北京地区动物实验设施动物饮水无菌检测 及绿脓杆菌污染菌株鉴定

摘要: 目的了解北京地区动物实验设施动物饮水微生物控制状况。方法根据实验动物无菌动物生活环境国家 标准检测方法,对实验动物设施内的动物饮用水样品进行分离培养。扩增分离绿脓杆菌的16SrRNA,并进行测序, 绘制发育树。结果在27 个参与检测的单位中,未经过处理的饮水无菌生长率41% ; 经过处理的饮水无菌生长率 仅29% 。绿脓杆菌检出率30% ,主要为RHH13 和ZAQ22 两个菌株。结论本次检测的实验动物饮水在处理后的 效果普遍不甚理想,存在绿脓杆菌的污染,直接影响实验动物质量。     

Purification and Partial Characterization of a Collagenolytic Enzyme Produced by Pseudomonas aeraginosa SCU Screened from Rotten Hides

Strain Pseudomonas Aeraginosa SCU isolated from rotten hides is shown to produce various gelatinolytic enzymes with molecular masses ranging from - 50 to - 200 kD. A gelatinolytic enzyme called PAC exhibiting collagenolytic activity is purified by SP sepharose fast flow, Sephadex G-200 gel filtration and native PAGE cutting method. The purified enzyme has an apparent molecular weight of about 110 kD by SDS PAGE without β-mercaptoethanol. Treatment withtβ-Me suggests that PAC is dissociated into three subunits approximately 33 kD, 25 kD and 20 kD with a ratio of 2:1:1, named sub A, sub B and sub C repectively. EDFA and EGTA display a significant inhibitory effect on the enzyme activity while PMSF, leupeptin and pepstain do not appreciably inhibit it. The first 15 amino acid residues of the major subunit (subA) are determined and the sequence is Ala-Glu-Ala-Gly-Gly-Pro-Gly-Gly-Asn-Gln-Lys-Ile-Gly -Lys-Tyr. This sequence is identical to that of elastase of P. aeruginosa. The fragment of encoding mature sub A is cloned and its sequence is determined, which has a high homology with the gene of elastase. These results indicate that PAC is a novel collagenolytic metalloprotease composed of three kinds of subunits, of which elastase is the major one.     

50味中药与8种抗生素对变形假单胞菌的体外抑菌作用

用牛津杯法选择诃子、大黄、黄连等50种单方中药对变形假单胞菌、金黄色葡萄球菌进行体外抑菌试验,并选用抑菌作用较好的药物组成复方二联、三联中药,药物质量浓度为60,240 mg/mL。结果表明:单方中药中,诃子对变形假单胞菌抑菌作用明显,为极敏感;复方二联中药中,240 mg/mL的诃子+白芍等4种对变形假单胞菌的抑菌作用明显,为极敏感;复方三联中药中,60 mg/mL的黄连+板蓝根+黄柏等5种对变形假单胞菌的抑菌效果为极敏感,240 mg/mL的黄连+连翘+黄芪等6种对变形假单胞菌的抑菌作用为极敏感;变形假单胞菌对恩诺沙星、盐酸多西环素、硫酸新霉素及硫酸庆大霉素4种药物高度敏感。由此认为黄连+板蓝根+黄柏等11个复方三联中药与恩诺沙星对变形假单胞菌抑菌作用最强,同时筛选出多个抑菌作用效果良好的复方三联中药和抗生素,可用于鱼病防治研究。     

大亚湾海洋细菌Pseudomonas sp.中的红色素

海洋细菌Pseudomonas sp.，采集于大亚湾，从该菌的菌体提取物分离得到2个红色素：灵菌红素A和新的衍生物二甲基灵菌红素B。它们的结构通过NMR，LC-MS等确定，并得出灵菌红素是具有强烈抗微生物和细胞毒性的化合物，同时研究了它的培养条件。     

Cloning and expression of ophc2, a new organphosphorus hydrolase gene

The amino acid sequences of N-terminal and internal peptide of OPHC2, purified from Pseudomonas pseudoalcaligenes strain C2-1 in our lab, are determined. The full-length organphosphorus hydrolase gene ophc2 is cloned by PCR using the degenerate primers designed according to the sequences and future inverse PCR. The ophc2 gene is 975 bp long with G C content of 63%, comprising one open reading frame encoding a polypeptide of 324 amino acids with a molecular weight of 36 kD. The nucleotide sequence of ophc2 shows low homologies with those organphosphorus hydrolase genes deposited in GenBank, one of which exhibits the highest homology of 46.4% with ophc2. The organphosphorus hydrolase protein expressed in E. coli bears normal bioactivity.     

微生物降解速灭菊酯的遗传毒性效应

从生产速灭菊酯的工厂排污口附近采泥样,从中分离降解速灭菊酯的细菌,接种于以速灭菊酯为唯一碳源的合成培养液中。培养48小时后,用人Ames试验检测其遗传毒性。结果,在高剂量时代谢产物有抑茵作用,低剂量时有致突变效应。而未经微生物降解的对照(其速灭菊酯浓度与降解试验初始浓度同)则均呈阴性。另外,对分离所得菌株进行了鉴定,主要降解菌为假单胞茵(Pseudomnas)。     

平菇黄腐病病原菌分离与鉴定

本实验主要是对平菇黄腐病病原菌进行研究。从感染黄腐病的平菇幼菇子实体中分离得到2种具平板生长优势的革兰氏阴性杆状细菌(编号为G1和G2),通过对平菇子实体回接感染实验,发现细菌G1感染的平菇子实体病灶与自然感染的黄腐病状况一致,因此认为该菌就是导致平菇黄腐病的主要病原菌。经采用传统的生理生化实验与16s rDNA序列分析以及Biolog微生物自动鉴定系统分析鉴定,确定该菌为荧光假单胞菌(Pseudomonas fluorescens)。     

铜绿假单胞菌液体发酵产生絮凝现象的研究

对铜绿假单胞菌液体发酵产生的絮凝现象进行了研究。初步确定其由铜绿假单胞菌产生的生物膜引起,测得生物膜中多糖含量约为6.3%,蛋白质含量约为7.7%。考察了添加颗粒营养物质豆饼粉对絮凝现象的影响,分析了天然群体感应抑制剂水杨酸、丁香酚、儿茶素、姜黄素和大蒜提取物对生物膜和菌浓的影响以及群体感应淬灭酶对菌浓的影响。结果显示,添加颗粒营养物质对絮凝物影响不显著;天然群体感应抑制剂不能显著提高铜绿假单胞菌液体发酵菌浓,但对生物膜的分散具有一定作用;群体感应淬灭酶对铜绿假单胞菌液体发酵浓度无显著影响。     

用全DNA转化法构建多功能石油降解菌

以嗜蜡的不动杆菌４－１ 作为受体菌,经溶菌酶破壁后,用嗜胶的假单胞菌Ｚ１７ 的全ＤＮＡ 转化,经再生后用只含高胶油的选择培养基筛选出转化子不动杆菌ＺＨ２０．破壁及再生的最佳条件为：溶菌酶浓度１．５ｍ ｇ／ｍ Ｌ,３７℃水浴保温４０ｍ ｉｎ,破壁率为９３．８％ ,再生率达到３０．２％ ;转化条件为：３０％ ＰＥＧ６０００,５０ｍ ｍ ｏｌ／ＬＣａＣｌ２ ,于３７℃保温２０ｍ ｉｎ．ＺＨ２０ 具有嗜高胶油和高蜡油的双重性状,５０℃条件下使高胶油粘度降低２２．２％ ,使高蜡油的凝固点降低２．５℃,且保持了受体菌的耐热和耐盐性能．液蜡培养基连续传代１０ 次,性能稳定．