致肾盂肾炎大肠杆菌粘附基因群的克隆及其抗血清的制备

以柯斯质粒pHC79为载体构建致肾盂肾炎大肠杆菌代表株E.coli J96染色体基因文库,获得两个能够表达粘附特性的重组柯斯质粒.进一步用鸟枪法亚克隆到载体pACYC184,得到三个阳性重组体.其中pCT10/E.coli K-12 p678-54能够稳定表达P菌毛和MRHA,分子量为14.6kb.用该克隆子免疫新西兰白兔,抗血清用带有载体质粒的受体菌(pACYC184/E.coli K-12 P678-54)吸收后,经菌毛粗提物的SDS-聚丙烯酰胺凝胶电泳及Western blotting和血凝抑制试验检测,其特异性仅针对致肾盂肾炎大肠杆菌的粘附基因群,可用于临床菌株的鉴定.     

毒素源性大肠杆菌K88ac抗原基因的次级克隆和重组质粒的限制图

从我国引起仔猪腹泻的野生株E. coli 79-1454克隆了K88ac抗原基因,获得了重组体E. coli RR1(pNZ8801),分子量为10 Md。为了得到分子量更小的重组体,用E．coRI消化重组质粒,除去中间约3.2 Md的片段,得到次级克隆株E. coli RR1(pNZ8802),质粒分子量为6.8 Md。测定其K 88ac抗原为阳性,而且其产生的K88at抗原量略高于初级克隆株。电镜照片表明重组体表面长有菌毛。萤光抗体染色和猪刷状缘细胞粘附试验亦表明有良好的粘附生物活性。此重组体可以与本人克隆的K99抗原基因构建成复合重组体。本文还作了重组体的限制性酶切图。     

致肾盂肾炎大肠杆菌粘附特性的研究

本文对临床肾盂肾炎病人尿标本中分离的大肠杆菌１３２和１３６的粘附特性进行了系统的研究。受试菌的Ｐ血型阳性红细胞血凝试验阳性,能够与人的尿道上皮细胞粘附。利用致肾盂肾炎大肠杆菌Ｐ菌毛粘附基因群抗血清进行免疫学检测,两株菌的全菌ＥＬＩＳＡ结果阳性,免疫电镜证实该抗血清能与受试菌株的菌毛特异性结合。提取临床分离株的菌毛蛋白进行免疫印迹测定,仅有一条蛋白带显色,其分子量为１６．６ｋｄ。致肾盂肾炎大肠杆菌的粘附特性是区别于其他大肠杆菌的重要特征,上述结果表明本文报告的两株大肠杆菌为致肾盂肾炎大肠杆菌。     

大肠杆菌K99菌毛fan操纵子的克隆、表达及活性

[目的]在体外克隆和表达猪产肠毒素大肠杆菌(ETEC) K99菌毛操纵子fan结构基因,并检测重组菌毛的相关生物学活性.[方法]以猪源分离的表达K99菌毛ETEC C83907株制取模板,成功PCR扩增出编码K99菌毛的fan操纵子,约5.7 kb.将fan操纵子克隆人表达质粒载体pBR322,筛选出含正确阳性质粒的重组菌.进一步将上述的重组质粒DNA转化至不含任何菌毛的大肠杆菌SE5000株,同时将空载体pBR322质粒转化入SE5000构建阴性对照菌株.[结果]该重组菌能与鼠抗K99菌毛单克隆抗体发生明显的凝集反应,与新生仔猪小肠上皮细胞刷状缘BBV分子有强烈凝集反应.电镜观察到上述重组菌表面大量表达K99菌毛,用热抽提法提纯其表达的K99菌毛,并经SDS-PAGE电泳和考马斯亮蓝染色,可以得到分子量约为18.5kDa的主要蛋白条带.纯化菌毛免疫小鼠后制备出高效价的鼠抗血清,能与携带K99菌毛的C83907、C83914、C83260野生株发生强烈的凝集反应,而与携带其他菌毛的ETEC不反应.玻板凝集试验和Western blot结果表明:体外表达的K99菌毛具有和野生K99菌毛相同的抗原性.用表达K99菌毛的重组菌进行HeLa细胞体外黏附试验和黏附抑制实验,结果表明:重组菌和野生菌株一样具有较强的粘附性,而且用重组菌毛制备的鼠抗血清能有效地抑制上述重组菌或野生菌株对细胞系的黏附结合.[讨论]本研究为进一步研究K99菌毛生物学作用建立了良好的实验平台.     

通过基因工程对赖氨酸生产菌B66构建的研究

从大肠杆菌E.coli K-12中通过PCR扩增出磷酸果糖激酶编码基因(pfkA),将其与表达载体pCMVTNTTMvector连接构建成重组质粒pKu-2,转化谷氨酸棒杆菌B10,并得到表达。酶活性测定表明pfkA基因在受体菌B66中得到表达水平为(126.6±0.76)U/g蛋白,解除了磷酸果糖激酶对已经改造的赖氨酸的整个代谢途径的限制。同时,转化菌对糖转化率比B10高11.59%,产酸率高16.52%。     

基于大肠杆菌K-12MG1655的narG基因缺失荧光工程菌的构建

本研究应用PCR扩增两翼与靶基因上下游同源含有Cmr(氯霉素抗性基因)的片段,经电击转化至E.coli K-12 MG1655中。在λRed重组系统的帮助下,通过氯霉素抗性基因两侧的同源序列在体内与narG基因发生同源重组,随后利用Cmr两端的FTP位点,通过FLP酶专一性重组将Cmr删除,获得narG基因精确敲出且不带Cmr的E.coli K-12 MG1655(ΔnarG)突变株。最后转化NO诱导的GFP(绿色荧光蛋白基因)报告基因质粒p SB1C3_BBa_K 381001,获得具有对(亚)硝酸盐荧光应答的重组发光工程菌株K-12 MG1655(ΔnarG)::GFP。生长曲线测定结果表明,narG基因的敲出对大肠杆菌正常生长无显著影响(p0.05)。荧光工程菌对(亚)硝酸盐的应答结果表明,E.coli K-12 MG1655(ΔnarG)::GFP对(亚)硝酸盐的敏感性相对于E.coli K-12 MG1655::GFP提高了2.5倍左右。且在0~8μmol/L的硝酸盐浓度下符合线性关系:(R2=0.968 3);在0~4μmol/L的亚硝酸盐浓度下符合线性关系:(R2=0.997 5)。本研究所构建的E.coli K-12 MG1655(ΔnarG)::GFP能够有效检测痕量(亚)硝酸盐,可为检测食品和水质中(亚)硝酸盐提供新的方法选择。     

反义RNA介导的大肠杆菌非必需基因rpsF的基因沉默

【目的】探讨反义RNA技术介导的大肠杆菌非必需基因rpsF基因沉默导致菌体生长受抑制的原因。【方法】将rpsF基因5'端41-230 bp的片段反向插入带有末端配对结构的反义表达载体pHN678,获得重组质粒,导入大肠杆菌宿主获得反义RNA菌株Escherichia.coli/pHNF,并用诱导剂IPTG诱导反义RNA表达,通过与对照菌E.coli/pHN678的液体生长状态差异判断菌体生长表型;采用Real time RT-PCR方法跟踪分析转录水平。【结果】构建了针对rpsF的反义RNA菌株,且其生长受抑制程度与IPTG浓度呈正相关。IPTG浓度为100μmol/L时,菌体生长未受抑制,但靶基因rpsF的mRNA量降低了36%,而rpsR是位于同一操纵子下游的必需基因,其转录水平却未受影响;IPTG浓度为200μmol/L时,菌体生长明显受抑制,经分析发现rpsR转录水平降低了12%。【结论】反义RNA菌株E.coli/pHNF生长受抑制的原因是由于此反义RNA引起了同一操纵子下另一必需基因rpsR的转录水平降低。     

四环素与3,4-苯并芘复合污染对抗性基因tetA(C)产生高抗性突变的影响

【目的】探究环境中同时存在低浓度四环素(tetracycline,TC)和3,4-苯并芘(benzo [a] pyrene,Bap)对抗性基因tetA(C)产生高抗性突变的影响。【方法】以大肠杆菌(Escherichia coli,E. coli)为宿主菌株,pACYC184质粒作为载体,四环素抗性基因tetA(C)作为研究对象,采用易错PCR构建基因文库的方法,建立基因突变位点对应高抗性的关系密码表。同时设置添加低浓度TC且添加0–30 mg/L Bap以及仅添加0–30 mg/L Bap的处理组,培养携带pACYC184质粒的大肠杆菌14 d,每组中随机挑选10株获得高抗性的菌株,对其中的tetA(C)基因片段进行测序,再结合突变位点密码表,计算高抗性菌株中由基因突变产生高抗性菌株的比例。【结果】测序结果显示在低浓度TC选择压力下,Bap浓度越高时,高抗性基因突变株占的比例也越高(P≤0.01),而不添加TC时,Bap浓度与高抗性基因突变株占比之间无变化规律(P0.05)。【结论】当环境中同时存在Bap和低浓度TC时,高抗性突变基因易于通过选择压力保存下来。     

肺炎支原体P1粘附蛋白基因的克隆

目的 在大肠杆菌中克隆含肺炎支原体P1粘附蛋白基因的重组质粒,为实现肺炎支原体P1粘附蛋白基因的大量扩增及表达奠定基础。方法 通过PCR方法获取肺炎原体P1粘附蛋白基因,用限制性核酸内酶EcoR Ⅰ切割后与pUC19载体DNA连接,转入大肠杆菌JM109菌株。用X－gal平板筛选转化子,应用P1基因区特异重复序列(RepMP2/3）引物对重组质粒进行PCR扩增和限制性核酸内酶切图谱分析鉴定。结果 PCR和限制性核酸内切酶图谱分析均证实所获重组质粒中含有P1粘附蛋白基因。结论 获得含有肺炎支原体P1粘附蛋白基因的重组克隆。     

大肠杆菌F18菌毛操纵子全基因克隆、表达及生物学活性

利用PCR技术以猪产肠毒素大肠杆菌F18标准菌株107/86和2134P基因组DNA为模板成功地扩增出编码F18ab和F18ac完整菌毛操纵子fed基因。将它们分别克隆入表达质粒载体pET-22b( ),结合酶切和核苷酸序列分析证明了PCR预期扩增产物的正确性。然后将克隆的重组载体DNA转化至大肠杆菌BL21(DE3),构建和筛选出分别含F18ab和F18ac完整fed基因的重组菌,经过IPTG诱导表达,在电镜下观察到上述两种重组菌能分别大量表达F18ab和F18ac菌毛。用热抽提法提纯其诱导表达的F18ab和F18ac菌毛,经SDS-PAGE电泳和考马斯亮蓝染色发现提纯后菌毛获单一分子量约为15kDa蛋白条带,免疫家兔后制备出高效价的兔抗血清,玻板凝集试验和Western blot结果表明:体外诱导表达的F18ab和F18ac菌毛具有和野生F18菌毛相同的抗原性。用表达F18ab和F18ac菌毛的上述2株重组菌分别进行小肠上皮细胞体外吸附试验和吸附抑制试验,结果表明:2株重组菌和野生菌株一样具有较强的粘附易感仔猪小肠上皮细胞的能力,而用表达F18ab和F18ac重组菌提纯的菌毛制备出兔抗血清都能有效地抑制上述重组菌或野生菌株对易感仔猪小肠上皮细胞的吸附结合。     

Localization of a genetic region involved in McrB restriction by Escherichia coli K-12.

A 5,500-base-pair BglII-EcoRI fragment proximal to the hsd genes of Escherichia coli K-12 has been cloned in the plasmid vector pUC9. The resultant hybrid plasmid was shown to complement the mcrB mutation of E. coli K802. The presence of the hybrid plasmid in strain K802 caused an 18.3-fold drop in transformation efficiency with AluI-methylated pACYC184 relative to unmethylated pACYC184. These results indicate that the cloned DNA is involved in the McrB system restriction of 5-methylcytosine DNA.     

Identification of two new genetically active regions associated with the osmZ locus of Escherichia coli: role in regulation of proU expression and mutagenic effect of cya, the structural gene for adenylate cyclase.

The Escherichia coli K-12 gene coding for the nucleoid-associated protein HNS was cloned together with 5.6 kb of downstream DNA in the vector pACYC184. The cloned DNA complemented a mutation in the osmZ locus of E. coli, which codes for HNS. However, the multicopy plasmid harboring the cloned sequence was found to be mutagenic and to produce at high frequency mutations that mapped to the E. coli cya gene, which codes for adenylate cyclase. Acquisition of the cya mutations was independent of RecA. These mutations were phenotypically suppressed by providing the cells with exogenous cyclic AMP and were complemented in trans by a plasmid carrying an active copy of the cya gene. A deletion analysis of the cloned sequences showed that DNA downstream of the gene coding for HNS was also required for the mutagenic effect of cya and had a role in regulating the expression of the osmZ-dependent proU locus. These sequences appear to contain at least two genetically active regions.     

Stabilization of the cloning vector pACYC184 by insertion of F plasmid leading region sequences

The leading region of the F plasmid is, by definition, the first part of the plasmid DNA to be transferred to the recipient cell during conjugation. Restriction fragments of the leading region, when cloned into the plasmid vector pACYC184, extended the maintenance of the normally unstable p15A-derived vector replicon in rec+ Escherichia coli K-12 cells. Mutations in the host's general recombination systems were found to influence the maintenance of these hybrid plasmids.     

Type 1C fimbriae of a uropathogenic Escherichia coli strain: cloning and characterization of the genes involved in the expression of the 1C antigen and nucleotide sequence of the subunit gene

The genes responsible for expression of type 1C fimbriae have been cloned from the uropathogenic Escherichia coli strain AD110 in the plasmid vector pACYC184. Analysis of deletion mutants from these plasmids showed that a 7-kb DNA fragment was required for biosynthesis of 1C fimbriae. Further analysis of this DNA fragment showed that four genes are present encoding proteins of 16, 18.5, 21 and 89 kDal. A DNA fragment encoding the 16-kDal fimbrial subunit has been cloned. The nucleotide sequence of the structural gene and of the C- and N-terminal flanking regions was determined. The structural gene codes for a polypeptide of 181 amino acids, including a 24-residue N-terminal signal sequence. The nucleotide sequence and the deduced amino acid sequence of the 1C subunit gene were compared with the sequences of the fimA gene, encoding the type 1 fimbrial subunit of E. coli K-12. The data show absolute homology at the N- and C-termini; there is less, but significant homology in the region between the N- and C-termini. Comparison of the amino acid compositions of the 1C and FimA subunit proteins with those of the F72 and PapA proteins (subunits for P-fimbriae) revealed that homology between these two sets of fimbrial subunits is also maximal at the N- and C-termini.     

Presence of Nuclear Bodies in Some Minicells of Escherichia coli

Electron microscopy of serial sections of Escherichia coli K-12 (P678-54) revealed that some "minicells" contain nuclear bodies.     

Inhibitory effect of adenosine 3'',5''-phosphate on cell division of Escherichia coli K-12 mutant derivatives

Cell division of Escherichia coli K-12 strain PA3092 was inhibited by the addition of adenosine 3',5'-phosphate (cAMP), and the cellular morphology was changed from rods into filaments. Nucleoids in the filaments were regularly distributed and septum formation was perfectly inhibited. This inhibition of cell division by cAMP was reversed by the addition of guanosine 3',5'-monophosphate. To examine whether the inhibitory effect of cAMP on cell division in E. coli PA3092 was specific, its effect in several parental strains was investigated. Induction of cell filamentation by cAMP was observed in E. coli PA309 and P678, but not in E. coli W505, W1, Y10, or the wild-type strain. This result suggests that filamentation by cAMP in E. coli PA3092, PA309, and P678 was due to the mutagenesis by which E. coli P678 was derived from E. coli W595.     

Escherichia coli bind to urinary bladder epithelium through nonspecific sialic acid mediated adherence

The first step in the bacterial colonization and infection of uropathogenic Escherichia coli is adherence to uroepithelium. Over 80% of all urinary tract infections are caused by E. coli. Uropathogenic E. coli express several adherence factors including type 1 and P fimbriae, which mediate attachment to the uroepithelium through specific binding to different glycoconjugate receptors. We showed that P and type 1 fimbriae are not the sole adhesins on uropathogenic E. coli and sialic acid also mediates nonspecific bacterial adherence of uropathogenic E. coli and urinary bladder epithelium.     

Cloning of the chromosomal determinants encoding hemolysin production and mannose-resistant hemagglutination in Escherichia coli.

We have cloned the chromosomal hemolysin determinants from Escherichia coli strains belonging to the four O-serotypes O4, O6, O18, and O75. The hemolysin-producing clones were isolated from gene banks of these strains which were constructed by inserting partial Sau3A fragments of chromosomal DNA into the cosmid pJC74. The hemolytic cosmid clones were relatively stable. The inserts were further subcloned either as SalI fragments in pACYC184 or as BamHI-SalI fragments in a recombinant plasmid (pANN202) containing cistron C (hlyC) of the plasmid-encoded hemolysin determinant. Detailed restriction maps of each of these determinants were constructed, and it was found that, despite sharing overall homology, the determinants exhibited minor specific differences in their structure. These appeared to be restricted to cistron A (hlyA), which is the structural gene for hemolysin. In the gene banks of two of these hemolytic strains, we could also identify clones which carried the genetic determinants for the mannose-resistant hemagglutination antigens Vb and VIc. Both of these fimbrial antigens were expressed in the E. coli K-12 clones to an extent similar to that observed in the wild-type strains. These recombinant cosmids were rather unstable, and, in the absence of selection, segregated at a high frequency.