广西黑山羊羊种布鲁氏菌的分离和鉴定

为了对一起黑山羊流产死胎的病原进行诊断,采集10份黑山羊血清进行布鲁氏菌病抗体检测,并采集流产胎儿进行细菌的分离。虎红平板凝集试验结果表明10份血清全为阳性,试管凝集试验检测有8份血清为阳性;对流产死胎分离出的细菌进行PCR鉴定证实该细菌为羊种布鲁氏菌。     

羊种布鲁氏菌分子生物学研究进展

综述了近年来羊种布鲁氏菌的研究概况,着重介绍了羊种布鲁氏菌基因组、致病因子、致病机理以及分子生物学检测方法等方面的研究进展,并对未来的研究发展趋势提出见解,为最终使该菌所引发的相关疾病得到有效控制提供参考.     

羊种布鲁氏菌的分离鉴定及其ugpB基因的原核表达

对新疆某羊场流产胎儿进行布鲁氏菌病原分离、培养,采用细菌群体形态观察、PCR、生化试验进行鉴定,结果分离得到了羊种布鲁氏菌生物3型,命名为027株.应用常规分子生物学方法克隆羊种布鲁氏菌生物3型027株的ugpB基因,构建原核表达载体pET-ugpB,转化E.coli BL21(DE3),IPTG诱导表达重组蛋白UgpB,进行SDS-PAGE和western blot分析,结果表明ugpB融合基因在大肠杆菌中得到了表达;采用Ni-NTA Agarose试剂盒进行蛋白纯化,获得了纯化的融合蛋白.     

羊种布鲁氏菌疫苗株PCR-RFLP鉴定方法的建立

为建立一种羊种布鲁氏菌Rev.1疫苗株PCR-RFLP鉴定方法,利用羊种布鲁氏菌Rev.1具有链霉素抗性的特性,选取与链霉素抗性相关编码核糖体蛋白S12的rps L基因为模板设计引物,经PCR扩增后,将产物进行Nci I酶切,发现羊种布鲁氏菌疫苗株Rev.1出现约500 bp条带,而不具有链霉素抗性的羊种布鲁氏菌株16M则出现384 bp条带。结果表明,PCR-RFLP方法能够用于羊种布鲁氏菌Rev.1疫苗株的鉴定,这为今后区分羊种布鲁氏菌疫苗株Rev.1免疫与野株感染提供了基础。     

牦牛流产胎儿中流产布鲁氏菌的分离与鉴定

牦牛流产胎儿中流产布鲁氏菌的分离与鉴定杨建梅（四川省金川县农牧局６２４１００）流产布鲁氏菌病的特征是在怀孕的母牛中引起流产。过去一般依据流产胎儿的消化道及肺组织内可找到布菌,其他组织则无菌,认为流产布鲁氏菌病垂直感染是细菌通过吞咽羊水进入胎儿所致,可...     

羊种布鲁氏菌Wzt基因的克隆及原核表达

为进一步研究Wzt蛋白在光滑型脂多糖合成路径中的作用,本试验利用PCR技术,以羊种布鲁氏菌16 M株基因组为模板,扩增出大小为759 bp的Wzt基因片段,将其连入pMD20-T载体,测序正确后构建重组质粒pET-28a-Wzt,转化E.coli BL21(DE3)工程菌,IPTG诱导其表达,最后用Western blotting鉴定蛋白。结果显示,扩增出的Wzt基因片段大小为759 bp,与GenBank中登录的羊种布鲁氏菌16 M株Wzt基因序列(登录号:AF047478.1)同源性为99.87%,证明成功克隆了Wzt基因,同时成功构建了pET-28a-Wzt原核表达载体,并在E.coli BL21(DE3)工程菌中表达了Wzt蛋白,诱导得到的融合蛋白大小约为30 ku,位于25～35 ku之间,与目的蛋白大小一致,结果表明成功表达了目的基因。     

呼和浩特地区牛羊布鲁菌流行株种及生物型鉴定

应用常规细菌学方法,对呼和浩特部分地区疑似布鲁菌病的牛、羊流产胎儿病料进行了布鲁菌分离,共分离到4株布鲁菌.进一步根据细菌形态、培养特性、生化特性以及平板凝集试验等对分离到的细菌进行种型鉴定.结果表明,其中3株为羊种布鲁菌生物3型,1株为牛种布鲁菌生物3型.本研究结果为内蒙古地区布鲁菌病的防控奠定了病原学基础.     

布鲁氏菌Ⅳ型分泌系统效应子VceC功能的初步研究

为研究布鲁氏菌(Brucella)Ⅳ型分泌系统效应子VceC的功能,本研究从羊种布鲁氏菌16M株基因组中克隆vceC基因片段,插入pEGFP-N1中,构建真核表达重组pEGFP-vceC。经脂质体转染HPT-8细胞,荧光显微镜观察、western blot鉴定目的蛋白在HPT-8细胞中表达情况,检测细胞凋亡、NO释放量及LDH活力。Western blot检测显示VceC在细胞中表达,转染pEGFP-vceC后的细胞凋亡、细胞上清液中NO释放量及LDH活力比对照组显著增加(p<0.05)。本研究初步表明VceC蛋白具有细胞毒性作用,为进一步研究VceC的功能奠定了基础。     

猪布鲁氏菌病病原分离及种型鉴定

猪布鲁氏菌病病原分离及种型鉴定易奇珍，左婉顺，周国基，林琼华，刘琪，郑列丰，韦庆昌，陈泽祥，赖家凯，高东升（广西兽医研究所，南宁５３０００１）我区猪布鲁氏菌病病原子１９５４年在桂林良丰农场分离出菌株，１９８１年以来，我区结合布病疫区面上综防工作，采集...     

The genome of Brucella melitensis

The genome of Brucella melitensis strain 16M was sequenced and contained 3,294,931 bp distributed over two circular chromosomes. Chromosome I was composed of 2,117,144 bp and chromosome II has 1,177,787 bp. A total of 3198 ORFs were predicted. The origins of replication of the chromosomes are similar to each other and to those of other -proteobacteria. Housekeeping genes such as those that encode for DNA replication, protein synthesis, core metabolism, and cell-wall biosynthesis were found on both chromosomes. Genes encoding adhesins, invasins, and hemolysins were also identified.     

粗糙型布鲁菌M111菌壳的制备

将含有裂解酶基因重组温控裂解质粒pBBR1MCS：：PR-PL-E电转化至粗糙型布鲁菌M111中,构建重组布鲁菌M111（pBBRlMCS：：PR-PL-E）。重组菌株在28℃培养,42℃诱导表达裂解酶E,从而制备布鲁菌菌壳。绘制布鲁菌生长曲线及裂解曲线,计算裂解率并用透射电镜观察布鲁菌菌壳的形态。结果显示,成功制备了布鲁菌菌壳,温控裂解质粒pBBRIMCS：：PR-PL-E对布鲁菌的裂解率为100％。透射电镜观察可见细菌内容物部分流出,细菌表面出现不同程度的皱缩,细胞形态发生变化。结果表明,本试验成功制备了粗糙型布鲁菌菌壳,初步研究了其基本特性,为下-步开展布鲁菌菌壳疫苗的研究奠定了基础。     

羊种布鲁氏菌Omp10基因的克隆及原核表达

根据GenBank公布的羊布鲁氏菌(B.melitensis) M5-90株外膜蛋白(outer membrane protein,Omp)基因序列,设计1对引物,以其全基因组为模板,采用PCR技术对其进行扩增,得到381 bp的目的片段,连接入pMD20-T载体,转化E.coli DH5α感受态细胞;测序正确后,构建pET-28a-Omp10原核表达质粒,再将该质粒转化入E.coli BL21(DE3), IPTG诱导表达融合蛋白His-Omp10,用SDS-PAGE和Western blotting进行分析.结果表明, 成功构建了含Omp10基因的原核表达载体,并在E.coli BL21(DE3)中表达了Omp10基因,诱导得到的融合蛋白经鉴定与目的蛋白大小一致,证明Omp10得到成功表达.该试验为布鲁氏菌病的进一步研究奠定基础.     

Control of small ruminant brucellosis by use of Brucella melitensis Rev.1 vaccine: laboratory aspects and field observations

Brucellosis vaccines are essential elements in control programs. Since first developed in the mid-1950s, the Brucella melitensis vaccine strain Rev.1 has been used worldwide and its significant value in protecting sheep and goats in endemic areas recognized. This review provides historical background on the development of the vaccine, its use and field complications arising in Israel following changes in the strain’s pathogenicity. The urgent need for resolving cases of vaccine strain excretion in the milk, horizontal transfer and a unique case of human infection has led to identification of an atypical B. melitensis biovar 1 strain that resembles strain Rev.1 in susceptibility to penicillin and dyes. An omp2 based PCR method has been developed that traced the lineage of Israeli B. melitensis biovar 1 strains. This locus serves as an epidemiological tag for the Rev.1 vaccine strain. Despite the rapid development of new approaches in the field of vaccination, it is anticipated that in the near future the Rev.1 vaccine would remain the only accepted vaccine in national control programs.     

山羊流产胎儿绵羊附睾种布鲁菌(Brucella ovis)的分离和鉴定

从山羊流产胎儿中分离出1株绵羊附睾种布鲁菌(Brucella ovis),经培养发现该菌需要CO2,不产生H2S,且经硫堇、碱性复红和A、M、R因子血清凝集试验及噬菌体裂解试验等方法鉴定,该菌株与国际标准菌株绵羊附睾种特性完全一致。在山羊中分离出绵羊附睾种布鲁菌株(Br.ovis)在国内尚属首次发现。     

羊种布鲁氏菌LpxB基因的克隆、原核表达及其蛋白的生物信息学分析

试验旨在克隆羊种布鲁氏菌LpxB基因并进行原核表达和蛋白的生物信息学分析。以布鲁氏菌M5-90株基因组为模板,参照GenBank中M5-90株基因组DNA序列,用DNAMAN软件设计1对引物,通过聚合酶链式反应（PCR）扩增得到大小为1 188 bp的LpxB基因,将其连接入pMD20-T载体上,构建pMD20-T-LpxB重组质粒,将其转化到E.coli DH5α感受态细胞中,经BamH Ⅰ和 Xho Ⅰ双酶切鉴定正确后扩大培养。将BamH Ⅰ和 Xho Ⅰ双酶切获得的LpxB片段连接入pET-28a,构建重组质粒pET-28a-LpxB,转化到E.coli BL21（DE3）中,双酶切鉴定正确后扩大培养。经IPTG诱导其表达,用SDS-PAGE和Western blotting对蛋白进行鉴定。运用DNAMAN、BioEdit等软件对LpxB基因编码的氨基酸序列进行分析。结果表明,本研究成功克隆了LpxB基因并进行了蛋白表达,在LpxB蛋白二级结构中,α-螺旋、伸展链、β-折叠和无规卷曲分别占52.41%、14.94%、8.10%和24.55%。     

Cloning,Prokaryotic Expression and Bioinformation Analysis of dhbC Gene of Brucella melitensis

This study was aimed to clone and express dhbC gene of Brucella melitensis, and analyze the bioinformatics of its expressed protein. A pair of primers were designed by referring to dhbC gene sequence information of Brucella melitensis M5-90 strain in GenBank, and the dhbC gene fragment was amplified by PCR method. The obtained dhbC gene was ligated into pMD20-T vector to construct pMD20-T-dhbC recombinant plasmid and transformed into E.coli DH5α competent cells. The plasmid was identified by restriction enzyme digestion. The recombinant plasmid pET28a-dhbC was constructed and transformed into E.coli BL21 (DE3) competent cells. The expression was induced by IPTG. The expressed product was analyzed by SDS-PAGE and Western blotting. Bioinformatics analysis of the amino acid sequence encoded by dhbC gene was carried out using bioinformatics software DNAMAN and related online sites ProtParam, SOPMA and Protscale. The results showed that dhbC gene was cloned with the length of 1 093 bp, and protein expression was expressed. The expressed fusion protein was about 47 ku, and was mainly in the form of inclusion body. The molecular weight of the dhbC protein was C₁₈₆₆H₂₉₆₈N₅₄₄O₅₆₂S₁₅, the molecular mass was 42 496.3 u, the theoretical isoelectric point (pI) was 5.81, the extinction coefficient was 33 835, the instability coefficient was 36.76, the hydrophobic index was 86.19, the total average hydrophobicity (GRAVY) was -0.215. The half-life of reticulocytes in mammals was predicted to be 30 h, and the secondary structure was dominated by α-helix (41.94%) and random coil (31.46%).     

Cloning,Prokaryotic Expression and Bioinformatics Analysis of LpxB Gene of Brucella melitensis

The study was aimed to clone and express LpxB gene,and perform the bioinformatics analysis of protein.The genomic DNA of Brucella melitensis M5-90 was used as template.According to the genome sequence of M5-90 on GenBank,a pair of primers was designed.LpxB gene,which was 1 188 bp,was amplified by PCR,and was ligated into pMD20-T vector.The constructed recombinant plasmid pMD20-T-LpxB was transformed into E.coli DH5α.The recombinant plasmid was confirmed by endonuclease digestion and sequencing.The coding region of LpxB from pMD20-T was digested by BamHⅠ and XhoⅠ.Then,the fragment was inserted into prokaryotic expression vector pET-28a,and the positive plasmid was named pET-28a-LpxB.The pET-28a-LpxB was transformed into E.coli BL21 (DE3).The expressed protein was identified by SDS-PAGE and Western blotting.DNAMAN and BioEdit softwares were used to analyze the sequence of amino acids encoded LpxB gene.The results showed that the CDS of LpxB was successfully cloned and expressed.The secondary structure of LpxB protein consisted structure α -helix,extended strand,β-turn and random coil which accounted for 52.41％,14.94％,8.10％ and 24.55％,respectively.