血浆凝集降低筛选金黄色葡萄球菌促凝相关基因

【背景】金黄色葡萄球菌是重要的致病菌,其中促凝聚是重要的致病机制之一,可能存在新的基因参与其中。【目的】通过采用血浆凝集降低方法筛选及鉴定促凝相关基因。【方法】利用转座子随机插入突变技术建立金黄色葡萄球菌转座子突变文库,采用动态比浊及试管凝集技术筛选凝集能力降低的突变株;应用抗性标记挽救法鉴定突变基因并应用生物信息学预测基因的功能。【结果】通过观察血浆凝集能力降低共计筛选到突变菌株82个。鉴定其中的76个突变菌株的转座子插入位点,涉及基因13个,包括报道的与促凝集有关基因4个(占筛选基因的30.8%)。【结论】从金黄色葡萄球菌凝集能力降低筛选与促凝集可能有关的基因,为金黄色葡萄球菌促凝集基因的筛选提供了新策略,同时为了解该菌促凝集过程提供了候选基因。     

IncFII-FIA-FIB型多重耐药质粒pBTR-CTXM的结构基因组学分析

【背景】IncFII-FIA-FIB型质粒广泛存在于肠杆菌科细菌中,介导了许多耐药基因的水平转移,并导致细菌多重耐药问题日益严重。【目的】分析IncFII-FIA-FIB型多重耐药质粒pBTR-CTXM的基因组结构,并研究其介导大肠杆菌BTR株的耐药基因水平转移机制。【方法】利用PCR进行耐药基因筛查;接合转移和电转化实验验证质粒pBTR-CTXM是否具备自主接合转移的特性;VITEK 2 Compact全自动细菌鉴定及药敏分析仪测定相关菌株对抗生素的药物敏感性;构建MatePair文库并进行细菌全基因组高通量测序和质粒结构基因组学分析。【结果】菌株BTR是携带blaNDM-1、blaCTX-M-15、blaTEM、qnrD、qnrS1、mph(A)、erm(B)和tetA(B)等耐药基因的多重耐药大肠杆菌,其中blaCTX-M-15、mph(A)、erm(B)和tet A(B)等耐药基因均位于大小为144 939 bp的质粒p BTR-CTXM (GenBank登录号MF156697)上,该质粒可与菌株BTR内质粒pNDM-BTR接合共转移到受体菌大肠杆菌EC600中。pBTR-CTXM具备IncFII-FIA-FIB型质粒典型的骨架区结构,其多重耐药(Multidrug-resistant,MDR)区由新的复合型转座子Tn6492、Tn2残余、Tn10残余、ISEcp1-blaCTX-M-15-Δorf477转座单元和一些插入序列组成。【结论】pBTR-CTXM中新复合型转座子Tn6492与Tn10残余和ISEcp1-blaCTX-M-15-Δorf477转座单元共同介导大肠杆菌BTR株的多重耐药与耐药基因的水平传播。     

嗜水气单胞菌若干毒力因子同时缺失的突变株的构建及特性分析

以携带质粒pAM12 0 (Tcr Tn916 )的大肠杆菌CG12 0株为供体菌 ,采用滤膜接合法与受体菌嗜水气单胞菌J_1株 (cfzr)进行接合转移 ,在含Tc和cfz选择平板上进行筛选。共获接合转移菌落 380 0个 ,其接合频率为 3× 10 - 5(按供体细胞计算 )。任取 38个接合子 ,提取基因组DNA ,以嗜水气单胞菌特异性 16SrDNA引物进行PCR扩增 ,所有接合子均阳性。为证明Tn916确实插入基因组 ,以四环素基因 (tet)引物进行PCR扩增 ,结果所有抗性接合子均扩增出一条特异条带。与亲本J_1株相比 ,所有接合子的主要毒力因子如蛋白酶、溶血素、DNA酶和淀粉酶等均不表达 ,对小鼠失去致病力 ,其LD50 大于 10 9CFU。接合子连传 10次后 ,四环素抗性消失 ,但毒力未恢复 ,说明通过转座子Tn916的插入可获得稳定的无毒嗜水气单胞菌突变株。Tn916引起嗜水气单胞菌毒力性状改变的机制有待研究 ,推测可能与该菌染色体上存在Tn916的热点或毒力岛有关。     

阴沟肠杆菌基因nifA经转座子Tn5整入催娩克氏杆菌NG13的基因组

根际固氮细菌与禾谷类作物如水稻、小麦、玉米等进行联合固氮,能使作物有不同程度的增产（APp等1980,Watanable和hn1984）。水稻是世界上主要粮食作物之一,因此,研究增强水稻根际细菌的联合固氮作用,吸引着科学家们的兴趣。催娩克氏杆菌（KMFithemp）NG13与水稻能进行有效的联合固氮（Yoo等1986）。但是在有氨的生长条件下,细菌的固氮活力受到阻遏。ZhU等将带有nifA的重组质粒引进阴沟肠杆菌EZ6后,观察到有氨存在条件下固氮酶组成型生成。我们实验室构建了具有广泛接合转移特性的阴沟肠杆菌Tns－nifA嵌合质粒PBF101,基因nif…     

AFP阴性与阳性肝细胞癌免疫组化和免疫电镜的比较性研究

目的比较性研究AFP阴性与阳性原发性肝细胞癌超微结构特征及AFP和Tn (Thomsen-Friedenreich-related antigen)蛋白表达及意义.方法 43例原发性肝细胞癌组织和5例正常肝组织分为三组:对照组(正常肝组织,5例);AFP阳性肝细胞癌组 (血清AFP>10ng/ml,22例);AFP阴性肝细胞癌组(血清AFP<10ng/ml,21例).应用透射电镜、免疫组织化学和细胞图象分析技术对AFP阴性与阳性肝癌细胞超微结构及AFP和Tn蛋白表达进行观察,并进行AFP和Tn蛋白免疫电镜标记.结果 1. 免疫组织化学结果显示:在AFP阴性肝细胞癌组癌细胞中(1)Tn蛋白表达强度(0.1498±0.0371)明显高于AFP阳性肝细胞癌组(0.0685±0.0156)(P<0.01);(2)AFP蛋白表达强度(0.1269±0.0347) 低于AFP阳性肝细胞癌组(0.1852±0.0234)(P<0.01).2.透射电镜观察:在AFP阴性组肝癌细胞中,癌细胞最突出的形态特征是胞质内细胞器大多十分简单,唯游离多聚核糖核蛋白体十分丰富.而在AFP阳性组肝癌细胞中,癌细胞胞质内细胞器相对较多,特别是粗面内质网尤为丰富.此外,线粒体及高尔基器也较明显.3.免疫电镜标记显示:AFP蛋白阳性标记主要位于粗面内质网,Tn蛋白阳性标记多位于游离多聚核糖核蛋白体,粗面内质网仅见有散在阳性分布.结论 (1)AFP和Tn蛋白在AFP阴性与阳性肝细胞癌组织中具有差异性分布特征,Tn蛋白有望成为AFP阴性肝细胞癌诊断辅助指标之一.(2)透射电镜和免疫电镜观察表明:AFP和Tn蛋白在肝癌细胞中的合成部位明显不同.     

荧光假单胞菌Tn5转座诱变及对青枯病生防特性

目的：利用Tn5转座诱变荧光假单胞菌PF20001,研究所获得的突变株对青枯病的生防效果。方法：利用三亲本杂交方式,将带有转座子Tn5的Tn5-102（含luxAB）的质粒pTR102成功地转入PF20001,利用平板相互拮抗法分析突变株对青枯病致病菌的拮抗作用。结果：通过诱导Tn5转座,得到荧光假单胞菌PF20001的Tn5插入突变库。经平板相互拮抗实验发现,菌株PF20001-lux-48拮抗圈明显大于野生型（半径达0.35cm）。用Tn5-lux特异引物进行PCR扩增,结果显示只有以该突变株的DNA为模板才能得到300bp的扩增产物,证实该菌株基因组中有Tn5插入。结论：Tn5的插入使菌株PF20001对青枯病生物防治能力增强。     

转座子Tn916对固氮螺菌Azospirillum brasilense的接合诱变及氨分泌菌株的筛选

以携带质粒pAM120(Ap~r,Tc~r/Tn916)的大肠杆菌(E.coli CG120)为供体菌株,与受体菌巴西固氮螺菌(Azospirillum brasilense)采用滤膜接合法进行接合转移,在选择平板上得到具较高频率的接合子(10~(-5)/每个供体菌,选择四环素抗性)。从846株四环素抗性接合子中进一步用奈氏法筛选得到氨分泌突变株3株。在无氮培养基上,其氨分泌量可达7.5～14.0mmol/L。用乙炔还原法分析氨分泌突变株在不同浓度氮源上的固氮活力,发现20mmol/L NH Cl的存在不抑制其固氮活性,固氮活力与无氮条件下野生株的活力相差不多。无选择压力下细胞分裂50代后的稳定性实验证明,转座子Tn916在氨分泌突变株中的稳定性在50％～80％之间。以固氮螺菌氨分泌突变株为供体菌株,对E.coli HBX1进行反向接合转移实验,证实Tn916确实存在于氨分泌固氮螺菌接合子中。     

Characterization of Tn5-induced symbiotically defective mutants of cowpea rhizobia

Symbiotically defective mutants of cowpea rhizobia strain IRC256 were isolated by random Tn5 mutagenesis and characterized. One auxotroph (MS1) requiring adenine and thiamine was a non-nodulating mutant (Nod⁻) and three prototrophic mutants were Nod⁺ Fix⁻ which formed small and ineffective nodules on cowpeas (Vigna unguiculata). Acetylene reduction activity of the Nod⁺ Fix⁻ mutants was reduced to 80–94% of that of the wild-type strain. The non-nodulating mutant (MS1) induced root-hair curling but did not show any nodule initiation or nodule development. Ultrastructural examination of nodules formed by Fix⁻ mutants showed that these contained few bacteroids, indicating either early senescence or a reduction in bacterial release into the cytoplasm of the host cell. DNA hybridization of total DNAs from a representative number of Tn5 mutants showed that each of them had one copy of the transposon Tn5 which was randomly inserted into the genome of cowpea rhizobia.     

Specificities of anti-sialyl-Tn and anti-Tn monoclonal antibodies generated using novel clustered synthetic glycopeptide epitopes

The fine specificities of MAbs generated using novel synthetic clustered STn and Tn glycopeptides as immunogens were compared with the anti-TAG-72 antibodies B72.3 and CC49. Hapten inhibition experiments demonstrated the specificity of several of the MAbs for STn and Tn expressed on ovine submaxillary mucin and tumor derived MUC-1 mucin. Amongst the STn specific MAbs only the B195.3 MAb shows absolute dependence on the presence of sialic acid and specificity to the simple disaccharide NANAA α2-6-GalNAc. Identification of tumor associated carbohydrate epitopes in cluster and monomer configurations are possible using MAbs detecting the defined structure specificities described herein. This revised version was published online in November 2006 with corrections to the Cover Date.     

The construction and preliminary analysis of a Tn5 transposon based random mutant library of baculovirus

A transposon-based random mutation library of AcMNPV, the type species of baculovirus, was constructed using a Tn5 transposon. The green fluorescence protein gene under the control of the Drosophila hsp 70 promoter was inserted into the transposon for easy tracking in insect cells. In vitro transposition was carried out using the transposon and AcMNPV genomic DNA to allow the random insertion of the transposon into the virus genome. The transposed genome was then used to transfect Sf21 insect cells, and a library of mutant viruses capable of expressing green fluorescence protein was obtained. Two mutant viruses, B9F and Li6A were isolated, and the sites of transposon insertion were determined to be within the coding regions of the 94k and p10 genes, respectively. Both genes were determined to be nonessential in viral replication and infection. This technique will be very useful in the functional study of baculovirus genes. __________ Translated from Journal of Fudan University(Natural Science), 2005,44(4) [译自: 复旦学报(自然科学版),2005,44(4)]