An RTX operon in hemolytic Moraxella bovis is absent from nonhemolytic strains

Pathogenic isolates of Moraxella bovis express a calcium-dependent transmembrane pore forming cytotoxin that is an RTX toxin encoded by mbxA. The DNA flanking mbxA was cloned and sequenced to determine if M. bovis contained a classical RTX operon. Open reading frames (ORFs) with deduced amino acid sequence homology to putative activation (RTX C) and transport (RTX B and D) proteins were identified and have been designated MbxC, MbxB, and MbxD, respectively. Thus, hemolytic M. bovis contains a typical RTX operon comprised of four genes arranged (5'-3') mbxCABD. In addition, the deduced amino acid sequences of DNA flanking mbxCABD revealed ORFs with amino acid sequence similarity to transposases (5'). At the 3' end of the mbx gene cluster, an ORF with homology to bacterial tolC genes was identified. Thus, as with the cya RTX operon of Bordetella pertussis, M. bovis appears to have a secretion accessory protein linked to RTX genes. Analysis of genomic DNA isolated from 5 nonhemolytic M. bovis strains by PCR and Southern blotting revealed the absence of mbxCABD. These strains did, however, amplify with primers specific for the 5' region flanking mbxC. M. bovis harbors a classical RTX operon that is absent in nonhemolytic strains.     

Identification and characterization of complete RTX operons in Moraxella bovoculi and Moraxella ovis

To determine if Moraxella bovoculi (M. bovoculi), a recently characterized coccoid Moraxella that was isolated from the eyes of calves affected with infectious bovine keratoconjunctivitis (IBK), and Moraxella ovis (M. ovis), originally isolated from sheep with conjunctivitis, possessed genes encoding RTX proteins, genomic DNA was amplified with oligonucleotide primers targeting RTX operon genes of Moraxella bovis (M. bovis). Complete classical RTX operons composed of RTXCABD genes closely linked to a putative secretion accessory protein encoding gene (tolC) were identified in M. bovoculi and M. ovis and were designated mbvCABDtolC and movCABDtolC, respectively. These genes were closely related to M. bovis mbxCABDtolC. Polyclonal rabbit antiserum against the carboxy terminus of M. bovoculi MbvA neutralized hemolytic activity of both M. bovoculi and M. ovis; this antiserum did not neutralize the hemolytic activity of M. bovis. M. bovoculi and M. ovis possess genes that encode proteins related to pathogenic factors of M. bovis.     

Relatedness of cytotoxins from geographically diverse isolates of Moraxella bovis

To determine whether amino acid sequence variation exists in the Moraxella bovis (M. bovis) cytotoxin (MbxA) from geographically diverse M. bovis isolated in the United States, mbxA was amplified and sequenced. The MbxA deduced amino acid sequence from M. bovis originally isolated in California, Washington, North Carolina, and Georgia, as well as reference strains of M. bovis isolated at the National Animal Disease Laboratory, Ames, IA, USA, all encoded a nearly identical 927 amino acid protein. MbxA from two of the four California isolates (SFS 9a and SFS 100a) differed from all other isolates at two sites at which the polar amino acids glutamine (position 666) and asparagine (position 823) were replaced by ionized amino acids glutamic acid and aspartic acid, respectively. Rabbit antiserum to the expressed carboxy terminus (amino acids 590-927) of MbxA from M. bovis (Tifton I) neutralized the hemolytic activity of SFS 9a and SFS 100a. The M. bovis cytotoxin appears to be conserved amongst geographically diverse isolates of M. bovis from the USA. Antiserum against the carboxy terminus of MbxA common to the majority of isolates neutralized the hemolytic activity of two strains with a divergent MbxA deduced amino acid sequence. Vaccines against IBK that incorporate MbxA as antigen may offer protection against geographically diverse strains of M. bovis.     

8株牛支原体分离株P81表面膜蛋白基因的克隆与序列分析

本研究对我国采集自8个省份患有牛呼吸道疾病的肺脏组织病料进行了病原分离鉴定,得到8株牛支原体(M.bovis)分离株。参考GenBank中已发表的M.bovisPG45株的P81基因序列,设计引物扩增P81基因,将其克隆到pMD-18T上,筛选重组质粒并对其进行序列测定及分析。结果显示,P81基因全长2097bp,含有1个开放性阅读框,编码699个氨基酸。这8株M.bovis分离株的P81同源性为99.4%～99.9%,与PG45株同源性94.6%～94.9%,与无乳支原体(M.agalacia)PG2株同源性仅为73.8%～74%,结果表明,M.bovisP81基因基因高度保守,种间差异较大,具有研究前景。     

Identification of a new Escherichia coli She haemolysin homolog in avian E. coli

Haemolysin is one type of virulence factor that assists in the pathogenesis of Escherichia coli. Currently, hemolytic activity in E. coli has been attributed to haemolysin genes found in either uropathogenic or enterohemorrhagic E. coli. Both haemolysins are classified as RTX toxins because they both have repeats in toxin domains and share similar operon organization, sequence homology, and mechanisms of action. Haemolytic avian E. coli isolates, however, lack either E. coli haemolysin gene. To investigate the avian E. coli haemolysin, a genomic library was made from an avian pathogenic E. coli. A haemolytic clone that was isolated was shown to contain homology with sheA, an E. coli K- 12 gene which causes haemolysis when present in high copy number. The cloned haemolysin gene, hlyE, lacked the conserved amino acid sequence and accessory genes common to all RTX toxins. DNA hybridizations and polymerase chain reaction amplifications showed that the nucleotide sequences homologous to hlyE were not present in a collection of three O157: H7 E. coli, five haemolytic canine uropathogenic E. coli, one haemolytic O26 E. coli, and three haemolytic avian pathogenic E. coli. Thus we have identified a new E. coli haemolysin distinct from the RTX haemolysins and have shown that some avian pathogenic E. coli possess a haemolysin with no apparent homology to hlyE or RTX haemolysins.     

Stability, antigenicity, and aggregation of Moraxella bovis cytolysin after purification and storage

OBJECTIVES: To compare stability, antigenicity, and aggregation characteristics of Moraxella bovis cytolysins among isolates from geographically diverse areas. STUDY POPULATION: 8 isolates of M. bovis. PROCEDURE: Filter-sterilized broth culture supernatants of M. bovis were concentrated, diafiltered, and chromatographed. The endotoxin and cytolysin activities in samples were measured. Chromatographed cytolysins of M. bovis were examined by immunoblotting. Hemolytic and leukotoxic activities were measured from samples collected at each step of purification and before and after storage. Hemolysis was measured directly by use of washed bovine erythrocyte targets. Leukotoxicity was measured by use of a 51Cr release assay. RESULTS: Cytolysin was retained by a filter with 100-kd nominal molecular weight limit. Hemolytic activity, leukotoxic activity, and endotoxin were eluted together in void volume of a gel-filtration column (molecular mass exclusion limit = 4 X 10(7) d). Gel-column chromatographed diafiltered retentate had the greatest specific cytolytic activity and the highest endotoxin-to-protein ratio. Frozen diafiltered retentate(-80 degrees C, 4 months) was cytolytic after thawing. Immunoblots of gel-column chromatographed cytolysin contained 4 proteins with molecular masses between 90 and 68 kd. Fractions with high lytic activities also had additional protein bands with molecular masses of 98 and 63 kd. Immunoblots of gel-column chromatographed diafiltered retentate revealed proteins with molecular masses between 90 and 68 kd. CONCLUSIONS AND CLINICAL RELEVANCE: Diafiltered M. bovis cytolysin is aggregated with endotoxin. Antigenicity and cytolytic activities in diafiltered retentate are conserved among M. bovis isolates. Diafiltration could be useful for bulk semipurification of M. bovis cytolysin. Cytolysin-enriched vaccines of M. bovis could be contaminated by endotoxin.     

Prevention of naturally occurring infectious bovine keratoconjunctivitis with a recombinant Moraxella bovis pilin-Moraxella bovis cytotoxin-ISCOM matrix adjuvanted vaccine

To evaluate the efficacy of a recombinant Moraxella bovis pilin-M. bovis cytotoxin subunit vaccine to prevent naturally occurring infectious bovine keratoconjunctivitis (IBK; pinkeye), a randomized, blinded, controlled field trial was conducted during summer 2005 in a northern California herd of beef cattle. One hundred and one steers were vaccinated with ISCOM matrix (adjuvant control), recombinant M. bovis cytotoxin carboxy terminus+ISCOM matrix (MbxA), or recombinant M. bovis pilin-cytotoxin carboxy terminus+ISCOM matrix (pilin-MbxA); calves received secondary vaccinations 21 days later. Calves were examined once weekly for 18 weeks for the development of corneal ulcers associated with IBK. Overall, the pilin-MbxA vaccinated group had the lowest overall cumulative proportion of ulcerated calves. Calves that received MbxA, whether alone or with pilin had significantly higher M. bovis cytotoxin serum neutralizing titers as compared to control calves. Results of ocular cultures suggested that vaccination with an M. bovis antigen affected organism type isolated from an ulcer: M. bovis was cultured more often from the eyes of control calves than from the eyes of calves vaccinated with MbxA and pilin-MbxA. In addition, vaccination of calves with MbxA and pilin-MbxA resulted in a higher prevalence of Moraxella bovoculi sp. nov. in ocular cultures. While no significant difference was observed between a cytotoxin versus pilin+cytotoxin vaccine against IBK, the reduced cumulative proportion of IBK in the pilin-cytotoxin vaccinated calves suggests it may provide an advantage over a cytotoxin vaccine alone. Efficacy of an M. bovis vaccine may be reduced in herds where IBK is associated with M. bovoculi sp. nov.     

Effectiveness of a cytolysin-enriched vaccine for protection of cattle against infectious bovine keratoconjunctivitis

OBJECTIVE: To determine the immunogenicity of a Moraxella bovis cytolysin-enriched vaccine for prevention of infectious bovine keratoconjunctivitis (IBK). ANIMALS: 104 mixed-breed beef calves ranging between 4 and 8 months of age. PROCEDURE: Vaccines were prepared by the diafiltration of broth culture supernatant from hemolytic M bovis or sterile media. The diafiltered retentate was combined with Quil A adjuvant. Calves were randomly assigned to receive either the cytolysin vaccine (n = 35) or, as controls, adjuvant (35) or saline (0.9% NaCl) solution (34). Eyes of all calves were examined weekly for signs of IBK for 15 weeks. Calves that developed severe IBK were treated SC with florfenicol. RESULTS: Cytolysin vaccine contained 4 proteins with molecular masses ranging between 65 and 90 kd. Cytolysin-vaccinated calves had fewer instances of IBK than control calves. The time of onset of corneal lesions in cytolysin-vaccinated calves that developed IBK was delayed, compared with that of calves in either control group. The cytolysin-Quil A vaccine contained endotoxin, but calves did not have clinical signs of illness after vaccination. CONCLUSIONS AND CLINICAL RELEVANCE: Calves that were vaccinated with a cytolysin-enriched vaccine had some resistance to IBK. Vaccines containing concentrated diafiltered M bovis cytolysin could protect beef calves against IBK.     

绵羊肝片吸虫谷胱甘肽硫转移酶基因的克隆与序列分析

本研究扩增肝片吸虫(Fasciola hepatica,Fh)谷胱甘肽硫转移酶(GST)基因,并进行同源性分析。根据GenBank发表的部分Fh GST基因序列设计并合成一对特异性引物,利用RT-PCR方法扩增出Fh GST基因完整的开放阅读框(open reading frame,ORF),测定序列,使用分子生物学软件进行同源性分析。获得Fh GST基因全长682 bp,编码218个氨基酸,与澳大利亚分离的肝片吸虫GST同源性较高,与大片吸虫和卫氏并殖吸虫的GST也有较高的同源性。不同虫株GST基因具有较高的同源性,因此Fh GST蛋白不适合用作诊断抗原,但由于其存在交叉反应,GST基因作为分子疫苗的候选基因具有重要意义。肝片吸虫GST基因的克隆,为进一步研究GST蛋白的功能和作用奠定了基础。     

Molecular cloning of a cDNA encoding a putative cuticle collagen of Trichinella spiralis

A 5-day-old adult stage-specific cDNA fragment from Trichinella spiralis was identified by suppression subtractive hybridization and was used as a probe to screen the cDNA library. The cDNA sequence coding for a putative T. spiralis cuticle collagen was isolated. The cDNA encoded an open reading frame of 343 amino acid residues with molecular weight of 35.1 k Da. The deduced protein contained an N-terminal signal peptide, a nematode cuticle collagen N-terminal domain and a collagen triple helix repeat domain. Searches in GenBank using BLASTP showed up to 47% identity to cuticle collagens from other nematodes. Southern blot analysis of genomic DNA indicated this gene was present as a single copy in T. spiralis genome.     

用cDNA末端扩增法克隆柞蚕攻击素(attacin)基因及序列分析

利用cDNA末端扩增(RLM-RACE)方法,克隆了柞蚕攻击素(attac in)基因cDNA序列(GenBank登陆号:AY960680)。柞蚕攻击素基因cDNA序列全长912 bp,其中699 bp的蛋白质编码区可编码233个氨基酸。预测蛋白质分子量为25 kD,等电点(pI)为7.54。柞蚕攻击素基因中包含2个内含子。S ignal P 3.0 Server程序分析结果显示,柞蚕攻击素第1-17位氨基酸为信号肽序列。蛋白质功能区分析预测,柞蚕攻击素在第47-112氨基酸之间为攻击素-N(attac in-N)功能区,第113-233位氨基酸之间为攻击素-C(attac in-C)功能区。以邻接法(ne igh-bor-join ing,NJ)建立了与其它昆虫攻击素的系统关系图。     

柞蚕溶菌酶基因的克隆和序列分析

采用cDNA末端扩增(RACE)方法及简并引物克隆了柞蚕溶菌酶(Antheraea pernyilysozyme)基因(Gen-Bank登录号:DQ353869)。该基因cDNA序列全长675 bp,由3个外显子和2个内含子组成,其开放读码框(ORF)长420 bp,编码140个氨基酸。生物信息学分析表明,该基因编码蛋白的1-20位氨基酸为信号肽序列,21-140位氨基酸为柞蚕溶菌酶成熟蛋白(分子质量14 kD,等电点8.46)。柞蚕溶菌酶氨基酸序列中含有c型溶菌酶分子所特有的活性中心Glu32、Asp50以及8个半胱氨酸残基,与鳞翅目昆虫溶菌酶的氨基酸序列同源性较高,属非钙结合型c型溶菌酶。柞蚕溶菌酶的模拟三级结构与印度柞蚕(Antheraea mylitta)溶菌酶的三级结构十分相似。     

牛支原体pdhc基因的克隆、表达及其亚细胞定位

试验旨在研究牛支原体（Mycoplasma bovis,M.bovis）武威株二氢硫辛酰胺转乙酰酶（PDHc-E2）基因序列特征及其在牛支原体细胞中的位置。参照GenBank中牛支原体HB0801株pdhc基因（登录号：CP002058.1）设计引物,应用PCR扩增获得牛支原体武威株pdhc基因,在测序及序列分析的基础上,应用Overlap PCR完成点突变后将其克隆至pET-28a（+）中,构建原核表达载体pET-pdhc。pET-pdhc转化大肠杆菌Rosetta（DE3）感受态细胞后经IPTG诱导获得融合蛋白,将纯化蛋白免疫新西兰兔制备多抗血清,应用iELISA和Western blotting对牛支原体武威株PDHc-E2在细胞内的分布进行初步研究。结果显示,牛支原体武威株pdhc基因CDS全长735 bp,编码244个氨基酸,与国内牛支原体分离株HB0801、Hubei-1、CQ-W70、NM2012等基因序列完全一致,与国际标准株PG45同源性为99.2%,与无乳支原体（M.agalactiae）同源性为90.9%~91.2%,与加利福尼亚支原体（M.californicum）ST6株的同源性仅为78.4%,基因序列非常保守;通过Overlap PCR将该基因中4个编码色氨酸的TGA密码子突变为TGG,且完成点突变后的基因在大肠杆菌中成功表达,重组蛋白大小约为29 ku,主要以可溶性形式存在,iELISA结果显示,重组蛋白PDHc-E2具有较高的免疫原性,可刺激新西兰兔产生高水平的抗体,血清效价高达1：100 000;亚细胞定位结果表明,制备的多抗血清与重组蛋白PDHc-E2、牛支原体全菌蛋白、牛支原体膜蛋白、牛支原体胞浆蛋白均能发生特异性结合,说明该蛋白在牛支原体细胞膜和细胞质中均有分布,为膜相关蛋白,但在细胞质中的分布多于细胞膜。本研究结果为进一步研究牛支原体的生物学功能提供了理论依据。     

家蚕新的卵色相关基因Bmwh2的克隆及组织表达与功能研究

腺苷三磷酸结合盒转运蛋白(ATP-binding cassette transporter,ABC转运蛋白)是古老而庞大的家族,广泛分布于从细菌到人类各种生物体中,主要利用ATP水解释放的能量实现多种底物的跨膜主动转运。通过RT-PCR扩增、克隆测序了家蚕ABC转运子Bmwh2完整的开放阅读框。用生物信息学方法分析发现,Bmwh2有14个外显子和13个内含子,编码689个氨基酸残基,分子质量77.38 kD,等电点8.42;Bmwh2含有1个核苷酸结合结构域和6个α螺旋构成的跨膜结构域,为半转运子,属于家蚕ABC转运子超家族G亚族成员。利用半定量RT-PCR方法分析Bmwh2在家蚕5龄第3天幼虫不同组织的表达水平,以精巢中的相对表达量最高。用合成的siRNA显微注射胚胎发育时期的蚕卵,对Bmwh2进行干涉研究,获得了白卵和嵌合体的干涉表型。根据实验结果,推测Bmwh2可能参与家蚕浆液膜色素前体的转运。     

家蚕BmLHep_59蛋白的表达和亚细胞定位

肝细胞癌相关抗原-59(hepatocellular carcinoma-associated antigen59,Hep_59)属于哺乳动物中一类含有约100个氨基酸残基的保守结构域家族,这类蛋白主要存在于真核生物细胞中,大多为假想蛋白。从家蚕蛹cDNA文库中筛选的一个新基因,其编码蛋白含有一个和Hep_59高度同源的保守结构域,将其命名为BmLHep_59(GenBank登录号:DN985181)。PCR扩增该基因的ORF,经EcoRⅠ/XhoⅠ双酶切重组到原核表达载体pET-28a(+),并转入E.coli Rosetta(DE3)感受态细胞中表达。通过镍柱亲和层析得到纯化的重组蛋白BmLHep_59,并以此纯化重组蛋白作为抗原免疫新西兰大白兔制备多克隆抗体,经ELISA检测抗血清效价可达1∶12 000以上。用Western blotting检测到家蚕5龄幼虫的表皮、丝腺和血淋巴中均有BmLHep_59表达,其中在血淋巴的表达量较高,暗示该蛋白可能参与调控家蚕的造血机能。该蛋白在蛹期中也有表达。亚细胞定位显示BmLHep_59在家蚕Bm5细胞的细胞质和细胞核中均有分布。     

分支杆菌分泌蛋白研究进展

对近年来分支杆菌分泌蛋白研究进展进行了简要综述。利用杂交技术将牛分支杆菌基因组和结核分支杆菌复合物基因组相比较,显示在牛分支杆菌基因组中共有11处基因缺失;早期滤液成分是分支杆菌在生长期间分泌的相关蛋白群,目前的研究倾向于将存在于结核分支杆菌培养滤液中的蛋白分为3个主要的群。结核分支杆菌全基因组测序的完成,使得分支杆菌分泌蛋白质研究从分离鉴定到功能研究都有了很大的进步。相信随着研究的深入,更多的分泌蛋白质将被发现。     

Proteins encoded in the 5' region of the pestivirus genome--considerations concerning taxonomy.

The first protein encoded within the pestivirus open reading frame is a nonstructural protein which removes itself from the polyprotein by autoproteolytic cleavage. The following nucleocapsid protein ends just before a putative signal sequence preceding three glycosylated proteins. All three glycoproteins are part of the viral envelope and exist in the form of disulfide-linked dimers. Pestiviruses have recently been reclassified as members of the family Flaviviridae which now comprises three genera, namely flavivirus, hepatitis C virus group and pestivirus. All members of the family have certain characteristics in common like the overall genome organization and the strategy of gene expression. Major differences exist, however, between the genera; the most obvious ones concern proteins encoded in the 5' region of the respective genomes.     

Brucella melitensis 16M: characterisation of the galE gene and mouse immunisation studies with a galE deficient mutant

The galE gene of Streptomyces lividans was used to probe a cosmid library harbouring Brucella melitensis 16M DNA and the nucleotide sequence of a 2.5 kb ClaI fragment which hybridised was determined. An open reading frame encoding a predicted polypeptide with significant homology to UDP-galactose-4-epimerases of Brucella arbortus strain 2308 and other bacterial species was identified. DNA sequences flanking the B. melitensis galE gene shared no identity with other gal genes and, as for B. abortus, were located adjacent to a mazG homologue. A plasmid which encoded the B. melitensis galE open reading frame complemented a galE mutation in Salmonella typhimurium LB5010, as shown by the restoration of smooth lipopolysaccharide (LPS) biosynthesis, sensitivity to phage P22 infection and restoration of UDP-galactose-4-epimerase activity. The galE gene on the B. melitensis 16M chromosome was disrupted by insertional inactivation and these mutants lacked UDP-galactose-4-epimerase activity but no discernible differences in LPS structure between parent and the mutants were observed. One B. melitensis 16M galE mutant, Bm92, was assessed for virulence in CD-1 and BALB/c mice and displayed similar kinetics of invasion and persistence in tissues compared with the parent bacterial strain. CD-1 mice immunised with B. melitensis 16M galE were protected against B. melitensis 16M challenge.     

Genomic and antigenic variations of porcine reproductive and respiratory syndrome virus major envelope GP5 glycoprotein.

The objective of the present study was to evaluate the importance of genomic and antigenic variations which may have affected the major envelope glycoprotein GP5 of porcine reproductive and respiratory syndrome virus (PRRSV) isolates responsible for outbreaks in Quebec and Ontario, in comparison with the modified-live U.S. vaccine strain (MLV) and the European prototype strain from Lelystad (LV). Nucleotide sequence analyses of the open reading frame (ORF)5 genes showed that all of the isolates studied were heterogenous, amino acid (aa) identities varied from 88 to 99% with the MLV strain, and between 51 and 54% with the LV strain. The aa substitutions were randomly scattered across the protein, although one region between residues 26 and 39 was found to correspond to a hypervariable region which involved 0 to 3 potential N-glycosylation sites. The ORF5 encoded products of 5 of these isolates, including the MLV and LV strains, were expressed in E. coli as recombinant proteins fused to the glutathione S-transferase (GST) protein and used to raise hyperimmune anti-ORF5 sera in rabbits. The reactivity patterns of strain-specific hyperimmune anti-ORF5 sera and a panel of 4 monoclonal antibodies directed against the ORF5 gene product of the Quebec IAF-Klop strain of PRRSV, indicated that GP5 of field isolates also underwent antigenic variations. The data suggest that neutralizing epitopes, independent of conformation and glycosylation, are also associated with antigenic variability of the GP5 of PRRSV.