F18菌毛粘附素F基因(fedF)在大肠杆菌中的表达及对小鼠的免疫保护效果

引起早期仔猪断奶腹泻(PWED)的主要致病菌是产肠毒素型大肠杆菌(ETEC),免疫预防是防治腹泻最主要的手段。本研究采用通过大肠杆菌(Escherichia coli)表达的重组F18菌毛粘附素F(FedF)作为抗原,在小鼠(Mus musculus)中探索以此为靶标免疫防治仔猪腹泻的可能性。根据相应F18菌毛粘附素基因(fedF)设计了一对添加EcoRⅠ和SalⅠ酶切位点的引物,以E.coli F18为模板扩增出全长908bp含20个信号肽序列的fedF片段,并与原核表达载体pET-30a(+)连接,导入大肠杆菌BL21,得到基因工程菌E.coli[pET-fedF],后经IPTG诱导及菌液蛋白电泳分析,结果表明,大小约为32.9kD的重组蛋白FedF可成功诱导表达;将重组工程菌口服免疫BALB/c小鼠,可有效地诱导小鼠血清中FedF特异性IgG的产生(P/N值>2.0)及小鼠肠粘膜sIgA显著增加了2.07倍(与空载体组pET-30比较)(P<0.05);攻毒试验表明,口服基因工程菌E.coli[pET-fedF]可显著地有效保护小鼠免予攻毒死亡,免疫保护率可达62.5%。研究结果提示,FedF黏附蛋白基因工程菌可通过口服诱导小鼠特异的体液和粘膜免疫反应,以此保护小鼠免受产肠毒素型大肠杆菌的进攻,因而有望作为一种口服疫苗防治产肠毒素型大肠杆菌引起的仔猪腹泻。     

中草药制剂治疗仔猪疥螨病的试验

本试验应用自制中草药制剂治疗仔猪疥螨病 ,结果表明 ,疗效良好 ,有效率和治愈率分别为86.6%、 68.7% ,与常规用药敌百虫的效果差异不显著 ( P>0 .0 5) ,且此制剂具有使用安全、原料来源广、成本低之优点。     

益生菌制剂对早期断奶仔猪生长性能和免疫指标的影响

分析益生菌制剂对早期断奶仔猪生长性能和免疫指标的影响,选择仔猪40头,将仔猪随机分为两组,基础饲料选择标准玉米-豆粕型,对照组饲喂基础饲料,观察组在基础饲料基础上添加益生菌饲料,仔猪断奶第0 d、第7 d、第14 d、第21 d采集新鲜粪样、前腔静脉血、回肠和空肠,测定仔猪生长性能以及血清、粪便、肠黏膜抗体含量。结果表明,在仔猪生长性能方面,观察组仔猪在最后重量、平均日增重量、腹泻率方面均明显高于对照组(P0.05);在仔猪血清、粪便、肠黏膜抗体含量方面,断奶后21 d观察组仔猪血清Ig G含量增加量明显更多(P0.05),粪便中Ig G含量两组对比差异不显著(P0.05),观察组回肠黏膜以及空肠黏膜SIg A含量明显更高(P0.05)。在早期断奶仔猪饲料中添加益生菌制剂,能够显著增强仔猪生长性能,提高仔猪血清Ig G、回肠黏膜以及空肠黏膜SIg A水平,增强仔猪机体免疫力。     

流感复合多表位疫苗的设计及小鼠免疫研究

鉴于目前出现了多种亚型禽流感病毒共存的局面,研究多价流感疫苗具有重要意义。根据各表位的免疫学特性,结合分子生物学信息软件的模拟功能,以H 3、H 9亚型流感病毒的HA抗原表位、流感病毒的其他主要抗原(NP、NA、M)表位的基因为基础,再附以K ozak序列和适当的酶切位点,设计并合成大小为765 bp的复合多表位基因盒-Ep。i将Ep i基因克隆入真核表达载体pIRES1neo中,构建DNA重组体pIR-Ep i;将H 7HA、Ep、iH 5HA基因以融合表达方式克隆到pIRES1neo中,构建了DNA重组体pIRE-H 57-Ep。i以上述构建的DNA重组体与鸡痘病毒重组株对BALB/c小鼠进行免疫接种后,检测体液与细胞免疫指标。研究结果表明,流感病毒复合多表位DNA重组体pIRE-H 57-Ep、ipIRE-Ep i免疫组小鼠均能产生针对H 3亚型流感病毒的特异性抗体,抗体效价(1∶1 600~1∶6 400)低于灭活疫苗免疫组(1∶12 800),但均高于其他对照免疫组(P<0.01)。pIRE-H 57-Ep i免疫组抗H 5、H 7 HA抗体效价分别为1∶12 800和1∶6 400,均高于其他免疫组(P<0.05)。pIRE-Ep i免疫组抗H 5、H 7HA抗体效价(1∶200,1∶100)较低,与其他对照免疫组差异不显著。pIRE-H 57-Ep i与pIRE-Ep i免疫组抗H 9亚型A IV的HA抗体效价(1∶6 400,1∶3 200)明显高于其他免疫组(P<0.05)。与PBS对照组及空质粒pIRES1neo对照组比较,用所构建的重组体免疫的各试验组小鼠的T淋巴细胞亚类CD 4 和CD 8 的数量显著提高(P<0.05)。pIRE-H 57-Ep i与pIRE-Ep i试验组的CD 4 与CD 8 淋巴细胞的数量较灭活苗显著增多(P<0.05),而各重组体免疫组之间差异不显著。此外,所有组的CD 4 /CD 8 比值均稳定在1.5~2.0,表明无异常免疫应答出现。EL ISPOT检测小鼠脾淋巴细胞分泌IFNγ-斑点数实验结果表明,pIRE-H 57-Ep、ipIRE-Ep i试验组的IFNγ-斑点数量(>70)比灭活苗(<40)显著增多(P<0.05),而各重组体免疫组之间差异不显著。上述结果说明,所构建的DNA重组体有良好的免疫原性,为最终获得能同时预防多种亚型流感病毒的多价疫苗奠定了基础。     

乳酸杆菌对断奶仔猪生长性能的影响

试验选用32头品种、胎次、饲养水平相同、健康的断奶仔猪随机分为4组,对照组饲喂剔除抗生素和抗菌素的仔猪基础日粮,试验Ⅰ组、试验Ⅱ组和试验Ⅲ组分别在仔猪基础日粮的上添加12、15、18mL／kg乳酸杆菌,通过30天的试验期后,测定4组的生长性能指标。结果表明:对照组的腹泻率明显高于试验组,腹泻率最低的是试验Ⅲ组;头平均日增重试验Ⅲ组与其它3组差异显著(P＜0．05),对照组和试验Ⅱ组差异显著(P＜0．05),其中试验Ⅲ组头平均日增重最高,为358g,对照组最低,为303g;试验Ⅲ组与其它3组料重比差异显著(P＜0．05),试验Ⅱ组与试验Ⅰ组差异不显著(P＞0．05),和对照组差异显著(P＜0．05),料重比较为理想的是试验Ⅲ组,其次是试验Ⅱ组。     

乳酸杆菌对断奶仔猪生长性能的影响

试验选用32头品种、胎次、饲养水平相同、健康的断奶仔猪随机分为4组,对照组饲喂剔除抗生素和抗菌素的仔猪基础日粮,试验I组、试验Ⅱ组和试验Ⅲ组分别在仔猪基础日粮的上添加12、15、18mL／kg乳酸杆菌，通过30天的试验期后，测定4组的生长性能指标。结果表明：对照组的腹泻率明显高于试验组，腹泻率最低的是试验Ⅲ组；头平均日增重试验Ⅲ组与其它3组差异显著（P<0.05），对照组和试验Ⅱ组差异显著（P<0.05），其中试验Ⅲ组头平均日增重最高，为358g，对照组最低,为303g；试验Ⅲ组与其它3组料重比差异显著（P<0.05），试验Ⅱ组与试验Ⅰ组差异不显著（P>0.05），和对照组差异显著（P<0.05）,料重比较为理想的是试验Ⅲ组，其次是试验Ⅱ组。     

蛤蚧口腔疾病病原研究(第二报)——预防及治疗试验

对蛤蚧口腔溃疡病主要病原——铜绿色假单孢菌进行药物预防及治疗对比试验。试验结果表明:15种药物敏感试验中,对磺胺、青霉素和卡那霉素等3种药物最为敏感,并将之配成软膏,作治疗对比试验。结果磺胺治愈率最为理想。重复治疗试验、田间扩大治疗试验,试验结果与实验室治疗试验结果差异不大。另外选用高锰酸钾溶液作为蛤蚧口腔、体表及房舍用具的消毒剂,对预防蛤蚧口腔溃疡病有较好效果。     

缩胆囊素抗体对猪生长性能的影响

本研究将CCK33基因四串体片段克隆到原核表达载体pBCX上,成功构建重组表达质粒pBCX-4CCK33。将其转化大肠杆菌BL21中表达,经SDS-PAGE可检测到分子量约为53 kDa的融合蛋白,最高表达量占菌体总蛋白的30.08%,蛋白可溶性分析表明,融合蛋白主要是以可溶性形式表达。Western-blotting证实,可溶表达的融合蛋白与CCK-8阳性血清具有良好的免疫反应性。将纯化的可溶性融合蛋白制成油乳剂疫苗,主动免疫蛋鸡,ELISA检测结果表明,该融合蛋白能在鸡体内引起免疫。用含CCK抗体的蛋黄粉饲喂82天龄肉猪,试验结果表明,在肉猪饲料中添加100g/t含CCK抗体蛋黄粉,试验期间平均日增重和采食量与阴性蛋黄粉组相比,分别提高6.64%和8%,差异显著（P<0.05）。     

发酵棉籽蛋白对仔猪生产性能的影响探究

选择160头保育出栏的杜×长×大三元杂仔猪,全面遵循性别、比例相接近的基本原则,将仔猪划分为试验组与对照组,并各自设置两个重复组,每组的仔猪头数为40头,保证公猪与母猪各占一半,并采取分头标号;对照组和试验组分别饲喂替代了0%和14%发酵棉籽蛋白的仔猪全价日粮。试验结果表明:试验组的饲料利用率得到明显提升,且在仔猪采食量、采食速度上都得到明显增加,日增重幅度为7.1%,降低了料肉比2.14%;试验组腹泻率降低了20.30%;试验组能够有效降低成本;试验组仔猪的精神状态和毛色优于对照组,粪尿恶臭减轻,猪场环境卫生得到改善。     

不同酿酒葡萄品种钾素营养特点及其吸收与利用研究

采用水培和大田试验对“干红”、“干白”2个葡萄品种的K素营养特点及其吸收与利用的研究结果表明，2个葡萄品种干物质积累最大值时的介质浓度不同，“干红”葡萄品种的介质浓度（120μmol/L）高于“干白”葡萄品种（800μmol／L），二者的生长速率、K素吸收效率、植株体内K浓度和极冠比均有显著性差异。“干红”酿酒葡萄品种的土壤K素依存率低，为53．1％－80．3％，K肥利用率较高；而“干白”葡萄品种依存率为56．5％－82．1％，K肥利用率低。K素对植株鲜物质量、根长、根数、百粒重、结果枝率等均有明显影响，且可增加酿酒葡萄含粮量和出汁率。     

抗速灭威单链抗体基因在巴斯德毕赤酵母中的表达及性质研究

选用巴斯德毕赤酵母（Pichia pastoris）系统表达特异性抗速灭威单链抗体（scFv）基因,以为速灭威特异性抗体的大量制备奠定基础。设计引物扩增阳性克隆scFv基因,亚克隆至表达载体pPICZαC,获得重组酵母表达质粒pPICZαC-scFv,线性化pPICZαC-scFv并高效电转P.pastoris（X-33）,对转化子进行抗性梯度筛选得到一株高效表达的X-33-Pp-SMW-12-6菌株。对获得的菌株先后进行表达条件的优化、优化条件下的诱导表达及单链抗体性质研究。结果表明：P.pastoris-scFv的质量接近其亲本E.coli-scFv的质量,X-33-Pp-SMW12-6在优化条件下的表达产量达28mg/L,比未优化前的产量提高了约8mg/L,经纯化后抗体纯度可达85%以上。因此,利用P.pastoris表达系统制备抗速灭威单链抗体比细菌表达系统更有效、更经济。     

检测多杀性巴氏杆菌毒素抗体的单抗竞争ELISA方法的建立

本研究以纯化的多杀性巴氏杆菌毒素基因片段的原核表达产物作为抗原免疫小鼠制备单抗,并利用表达蛋白和多杀性巴氏杆菌毒素单抗酶结合物建立了竞争ELISA方法检测多杀性巴氏杆菌毒素抗体。经过研究确定抗原包被浓度为223ng／mL,待检血清最佳稀释度为1：2,酶标单抗工作浓度为1：3200,血清抑制率大于50%为阳性。应用单抗竞争ELISA和细胞毒性中和试验同时对82份血清进行猪多杀性巴氏杆菌毒素抗体检测,竞争ELISA的检出率为40.2％,细胞毒性中和试验检出率为36.6％,两者符合率达91.5％。试验结果表明,该ELISA方法特异性强,敏感性高,稳定性和重复性好,操作简便。本方法的建立在实验室诊断的标准化、猪群萎缩性鼻炎疫苗免疫效果的评价及流行病学调查方面具有应用价值。     

Overexpression of the soluble form of chicken cystatin in Escherichia coli and its purification

A cDNA encoding chicken cystatin was cloned into the pET-23a(+) expression vector and then transformed into Escherichia coli AD494(DE3)pLysS expression host. An active soluble form of cystatin was expressed in the cytoplasm of E. coli induced by isopropyl beta-D-thiogalactopyranoside. The recombinant chicken cystatin was purified to electrophoretic homogeneity by a simple and rapid method involving heat treatment and Sephacryl S-100 gel filtration chromatography. The recombinant cystatin behaved as a thermal-stable protein and exhibited papain-like protease inhibition activity comparable to the natural chicken cystatin.     

Hydrophobic grid membrane filter/MUG method for total coliform and Escherichia coli enumeration in foods: collaborative study

Twenty-four laboratories participated in a collaborative study to validate a hydrophobic grid membrane filter (HGMF) method incorporating the use of 4-methylumbelliferyl-beta-D-glucuronide (MUG) for enumeration of total coliform and Escherichia coli bacteria in foods by comparing its performance against the AOAC 3-tube MPN method (46.013-46.016). Raw milk, raw ground poultry, whole egg powder, cheese powder, and ground black pepper were included in the study. The total coliform methods did not differ significantly, except that the 3-tube method detected a significantly higher level of total coliforms than did the HGMF method in the ground black pepper. Conversely, the HGMF/MUG E. coli method detected significantly higher numbers of E. coli present in the egg powder, cheese powder, and ground black pepper samples, while not differing significantly from the 3-tube method for the raw milk and raw ground poultry samples. The overall confirmation rate of MUG-positive colonies isolated using the HGMF method was 99.5%. The hydrophobic grid membrane filter/MUG method has been adopted official first action as an additional method to AOAC official final action method 46.030-46.034.     

Identification of antiadhesive fraction(s) in nonimmunized egg yolk powder: in vitro study

The presence of antiadhesive component(s) in the hen egg yolk against foodborne pathogens was anticipated from results of a previous animal study conducted by the authors. The previous work showed egg yolk powder without specific antibodies is effective in controlling Salmonella enteritidis,Salmonella typhimurium, and Escherichia coli O157:H7 colonization in laying hens. Therefore, this study was necessary to locate the activity and identify the effective component(s). In vitro experiments were conducted using confluent Caco-2 cell monolayers. S. enteritidis, S. typhimurium, and E. coli O157:H7 were investigated against the various extracted granule and plasma fractions in three different assays: adhesion elimination, adhesion prevention, and antimicrobial. This study revealed original findings and identified the protective yolk fraction against the foodborne pathogens as the granule component, high-density lipoproteins (HDL). The protective activity conveyed by HDL was confirmed to remain intact despite peptic and tryptic enzymatic digestion and to have antiadhesive but not antimicrobial effect.     

Genetic methods for the detection of microbial pathogens. Identification of enterotoxigenic Escherichia coli by DNA colony hybridization: collaborative study

Enteropathogenic Escherichia coli strains may produce a cholera-like, heat-labile enterotoxin (LT) as a virulence factor. The gene that codes for LT can be purified by recombinant DNA techniques and used as a genetic probe for DNA hybridization. These probes detect enterotoxigenic strains as well as strains that may not manifest toxin production but carry the genetic information to do so. In this study, 13 laboratories tested 3 known and 25 unknown (10 positive and 15 negative) cultures of E. coli for the presence of the LT gene. The isolates had been tested and classified by the mouse Y-1 adrenal cell test and an enzyme-linked immunosorbent assay. Cultures were spotted on nitrocellulose filters on MacConkey agar and incubated. Colonies were lysed in situ and their DNA was hybridized to 32P-labeled, purified LT gene DNA (provided to the collaborators). Positive colonies were identified by autoradiography. Of 325 samples, 315 (96.9%) were identified correctly and 10 were misclassified; there were 6 false negative and 4 false positive identifications. Chi-square values indicated that the method agreed with the previous classification and was equally efficient in distinguishing positive and negative samples (95.7 and 98.1%, respectively). The method has been adopted official first action.     

Isolation and characterization of immunoglobulin in yolk (IgY) specific against hen egg white lysozyme by immunoaffinity chromatography

Six hens were intramuscularly (im) immunized once a week for 3 weeks using chicken egg white lysozyme (LS) as antigen. Antibody (immunoglobulin in yolk, IgY) ELISA values of 10(3)-fold diluted yolk were almost as high as 1.879 in the sixth week and maintained a value of 0.756 in the eighth week after the initial immunization treatment. The purification efficiency (specific activity of purified IgY against LS/specific activity of antibody in yolk against LS) of IgY specific against LS isolated by laboratory-prepared LS-bound (IgY-) Sepharose 4 Fast Flow immunoaffinity column was approximately 3380. By applying various amounts (0-22 mg) of the thusly obtained IgY specific against LS to the immunoaffinity column, the binding capacity (q(m)) and dissociation constant (K(d), M(-1)) of such immunoaffinity gel for IgY against LS were found to be 0.68 mg of IgY/mL of wet gel (0.54 mg of IgY/mg of LS) and 7.13 x 10(-6) M, respectively, as determined by Langmuir-type adsorption isotherms.     

H5N1亚型AIVHA抗原表位的高效表达及其抗原活性分析

血凝素（hemagglutinin,HA）蛋白是禽流感病毒（avian influenza virus,AIV）的一个重要表面抗原性蛋白,在疾病诊断和防治上有重要意义。本研究为了探讨一种更为简便有效的HA重组蛋白表达途径,利用生物信息学软件,对H5N1亚型AIVHA基因编码的氨基酸序列进行分析,在分析其在大肠杆菌中的密码子偏好性、稀有密码子分布情况及有关蛋白的抗原性等重要特性后,构建了HA抗原表位重组表达质粒pET-32a（＋）-HA。经测试,该重组质粒在1mmol/LIPTG诱导剂作用下诱导过夜,能在大肠杆菌Rosetta-gami B（DE3）中高效表达,并得到48.1kD大小的目的重组表达蛋白。重组蛋白用6×His-tagged protein纯化试剂盒纯化后,与福氏佐剂等量混合制备成抗原,以200μg/鸡的剂量皮下注射2月龄SPF鸡3次,采血分离血清。Western-Blot试验结果表明,该重组表达蛋白能分别与所制备的高免鸡血清及H5N1亚型AIV阳性血清发生特异性反应,在硝酸纤维素膜上出现特异性杂交带。说明本试验研究的HA抗原重组表达蛋白具有良好的免疫原性和反应原性,保留了HA蛋白的抗原活性,提示该重组蛋白在H5亚型AIV的防治技术研究中具有重要的实际应用价值。     

抗对硫磷基因工程四价抗体在大肠杆菌中高效表达与鉴定

为提高抗对硫磷基因工程四价抗体在大肠杆菌中可溶性表达量,本研究利用克隆技术得到四价抗体基因sc-sa,将该融合基因连接至表达载体pTO-T7的Ω序列和T7启动子下游,构建成功的表达质粒导入大肠杆菌OrigamiB(DE3),经IPTG诱导表达,SDS-PAGE和Western Blot鉴定表达产物,Ni亲和层析纯化蛋白,间接非竞争ELISA法测定该四价抗体的亲和力。结果表明在大肠杆菌OrigamiB(DE3)中表达分子量约为46kDa的四价抗体,0.1mmol/L的IPTG在30℃条件下诱导原核表达,外源蛋白占菌体总蛋白含量近50%,纯化后可溶性蛋白纯度在90%以上,ELISA结果显示该抗体与对硫磷结合呈阳性,抗体效价在1∶1×106以上,亲和常数为6.84×108L/mol。与母源抗体和相应单链抗体相比,利用pTO-T7载体四价抗体在大肠杆菌OrigamiB(DE3) 中可实现高效表达,且抗体效价和亲和力均得到进一步提高。