流感疫苗中间品-鸡胚尿囊病毒液质量控制的研究

为建立流感疫苗中间品-鸡胚尿囊病毒液质量控制点提供依据。用鲎试剂法、微生物限度检查法、沙门菌检查法检测流感疫苗中间品一鸡胚尿囊病毒液的细菌内毒素含量、微生物限度及沙门菌。在300份鸡胚尿囊病毒液中,细菌内毒素含量大于5EU／mL的阳检率为5％;微生物限度检查中小于lO个／mL细菌菌落数的占78．67％,小于10个／mL霉菌菌落数的占80．67％,小于10个／mL酵母菌菌落数的占88．67％。沙门菌属的阳检率为4％,其中未检出A～F群的沙门菌。对流感疫苗中间品-鸡胚尿囊病毒液进行细菌内毒素,微生物限度及沙门菌检测可避免不合格尿囊病毒液进入后续生产,污染后续中间品。     

在鸡胚绒毛尿囊膜上评价血管生成技术的应用与优化

目的：用鸡胚绒毛尿囊膜（CAM）评价血管生成并进行优化,为抗血管生成药物筛选提供良好的体内实验模型。方法：对CAM技术的各个环节,包括合适日龄胚蛋选择、蛋壳开窗及暴露CAM的方法、载体选择、结果分析等进行了实验对比。结果：采取气室开窗,以甲基纤维素为药物载体,促进血管生成的药物在孵化11d以后、抑制血管生成的药物在孵化7～9d给药,鸡胚存活率为85.8%,生长状况良好,被检测药物有明显的抑制或促进血管生成的效应。结论：通过优化建立了操作简便、鸡胚存活率高的在CAM上评价血管生成的技术;应用CAM技术评价了若干影响血管生成的因子。     

满山香水煎液对流行性感冒病毒的抑制效果观察

一、满山香水煎液75％浓度,在鸡胚内对流感病毒亚．洲甲型赣防71-5株、京科68一1株(60EID50感染量)均有较明显的抑制作用。 二、“毒一药”或“药一毒”,虽感染和注药先后不同(相隔60分钟),其抑制效果无显著差异。 三、鸡胚尿襄腔感染病毒,60分钟后,经卵黄襄注药,对流感病毒赣防71-5株,京科68-1株也均有抑制作用。 四、阴性尿液经鸡胚尿囊腔盲传两代后,除个别血凝转阳外,大部分仍为阴性,药液在鸡胚内的抑毒作用比较显著。     

流感裂解疫苗生产中沙门菌和微生物限度的监控研究

通过对2008年流感裂解疫苗生产中的监控,结果显示,流感病毒接种鸡胚尿囊液中未检出沙门菌,除菌过滤前微生物限度均小于10CFU/ml、细菌内毒素均小于25EU/ml,纯化过程去除了99%的杂蛋白。表明生产中使用的健康鸡胚尿囊液是符合生物制品规程的要求,现行生产工艺对微生物限度和细菌内毒素的控制是有效的,现行纯化过程对病毒液的纯化是有效的。     

表达绿色荧光蛋白重组新城疫病毒 LaSota疫苗株的构建

新城疫病毒是理想的新型活病毒疫苗载体,具有巨大的优势和应用前景。采用生产实践中广泛应用、免疫效果良好的NDV LaSota弱毒疫苗株,建立了反向遗传操作系统。在此基础上,进一步构建了表达绿色荧光蛋白(GFP)的重组NDV基因组cDNA克隆,成功救获了重组病毒rLaSota-EGFP,病毒F1代尿囊病毒液按1×104EID50接种9~10日龄SPF鸡胚尿囊腔,接种后分别于24h、48h、72h及96h收获尿囊液,检测平均HA滴度分别为28、210.3、211.3和211,每mL尿囊液病毒量EID50分别为108.64、109.22、109.21和109.64,重组病毒与亲本株生长滴度在相近时间达到峰值,生长动力学特性与亲本株无明显差异。各代次重组病毒按1×106EID50病毒量接种9~10日龄SPF鸡胚,96h内完全不致死鸡胚。救获重组病毒保持了LaSota弱毒疫苗亲本毒株对鸡胚良好的高滴度生长适应和低致病特性,并且鸡胚连续传9代次仍保持GFP的稳定表达及生物学特性不变。重组病毒rLaSota-EGFP的成功救获为开展新城疫病毒活载体疫苗研制提供了可行的技术平台。     

火鸡新城疫病毒的分离与鉴定

1988年北京某火鸡场暴发新城疫,从病鸡脾、脑组织分离到新城疫病毒(NDV)强毒株。现简要报告如下。 将病火鸡的脾和脑研磨,用生理盐水1:5稀释,接种9日龄非免疫鸡胚3只,收集24小时后死亡的鸡胚尿囊液,即为待测病毒。低温冰箱保存备用,红细胞为非免疫鸡红细胞,生理盐水洗4次,配成0.5％浓度,4℃冰箱保存。     

表达绿色荧光蛋白重组新城疫病毒 LaSota疫苗株的构建

新城疫病毒是理想的新型活病毒疫苗载体,具有巨大的优势和应用前景。采用生产实践中广泛应用、免疫效果良好的NDV LaSota弱毒疫苗株,建立了反向遗传操作系统。在此基础上,进一步构建了表达绿色荧光蛋白（GFP）的重组NDV基因组cDNA克隆,成功救获了重组病毒rLaSota_EGFP,病毒F1代尿囊病毒液按1×10.4EID50接种9～10日龄SPF鸡胚尿囊腔,接种后分别于24h、48h、72h及96h收获尿囊液,检测平均HA滴度分别为2.8、2 10.3、2 11.3和2 11,每mL尿囊液病毒量EID50分别为108.64、109.22、109.21和109.64,重组病毒与亲本株生长滴度在相近时间达到峰值,生长动力学特性与亲本株无明显差异。各代次重组病毒按1×10.6EID50病毒量接种9～10日龄SPF鸡胚,96h内完全不致死鸡胚。救获重组病毒保持了LaSota弱毒疫苗亲本毒株对鸡胚良好的高滴度生长适应和低致病特性,并且鸡胚连续传9代次仍保持GFP的稳定表达及生物学特性不变。重组病毒rLaSota_EGFP的成功救获为开展新城疫病毒活载体疫苗研制提供了可行的技术平台。     

“乳珍”对体外培养细胞的作用

“乳珍”是我们利用牛初乳研制成的营养保健品,它不仅含有丰富的蛋白质、维生素和矿物质,而且含有多种生物活性物质。本实验研究了它对体外培养细胞的作用。 方法 一、鸡胚骨胳肌细胞的体外培养在无菌条件下,取9d龄鸡胚后肢肌肉,制成细胞悬液。用Eagle MEM(MEM中另加入右旋葡萄糖6g/L、青霉素钠盐10万U/L、硫酸链霉素     

SPF鸡盲肠复合微生态制剂代替抗生素预防肉仔鸡沙门茵感染

目的在雏鸡盲肠尽快建立完整健康菌群,通过“竞争排除（Competitiveexclusion,CE）”抑制病原菌定植感染。方法提取SPF鸡盲肠菌群,排除沙门菌感染的可能,制成复合微生态制剂,在鸡胚孵化19d啄壳时和21d雏鸡全出壳时进行两次喷雾接种,监测鸡群沙门菌抗体和粪便中沙门菌,测定成活率、生产性能。结果进行喷雾接种后的小鸡至12日龄可以完全不使用抗生素,成活率、采食量与体重略高于使用抗生素的对照组,能提高饲料报酬,饲养12、24、35d均未检出粪便沙门菌和血液沙门菌抗体,全程无沙门菌感染。结论SPF鸡盲肠复合微生态制剂能代替抗生素预防肉仔鸡沙门菌感染。     

短发夹结构RNA干扰新城疫病毒的增殖

 以新城疫病毒（NDV）NP基因为标靶,构建3个细胞内表达发夹样结构小干扰RNA（shRNA）的质粒载体,在鸡胚成纤维细胞（CEF）和鸡胚上进行了RNAi试验,筛选出一个有效抑制病毒复制的小分子ndv1.用阳离子脂质体转染试剂Silent-fect 将ndv1转染CEF,以不相关shRNA质粒载体HK为阴性对照,4 h后接种NDV,与对照相比,干涉组在病毒感染后3 h NP基因的表达量降低2.3倍,6 h 降低21.1倍,9 h降低9.8倍;ndv1能在48 h内完全阻断NDV在CEF中的增殖,延缓病变出现时间,减轻病变程度.将Silent-fect-ndv1混合物与NDV同时注入10日龄SPF鸡胚绒毛尿囊腔,能使10⁵ ELD₅₀NDV感染后17 h鸡胚尿囊液中病毒增殖量减少94.4%,使10⁶ ELD₅₀NDV感染后17　h鸡胚尿囊液中病毒增殖量减少62.5%.实验结果证实,在CEF中存在RNAi机制,抑制NDV NP基因的表达能有效阻断该病毒增殖,说明NP基因在NDV复制过程中起重要作用.实验结果为进一步利用RNAi技术在CEF和鸡胚中研究病毒基因组功能及筛选抗病毒小分子奠定了基础.     

Frequency of R Factor-mediated Multiple Drug Resistance in Klebsiella and Aerobacter

A comparative study was done on the transfer frequency of R factors from 90 strains of multiple drug-resistant Aerobacter and 81 strains of Klebsiella to Escherichia coli CSH-2 (F(-), met(-), pro(-), Nal-r). The most common resistance patterns for the Aerobacter isolants were ampicillin streptomycin chloramphenicol tetracycline and ampicillin streptomycin chloramphenicol tetracycline kanamycin neomycin; for the Klebsiella isolants, the most common resistance pattern was ampicillin kanamycin streptomycin tetracycline chloramphenicol neomycin. R factors were isolated from 14.1% of the Aerobacter strains; 61.5% of these R factors harbored R determinants for ampicillin streptomycin tetracycline. R factors were isolated from 79.1% of the Klebsiella strains; four R factors were isolated with significant frequency; streptomycin chloramphenicol kanamycin neomycin, 37.5%; ampicillin streptomycin tetracycline kanamycin neomycin, 14.1%; ampicillin streptomycin tetracycline, 12.5%; and streptomycin chloramphenicol tetracycline, 12.5%.Chloramphenicol, kanamycin, and neomycin resistance was rarely transferred from the Aerobacter strains, although over 50% of the clinical isolants possessed resistance to these antibiotics. In contrast, over 75% of the Klebsiella strains transferred resistance to chloramphenicol, kanamycin, neomycin. Highest frequency of transferred resistance to individual drugs in the Aerobacter strains was to streptomycin (14.8%), whereas in the Klebsiella group resistance to four drugs was transferred at a very high frequency: streptomycin (80.8%), chloramphenicol (78.5%), kanamycin (76.4%), and neomycin (75.9%).     

Availability of avidin-bound biotin to the chicken embryo.

Avidin, an exceptionally stable protein in egg white, binds the vitamin biotin with very high affinity and can induce biotin deficiency when fed to animals. To determine if biotin bound to avidin is available to the chicken embryo, the fate of [3H]biotin complexed to avidin was monitored during embryonic development. The majority (greater than 85%) of the [3H]biotin was extraembryonic until the day before hatching, when embryos swallow egg white and withdraw the yolk sac into their abdomen. Thus, biotin in the egg white of chicken eggs contributes little to the biotin status of the chick prior to hatching. After hatching, much of the [3H]biotin was assimilated. About 30% of the total was found in the liver and kidneys by 4 days of age. The biotin in liver was associated with large proteins and not with avidin. In a separate experiment, biotin injected into the egg white of biotin-deficient eggs failed to increase embryonic development or hatchability. Both experiments suggest that biotin in egg yolk is the primary and virtually sole source of biotin for the chicken embryo.     

Chloramphenicol Combined with Ampicillin in Treatment of Typhoid

Fifty out of 100 patients with typhoid fever, matched for age, stage of illness, and degree of fever, were treated with chloramphenicol and the other 50 were treated with chloramphenicol combined with ampicillin. The febrile period was shortened by up to 29% in patients treated with the combined drugs compared with those given only chloramphenicol. Moreover, no patient on combined therapy had a febrile relapse, whereas two relapses occurred among patients treated with chloramphenicol only. We conclude that chloramphenicol and ampicillin together are better than chloramphenicol alone in the treatment of typhoid fever.     

Site-directed mutagenesis of the gag-myc gene of avian myelocytomatosis virus 29: biological activity and intracellular localization of structurally altered proteins.

Transfection of chicken embryo cells with pMC29, a plasmid vector containing the sequences for the acute transforming virus MC29, and a cloned transformation-defective helper virus, p delta Mst, resulted in morphological transformation, the synthesis of P110gag-myc (the product of the gag-myc oncogene), and the production of infectious virus. MC29 mutants bearing site-directed deletions within the gag-specific sequences or within the middle portion of the myc sequences efficiently induced transformation of chicken embryo cells in culture. However, variants containing deletions of sequences in the amino-terminal half or carboxy-terminal portion of the myc gene were defective for transformation. The gag-myc proteins encoded by these variants efficiently localized to the cell nucleus. Premature termination mutants were isolated which encoded gag-myc proteins lacking the carboxy-terminal 185 residues; these truncated proteins localized to both the nucleus and the cytoplasm. Deletion of as few as 11 residues within the middle of the myc-specific sequences (residues Ile-239 to Glu-249) significantly reduced the efficiency of chicken hematopoietic cell transformation.     

High-performance thin-layer chromatography-bioautography for multiple antibiotic residues in cow's milk

An analytical method to identify and quantify multiple antibiotic residues (chloramphenicol, ampicillin, benzylpenicillin, dicloxacillin and erythromycin) in cow's milk by high-performance thin-layer chromatography (HPTLC) combined with bioautography was developed. The test microorganism used for bioautography was Bacillus subtilis ATCC 6633. Antibiotic residues were extracted with acetonitrile, fat eliminated with petroleum ether and residues isolated with dichloromethane The sensitivity of the method guarantees the detection of the above-mentioned antibiotics at levels below maximum residue limits (MRL) allowed for milk. Percentage recoveries ranged between 90 and 100%, with coefficients of variation between 7.2 and 21.3%. Some advantages of this methodology over thin-layer chromatography (TLC)/bioautography are also discussed.     

Interaction of the 47-kDa talin fragment and the 32-kDa vinculin fragment with acidic phospholipids: a computer analysis.

In recent in vitro experiments, it has been demonstrated that the 47-kDa fragment of the talin molecule and the 32-kDa fragment of the vinculin molecule interact with acidic phospholipids. By using a computer analysis method, we determined the hydrophobic and amphipathic stretches of these fragments and, by applying a purpose-written matrix method, we ascertained the molecular amphipathic structure of alpha-helices. Calculations for the 47-kDa mouse talin fragment (residues 1-433; NH2-terminal region) suggest specific interactions of residues 21-39, 287-342, and 385-406 with acidic phospholipids and a general lipid-binding domain for mouse talin (primary amino acid sequence 385-401) and for Dictyostelium talin (primary amino acid sequence 348-364). Calculations for the 32-kDa chicken embryo vinculin fragment (residues 858-1066; COOH-terminal region) and from nematode vinculin alignment indicate for chicken embryo vinculin residues 935-978 and 1020-1040 interactions with acidic phospholipids. Experimental confirmation has been given for vinculin (residues 916-970), and future detailed experimental analyses are now needed to support the remaining computational data.     

Rapid method for the determination of ampicillin residues in animal muscle tissues by high-performance liquid chromatography with fluorescence detection

A rapid and sensitive HPLC method was developed for the determination of ampicillin residues in muscle tissues of beef, pork, chicken and catfish. Muscle tissues were blended with a food processor into paste. A 5-g aliquot of the blended tissues was homogenized with 14 ml of 0.01 M phosphate buffer (pH 4.5) using a tissue homogenizer. Proteins were precipitated with the addition of 1 ml trichloroacetic acid (75%, w/v) followed by centrifugation. After filtration, 1 ml of the supernatant was reacted with formaldehyde under acidic and heating conditions. The ampicillin fluorescent derivative was then analyzed by reverse phase HPLC with fluorescence detection. Recoveries of spiked ampicillin at 5, 10 and 20 ng/g were >85%, with coefficients of variation <5%. The limit of detection and limit of quantitation for ampicillin in the tissues were 0.6 ng/g and 1.5 ng/g, respectively. The method is also applicable to the analysis of ampicillin residue in dry milk powder.     

Antimicrobial resistance and resistance plasmids in Salmonella from Ontario, Canada.

Collections of 589 human and 204 animal strains of Salmonella isolated in Ontario during the summer of1974 were examined for susceptibility to 12 antimicrobial agents. Many isolates were found to be resistant to both chloramphenicol (12.4% of the human and 38.2% of the animal sample) and ampicillin. The chloramphenicol resistance almost always occurred in strains which were also resistant to ampicillin and was usually due to a self-transmissible plasmid with a resistance pattern of CmKmSmTc (chloramphenicol, kanamycin, streptomycin, and tetracycline) or CmTc. Ampicillin resistance in these strains was mediated by a variety of plasmids with patterns ApSu (ampicillin and sulfa drugs) and ApSmSu, many of which were nonself-transmissible. Ampicillin resistance in chloramphenicol-sensitive strains was transferable from 21% of the strains, and it was associated with resistance patterns which were different from the self-transferable ampicillin patterns from the chloramphenicol-resistance strains.     

Resistance of some strains ofBacteroides to ampicillin,methicillin and cloxacillin

Twenty-three strains of thefragilis-convexus group ofBacteroides and two strains of thethetaiotaomicron group have been tested for sensitivity to ampicillin, methicillin and benzylpenicillin. Thirteen of these strains (twelve of thefragilis-convexus group and one of thethetaiotaomicron group) have been tested to cloxacillin, tetracycline, chloramphenicol and erythromycin.The strains proved to be resistant to benzylpenicillin, ampicillin, methicillin and cloxacillin.