于低毒力LPS的提取,为相关免疫增强剂的研究奠定基础。     

屎肠球菌荚膜多糖的提取纯化及其多糖含量的测定

为提取纯化屎肠球菌（E.faecium）荚膜多糖（CPS）并测定其多糖含量,试验采用高压法破碎菌体,加入核糖核酸酶（25 μg/mL）、脱氧核糖核酸酶（0.5 mg/mL）、溶菌酶（0.25 mg/mL）及蛋白酶K （1.25 mg/mL）等去除核酸和蛋白质,得到荚膜多糖粗提物,经过30 ku和100 ku超滤离心管超滤浓缩和DEAE Sepharose Fast Flow层析柱层析后,得到纯化的屎肠球菌荚膜多糖,再利用苯酚-硫酸法测定其含量。结果表明,成功从屎肠球菌中提取纯化出荚膜多糖,其含量为75.21%,初步确定了屎肠球菌荚膜多糖的提取纯化方法,为今后相关疫苗的研制奠定了基础。     

加米霉素注射液内毒素检查方法研究

参照《中华人民共和国兽药典》2015版(后简称兽药典)一部附录200页细菌内毒素检查法的有关规定,对加米霉素注射液内毒素限度及检查方法进行了研究。结果显示,加米霉素注射液在0.075～0.3mg/mL时对鲎试剂无增强或抑制作用,产品内毒素含量均小于限定值125EU/mL,符合规定。通过试验研究,建立了加米霉素注射液的内毒素检查方法,且采用凝胶法对其进行细菌内毒素检查是可行的。     

血液内毒素对哺乳仔猪腹泻的影响

前腔静脉采集试验组(腹泻组)与对照组(不腹泻组)母猪和仔猪血液,用LAL试剂(鲎试剂)测定血液内毒素含量,用分光光度法测定D-乳酸浓度和二胺氧化酶活性。结果表明,试验组母猪血液内毒素含量平均为1.978 EU/m L,比对照组1.007 EU/m L高96.43%,说明母猪体内高含量的内毒素是引起新生仔猪腹泻的原因之一。试验组仔猪血液内毒素含量为1.125 EU/m L,比对照组0.276 EU/m L高307.61%,说明仔猪体内较高含量的内毒素是引起仔猪腹泻的直接原因。试验组仔猪血浆D-乳酸含量达12.33μg/m L,比对照组仔猪血浆D-乳酸含量8.17μg/m L高4.16μg/m L。试验组仔猪血浆二胺氧化酶活性9.12 U/L,比对照组仔猪血浆二胺氧化酶活性5.63 U/L高3.49 U/L,说明腹泻仔猪肠黏膜屏障受到一定程度的破坏。     

硫酸链霉素细菌内毒素定量测定法的研究

应用鲎试剂动态浊度法，将硫酸链霉素制备成4、2、1、0．5mg／mL溶液，定量添加标准内毒素进行干扰预试验，计算回收率，确定不干扰浓度为0．5—2mg／mL。从中选出1mg／mL为最佳浓度，然后进行3个批次硫酸链霉素正式干扰试验。在供试品中定量添加标准内毒素，其回收率分别为125．8％、126．1％、127．3％，均在50％-200％之间，说明此浓度的供试品对鲎试剂反应无干扰作用。因此，内毒素含量在0．015—1EU／mL范围内，将硫酸链霉素制备成浓度为1．0mg／mL的溶液，可用于细菌内毒素的定量检测。     

鲎试剂检测乳品中内毒素浓度的研究

为研究乳品中内毒素的浓度范围,本研究检测了不同牛奶样品中内毒素的浓度和细菌总数,分析了牛奶中内毒素的浓度及其与细菌污染的关系,并对来自牛场的107份鲜奶样品和市售的乳制品进行了检测。结果表明,鲎试剂检测法可用于牛奶内毒素的检测,但不能用于判断牛奶中细菌含量的高低;85.1%(87/107)的鲜奶样品的内毒素浓度低于300EU/mL,5.6%(6/107)的鲜奶样品内毒素浓度超过500EU/mL;不同厂家、不同类别的乳制品内毒素浓度差异较大。本研究为奶制品的安全检验提供了参考。     

副鸡禽杆菌外膜囊泡提取及成分分析

为研究副鸡禽杆菌外膜囊泡(OMVs)的构成,提取A型副鸡禽杆菌的OMVs,纯化并透射电镜观察其形态结构,检测其内毒素含量和DNA含量,分析其是否具有血凝活性,测定OMVs蛋白含量,并用质谱法分析了OMVs的蛋白组成成分。结果显示,副鸡禽杆菌的OMVs呈球形、泡状,直径为20 nm～300 nm,含有内毒素和少量核酸物质。该OMVs不能凝集鸡红细胞,Western blot结果表明,OMVs蛋白与该菌外膜蛋白单克隆抗体有阳性反应,构成OMVs的蛋白分子质量大多集中于65 ku和13 ku处,蛋白成分主要有ATP依赖性RNA解旋酶等多种功能酶、铁链霉素B受体、膜蛋白等结构成分和一些未知功能的假定蛋白等,为进一步分析副鸡禽杆菌外膜囊泡在致病和免疫方面的作用奠定基础。     

Extraction,Purification and Activity Analysis of Lipopolysaccharides from Escherichia coli Isolated from Swine

In order to obtain higher purity and high yield of LPS isolated from Escherichia coli, and evaluate its virulence,excrement of diarrhea piglet was collected as pathological samples, of which, some strains of Escherichia coli were isolated and identified. And furthermore, a strain of Escherichia coli was acquired by 16S rDNA identification. The LPS from this strain of Escherichia coli was isolated with the hot-phenol water method and purified by DNaseⅠ, RNaseA, proteinase K treatment and alcohol precipitation. The polysaccharide, protein and nucleic acid content of purified LPS were detected by phenol-sulfuric acid method, Bradford method and UV points spectrophotometric method. And its bioactivity and acute median lethal dose was determinated by tachypleus amebocyte lysate (TAL) method and acute toxicity experiment method, respectively. The results showed that a strain of highly pathogenic Escherichia coli was successfully isolated. The average yield of purified LPS was 3.74%, the proportion of the polysaccharide was 13.40%, the protein was 0.48%, and the nucleic acid was 0.06%. SDS-PAGE electrophoresis and silver stain showed that the bands were mainly concentrated in the range of 14 to 160 ku. The LAL bioactivity of the prepared LPS was 1.21×10⁷ EU/mg, LD₅₀ of the mouse abdominal cavity was 31.86 mg/kg. The results revealed that the diarrhea piglets in the farm was caused by pathogenic Escherichia coli,purified LPS from this strain of Escherichia coli had the advantages of higher purity, high yield and strong virulence, which would provide theoretical data for clinical prevention and treatment of diarrhea piglets.     

Isolation and biological characterization of two lipopolysaccharides and a capsular-enriched polysaccharide preparation from Haemophilus pleuropneumoniae

A smooth-type lipopolysaccharide (HpS-LPS), a rough-type lipopolysaccharide (HpR-LPS), and a capsular-enriched polysaccharide preparation (HpC-PS) were extracted and purified from Haemophilus pleuropneumoniae serotype 5, strain J45, by the use of phenol-water (HpS-LPS) and phenol-chloroform-petroleum-ether (HpR-LPS) techniques. Chemical analysis of the HpS-LPS and HpR-LPS indicated that they contained 0.7% and 4.4% (w/w) 2-keto-3-deoxyoctonate, 11.8% and 10.4% phosphate, 0.8% and 0.8% nucleic acid, and 0.8% and 1.1% protein, respectively. The HpC-PS contained 0.3% 2-keto-3-deoxyoctonate, 1.4% phosphate, 0.2% nucleic acid, and 0.8% protein. With sodium dodecyl sulfate-polyacrylamide-gel electrophoresis, the HpS-LPS banded as a smooth-type LPS and the HpR-LPS banded as a rough-type LPS. Electrophoresis of HpC-PS indicated the presence of a broad high molecular weight band. Gelation of Limulus amoebocyte lysate developed at a minimum concentration of 8 ng/ml of HpS-LPS, 0.3 ng/ml of HpR-LPS, and 35 ng/ml of HpC-PS. The lipopolysaccharide preparations provoked a positive dermal Shwartzman reaction in rabbits and swine, a biphasic febrile response in rabbits, and a monophasic response in swine. Responses were typical of endotoxic activity with swine having greater sensitivity than rabbits. The chick embryo 50% lethal dose was calculated to be 7.3 ng for HpS-LPS, 1.6 ng for HpR-LPS, 5.1 ng for the lipopolysaccharide of Escherichia coli 0111:B4; and the HpC-PS was not toxic. In all assays, HpR-LPS was significantly more toxic than was HpS-LPS. The HpC-PS preparation was not toxic, even at high concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

原儿茶酸对LPS诱导的小鼠子宫内膜炎的作用机制研究

【目的】 探究原儿茶酸对脂多糖(LPS)诱导的小鼠子宫内膜炎的作用机制。【方法】 将60只小鼠随机分为对照组、LPS组、LPS+不同浓度原儿茶酸(20、40、80 mg/kg)组共5组,每组各12只。对照组小鼠用微量注射器经阴道灌注50 mL生理盐水,LPS组灌注50 mL LPS(1 mg/kg),原儿茶酸治疗组灌注50 mL LPS前1 h分别腹腔注射20、40、80 mg/kg原儿茶酸,注射24 h后,颈椎脱臼法处死所有小鼠。收集子宫组织,通过HE染色检测子宫组织病理变化,用试剂盒检测髓过氧化物酶(MPO)活性,ELISA法检测炎症因子肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)和IL-6含量;Western blotting法检测p65、核因子-κB(NF-κB)的抑制蛋白(IκB)以及磷酸化p65、IκB(p-p65、p-IκB)蛋白表达水平。【结果】 组织病理结果显示,对照组小鼠子宫组织形态正常,子宫内膜上皮结构正常;LPS组小鼠子宫组织出现严重的炎性细胞浸润和子宫内膜上皮充血及水肿。与LPS组相比,随着原儿茶酸浓度的增加,小鼠子宫组织中炎性细胞明显减少,子宫内膜上皮充血水肿情况也明显得到改善。MPO活性检测表明,与对照组相比,LPS组MPO活性极显著升高(P<0.01);与LPS组相比,原儿茶酸治疗组MPO活性均极显著降低(P<0.01),并呈剂量依赖性。ELISA结果表明,与对照组相比,LPS组小鼠子宫内膜组织中TNF-α、IL-1β和IL-6含量均极显著升高(P<0.01);与LPS组相比,原儿茶酸治疗组小鼠子宫内膜组织中炎性细胞因子TNF-α、IL-1β和IL-6含量均极显著降低(P<0.01),并呈剂量依赖性。Western blotting结果表明,与对照组相比,LPS组p-p65和p-IκB的表达水平均极显著升高(P<0.01);与LPS组相比,原儿茶酸治疗组p-p65和p-IκB的表达水平均极显著降低(P<0.01),且呈剂量依赖性。【结论】 原儿茶酸通过减轻子宫组织的病理变化,降低子宫组织MPO活性,抑制NF-κB信号通路及炎症细胞因子的产生,对LPS诱导的小鼠子宫内膜炎起到保护作用。     

大雁、斑头雁和天鹅源沙门氏菌血清及脉冲场凝胶电泳分型研究

为了解大雁、斑头雁及天鹅源沙门氏菌流行特征和脉冲场凝胶电泳（PFGE）分型,本研究对33株沙门氏菌进行血清型鉴定,采用PFGE对其进行基因分型,并用BioNumerics 6.6软件进行聚类分析。鉴定出丙型副伤寒沙门氏菌6株、汤卜逊沙门氏菌19株、乙型副伤寒沙门氏菌1株、伊鲁木沙门氏菌1株、奥斯陆沙门氏菌1株、5株未定型。PFGE聚类分型共分为16个型别,其相似度为53.7%~100.0%,且大多数菌株与人源菌株相似度超过80%。结果表明,大雁、斑头雁及天鹅源沙门氏菌相同血清型PFGE带型高度相似,可能来自同一克隆株,不同血清型PFGE带型差别较大。     

Immunochemical relationship of three antigens purified from Pasteurella multocida strain P-1059

Three antigens were prepared from a type-3 avian strain of Pasteurella multocida, and their chemical and immunologic characteristics were studied. An antigen, designated 2.5S, was extracted with 2.5% NaCl solution and purified by chromatography. Lipopolysaccharide (LPS) was extracted with phenol-water, and a third antigen, designated FS, was extracted in 0.3% formalin solution containing 0.85% NaCl and purified by differential centrifugation. The 2.5S and the FS antigens consisted of 40% protein and 15% carbohydrate, whereas LPS did not contain a substantial amount of protein. A major protein component with a molecular weight of 44,000 was detected in the 2.5S antigen, as well as in the FS antigen. Of the 3 antigens, LPS had the highest activity in mouse lethality and Limulus lysate tests. Antigenic cross-reactions among the 3 antigens were demonstrated by immunodiffusion tests. The 2.5S antigen was indistinguishable from the FS antigen, as both antigens contained the LPS component of approximately 45%. Treatments with various reagents indicated that the 2.5S and FS antigens contained at least 2 antigenic determinants. The first was a heat-stable protein sensitive to protease or phenol-water, and the second was a periodate-sensitive carbohydrate, which was a major antigenic determinant on the LPS antigen.     

PFGE Genotyping and Serotype Analysis of Salmonella Isolated from Goose,Anserindicus and Cygnus

To understand the Salmonella epidemic characteristics and pulsed field gel electrophoresis（PFGE）genotyping of goose,Anserindicus and Cygnus,33 strains of Salmonella were serotype typed by PFGE and genotyped with BioNumerics 6.6 cluster analysis software.The results showed that there were 16 PFGE patterns,which displayed 6 strains Salmonella paratyphi C,19 strains Salmonella thompson,1 strains Salmonella paratyphi B,1 strain Salmonella irumu,1 strain Salmonella oslo,5 undifferentiated.PFGE similarity is about 53.7% to 100.0%,and most of the strains and anthropogenic strains similarity was more than 80%.The results showed that goose,Anserindicus and Cygnus Salmonella serotypes same PFGE type with a high degree of similarity,and possibly from the same clone,quite different serotypes PFGE band pattern difference.     

华南地区肉鸽源沙门氏菌的鉴定及遗传进化分析

本试验从华南地区发病肉鸽肠道和肝脏中用BS培养基分离、纯化沙门氏菌,并进行多项生化试验和血清型鉴定试验,结果显示各项生化检测指标均符合沙门氏菌的特点;血清型鉴定试验结果表明该分离菌属沙门氏菌属A-F群。进一步对该分离菌的16S rRNA片段测序及遗传进化分析,确定该分离菌为沙门氏菌菌株,与鼠伤寒沙门氏菌同源性为100%。对该分离菌进行动物回归试验,结果表明该沙门氏菌菌株为华南地区肉鸽的致病菌株。该结果为进一步研究华南地区肉鸽沙门氏菌的致病机理和防控奠定了基础。     

1株猪腺病毒3型的分离培养及其fiber蛋白多克隆抗体的制备

本研究旨在对临床1份腹泻病料进行病原的分离培养并探究该病原编码的主要抗原蛋白的生物学特点。采用巢式PCR检测病料猪腺病毒3型（Porcine adenovirus type 3,PADV3）核酸并进行病毒分离培养,采用免疫过氧化物酶单层细胞染色法（IPMA）进行病毒血清学鉴定,PCR扩增fiber基因,将其ORF区克隆至pET-28a（+）载体并转化大肠杆菌BL21（DE3）感受态细胞,IPTG诱导重组fiber蛋白表达,采用SDS-PAGE和Western blotting鉴定重组蛋白,将纯化的重组蛋白免疫家兔,制备多克隆抗体并测定抗体免疫活性。结果表明,病料为PADV3核酸阳性,感染ST细胞产生典型的"葡萄串"细胞病变效应（CPE）,P5~P11代次中的PADV3核酸稳定,分离的毒株P5代为PADV3型血清学阳性,fiber基因开放阅读框（ORF）为1 296 bp（GenBank登录号：MT774498）,与PADV3毒株的核苷酸相似性最高（86.1%）,与其他毒株相似性均低于40%;原核表达的重组fiber蛋白分子质量为45.2 ku,fiber蛋白多克隆抗体与PADV3感染的细胞呈特异性的细胞核染色。本试验成功分离得到1株PADV3毒株,命名为PADV3-HY1812,所制备的fiber蛋白多克隆抗体具有良好的免疫活性。     

猪丁型冠状病毒N蛋白原核表达及其多克隆抗体的制备

【目的】表达与纯化猪丁型冠状病毒（Porcine deltacoronavirus,PDCoV）的核衣壳（nucleocapsid,N）蛋白,并制备其多克隆抗体（polyclonal antibody,PcAb）。【方法】以PDCoV CHN-HN-1601分离株基因组RNA为模板,利用RT-PCR扩增PDCoV全长的N基因编码序列,将其克隆至原核表达载体pET-28a（+）中构建重组质粒pET-28a-PDCoV-N。经酶切和测序鉴定后,将重组质粒转化大肠杆菌BL21（DE3）感受态细胞,利用0.5 mmol/L IPTG于37 ℃诱导表达12 h。在非变性条件下,利用Ni-NTA琼脂糖树脂从菌体裂解液上清中纯化N-端和C-端均携带6×His标签的重组N蛋白,并将其免疫新西兰白兔制备抗血清。利用Protein A/G琼脂糖树脂从免疫兔的抗血清中亲和层析纯化多克隆抗体,并对多克隆抗体进行Western blotting和间接免疫荧光试验（IFA）鉴定及抗体效价的间接ELISA测定。【结果】重组PDCoV-N蛋白以可溶性和包涵体两种形式表达,分子质量约为42 ku;上清可溶性N蛋白的纯化纯度可达90%、蛋白浓度为0.45 mg/mL;纯化后的多克隆抗体纯度较高,ELISA方法检测其效价为1∶6 400,能特异性地识别重组N蛋白和PDCoV,与可引起猪腹泻的猪流行性腹泻病毒（PEDV）、猪传染性胃肠炎病毒（TGEV）、猪轮状病毒（PRoV）无交叉反应。【结论】本研究成功制备重组PDCoV-N蛋白及其多克隆抗体,为后续深入研究N蛋白的功能、PDCoV优化血清学检测方法、免疫层析试纸条的制备及开展PDCoV相关基础研究提供参考。