幽门螺杆菌尿素酶B亚单位的克隆和序列分析

幽门螺杆菌是消化道疾病的主要致病菌。其有效抗原成份尿素酶B亚单位(UreB)可刺激机体产生保护性的免疫反应。用高保真PCR扩增系统扩增出UreB基因片段,将其克隆至质粒pUC19中,对其全序列进行了测定。克隆的UreB基因序列与报道的序列完全一致。这一研究获得了序列正确的UreB基因,为将来以UreB分子为抗原的疫苗研制工作打下了良好的基础。     

Identification of mixed genotypes in Helicobacter pylori from gastric biopsy tissue by analysis or urease gene polymorphisms

Abstract PCR-RFLP analysis of the urease A and B genes was used to investigate the frequency that Helicobacter pylori from gastric biopsy specimens comprised genotypically distinct strains. Seventy-five strains of H. pylori from 44 subjects from four geographically diverse locations were examined and 31 distinct urease gene profiles were identified. For most cultures, the totals of the RFLP sizes were within 4% of the 2.41-kb PCR product. Five strains were genomically more complex suggesting they contained a mixture of related genotypes. The validity of the test was confirmed by analysis of a known strain mixture and of single colony picks from biopsy cultures. Urease gene PCR-RFLP profiling was shown to be more sensitive than ribotyping for determining the genotypic homogeneity of H. pylori .     

Kinetic properties of Helicobacter pylori urease compared with jack bean urease

The urease proteins of the jack bean (Canavalia ensiformis) and Helicobacter pylori are similar in molecular mass when separated by non-denaturing gradient polyacrylamide gel electrophoresis, both having three main forms. The molecular mass of their major protein form is within the range 440-480 kDa with the other two lesser forms at 230-260 kDa and 660-740 kDa. These forms are all urease active; however, significant kinetic differences exist between the H. pylori and jack bean ureases. Jack bean urease has a single pH optimum at 7.4, whereas H. pylori urease has two pH optima of 4.6 and 8.2 in barbitone and phosphate buffers that were capable of spanning the pH range 3 to 10. The H. pylori Km was 0.6 mM at pH 4.6 and 1.0 mM at pH 8.2 in barbitone buffer, greater than 10.0 mM, and 1.1 mM respectively in phosphate buffer and also greater than 10.0 mM in Tris.HCl at pH 8.2. By comparison, the jack bean urease had a Km of 1.3 mM in Tris.HCl under our experimental conditions. The findings show that the urease activity of H. pylori was inhibited at the pH optimum of 4.6 in the phosphate buffer, but not in the barbitone buffer. This was shown to be due to competitive inhibition by the sodium and potassium ions in the phosphate buffer, not the phosphate ions as suggested earlier. Jack bean urease activity was similarly inhibited by phosphate buffer but again due to the effect of sodium and potassium ions.     

高效表达幽门螺杆菌UreB重组蛋白工程菌的构建

克隆表达幽门螺杆菌(Hp)的尿素酶B亚单位(UreB)重组蛋白,可为Hp疫苗开发和快速诊断试剂盒的研究奠定基础。用PCR方法由幽门螺杆菌染色体DNA扩增UreB基因片段,将其融合插入原核表达载体pQE30中,并在M15大肠杆菌表达。经酶切、测序分析,包括部分融合载体基因在内的重组UreB基因片段由1773bp组成。为编码591个氨基酸残基的多肽。SDS-PAGE分析显示重组表达的目的蛋白相对分子量约为66kD,表达量点菌体总蛋白的23．5％,并经免疫印迹分析证实被幽门螺杆菌感染的阳性血清可与纯化UreB重组蛋白发生特异性的结合反应。UreB重组蛋白具有良好的抗原性,将有可能成为一种有效蛋白质疫苗以及快速诊断试剂盒用于Hp感染的防治和检测。     

普通Taq DNA聚合酶扩增幽门螺杆菌尿素酶基因长片段

ＰＣＲ反应扩增较长的ＤＮＡ片段比较困难。本实验对幽门螺杆菌染色体ＤＮＡ上的２７Ｋｂ尿素酶基因用普通ＴａｑＤＮＡ聚合酶进行扩增,扩增结果以琼脂糖凝胶电泳证实,得到所需的２７Ｋｂ的片段。该实验为今后幽门螺杆菌工作的进一步研究提供了经济,有效,简捷的条件。     

Intrafamilial spread of Helicobacter pylori: a genetic analysis

Background. A high incidence of Helicobacter pylori among family members of children with H. pylori gastritis has previously been documented on biopsy material. The main objective of this study was the genetic clarification of H. pylori strains involved in intrafamilial dispersion. Materials and Methods. Formalin‐fixed, paraffin‐embedded material of antral mucosa from 32 members of 11 families was studied for the presence of genetic homogeneity. To achieve this goal, the entire genome of H. pylori was studied by the polymerase chain reaction (PCR)‐based random amplified polymorphic DNA (RAPD) fingerprinting method. Furthermore, the Urease A gene was analyzed using a multiplex PCR‐assay and an alternative mutation detection method based on the Hydrolink? analysis. Results. RAPD fingerprinting confirmed that closely related H. pylori strains were involved in the intrafamilial dispersion. Mutations and small deletions in Urease A gene were found in 22 out of 32 individuals. Conclusions. The homology of the H. pylori genome in members of the same family strongly supports the hypothesis of transmission of H. pylori from person‐to‐person or from a common source.     

Differentiation between isolates of Helicobacter pylori by PCR-RFLP analysis of urease A and B genes and comparison with ribosomal RNA gene patterns

Abstract Genetic diversity amongst 21 human gastric isolates of Helicobacter pylori was investigated by polymerase chain reaction amplification and Hae III digest (restriction fragment length polymorphism) analysis of an internal 2.4-kb segment of the urease A and urease B genes. H. pylori from 11 independent individuals yielded nine distinct restriction fragment patterns but only one pattern was common to H. pylori from two individuals. By contrast, multiple isolate sets of H. pylori from two patients each had common urease gene patterns. Most strains with the same urease gene patterns were distinguishable in their ribosomal RNA gene patterns. The study demonstrated diversity amongst H. pylori and established that PCR analysis of urease genes provided a novel method of identifying isolates. The profiles were reproducible and convenient to obtain and analyse, and were almost as discriminatory as Hae III ribopatterns.     

Mutational analysis of the Helicobacter pylori carbonic anhydrases

In the gastric microenvironment, Helicobacter pylori is exposed to bicarbonate, urea and acid. Here it is demonstrated that both H. pylori carbonic anhydrases (CAs) are required for maintaining urease activity and therefore influence H. pylori urea resistance at neutral pH. Furthermore, the beta-CA is required for acid resistance as indicated by a growth defect of the corresponding mutant at low pH. The alpha- and beta-CA mutants as well as the double mutant were more resistant to bicarbonate, indicating that both enzymes are involved in bicarbonate metabolism. These phenotypes support important CA-functions in H. pylori urea and bicarbonate metabolism and acid resistance. Thus, both CA enzymes might be required for survival in the gastric niche.     

幽门螺杆菌尿素酶单克隆抗体的研制及鉴定

应用杂交瘤技术 ,建立稳定分泌抗幽门螺杆菌尿素酶单克隆抗体 (HPU McAb)的细胞系。用纯化幽门螺杆菌尿素酶 (HPU)抗原加福氏佐剂免疫BALB/c小鼠 ,按常规方法进行细胞融合 ,以纯化HPU抗原包被 ,间接ELISA方法筛选 ,并经多次有限稀释法克隆。获得 6株抗HPU的杂交瘤 ,腹水效价达 1∶6 .4× 10 4～ 1∶2 .5 6× 10 5,特异性专一 ,并对其进行体内外连续传代 3个月 ,分泌抗体能力稳定。IgG亚类分型主要为IgG1型。该细胞系能稳定分泌幽门螺杆菌尿素酶单抗。     

幽门螺旋杆菌重组尿素酶B亚基的纯化及其免疫学性质的研究

目的:幽门螺旋杆菌(Hp)尿素酶是Hp重要的定制因子和致病因子,Hp尿素酶活性位点位于Hp尿素酶B亚基(UreB),研发基于UreB的Hp疫苗是一种很有前景的防治Hp感染的策略。方法:主要利用基因克隆技术从幽门螺旋杆菌标准菌株SS1(Hp SS1)获得Hp尿素酶B亚基基因,并构建含有重组Hp尿素酶B亚基(rUreB)基因的重组表达载体pET-rUreB及其重组菌株;重组菌株经蛋白表达和优化后,利用Ni-NTP镍离子亲和层析和DEAE Sepharose FF阴离子交换层析纯化重组尿素酶B亚基(rUreB),并进一步通过腹腔注射免疫BALB/c小鼠,研究rUreB的免疫学性质。结果:通过基因克隆技术成功获得了Hp尿素酶B亚基基因,并成功构建了重组表达载体pET-rUreB及其重组菌株BL21(DE3)/pET-rUreB,经蛋白表达优化及纯化,可获得高纯度(96.5%)的重组蛋白rUreB。重组蛋白rUreB辅以弗氏佐剂腹腔注射免疫BALB/c小鼠,经间接ELISA鉴定小鼠能够产生针对天然Hp尿素酶和UreB的高滴度特异性抗体,且能够显著性抑制Hp尿素酶的活性。结论:重组Hp尿素酶B亚基能够在大肠杆菌表达系统中获得较高水平的表达,具有较高的免疫学特异性,其抗体能够有效抑制Hp尿素酶活性。为研究基于尿素酶的防治Hp感染的Hp疫苗奠定了一定的实验基础。     

Urease-mediated destruction of bacteria is specific for Helicobacter urease and results in total cellular disruption

Abstract The survival of Helicobacter mustelae, Proteus mirabilis, Escherichia coli and Campylobacter jejuni in the presence of urea and citrate at pH 6.0 was examined. H. mustelae , which has urease activity similar to H. pylori , had a markedly reduced survival, median 2.5% (0–78%) ( P <0.001) when incubated nder these conditions. Only 7% of the ammonia produced by H. mutelae urease activity was recovered from the buffer, a similar percentage to that previously reported with H. pylori . None of the other organisms, all of which had lower urease activity, had impaired survival under these conditions. Electron microscopical studies demonstrated extensive structural damage to H. pylori following exposure to urea and citrate at pH 6.0. This structural damage to the organisms makes it unlikely that the low recovery of ammonia was due to retention of ammonia within the bacteria and suggests that the ammonia may have been incorporated into glutamate or other amino acids. Incorporation of ammonia into these compounds would deplete the cell of the key metabolic intermediate α-ketoglutarate and could thus explain the mechanism of the urease-dependent destruction of the organism.     

Helicobacter pylori utilises urea for amino acid synthesis

Abstract Helicobacter pylori has one of the highest urease activities of all known bacteria. Its enzymatic production of ammonia protects the organism from acid damage by gastric juice. The possibility that the urease activity allows the bacterium to utilise urea as a nitrogen source for the synthesis of amino acids was investigated. H. pylori (NCTC 11638) was incubated with 50 mM urea, enriched to 5 atom% excess ¹⁵N, that is the excess enrichment of ¹⁵N above the normal background, in the presence of either NaCl pH 6.0, or 0.2M citrate pH 6.0. E. coli (NCTC 9001) was used as a urease-negative control. ¹⁵N enrichment was detected by isotope ratio mass spectrometry. H. pylori showed intracellular incorporation of ¹⁵N in the presence of citrate buffer pH 6.0 but there was no significant incorporation of ¹⁵N in unbuffered saline or by E. coli in either pH 6.0 citrate buffer or unbuffered saline. The intracellular fate of the urea-nitrogen was determined by means of gas chromatography/mass spectrometry following incubation with ¹⁵N enriched 5 mM urea in the presence of either 0.2 M citrate buffer pH 6.0 or 0.2 M acetate buffer pH 6.0. After 5 min incubation in either buffer the ¹⁵n label appeared in glutamate, glutamine, phenylalanine, aspartate and alanine. It appears, therefore, that at pH and urea concentrations typical of the gastric mucosal surface, H. pylori utilises exogenous urea as a nitrogen source for amino acid synthesis. The ammonia produced by H. pylori urease activity thus facilitates the organism's nitrogen metabolism at neutral pH as well as protecting it from acid damage at low pH.     

Genetic complementation of the urease-negative Helicobacter pylori mutant N6ureB::TnKm

Helicobacter pylori produces urease composed of the structural subunits UreA and UreB. Isogenic mutants produced by shuttle mutagenesis from the wild-type strain N6 are widely used in the literature. We describe the genetic complementation of the mutant N6ureB::TnKm by stable transformation with the vector pHel2 containing the cloned genes ureA and ureB and their specific promoter sequence. The orientation of the cloned insert was found to be crucial for urease expression. The majority of complemented clones functionally expressed urease at higher levels than did N6. Homologous recombination between chromosomal and cloned genes occurred at a frequency of 5%.     

幽门螺杆菌在胃内定植的相关影响因素

胃内定植是引起幽门螺杆菌（Helicobacter pylori,H. pylori）感染的先决条件。H. pylori可穿过胃黏液层并与胃上皮细胞相互作用。这个定植过程主要受到H. pylori动力和尿素酶的影响。同时H. pylori形态、胃内pH、外膜蛋白及益生菌等也在其中扮演重要角色。该研究主要对H. pylori胃内定植过程中的相关影响因素进行综述。     

Contribution of dppA to urease activity in Helicobacter pylori 26695

BACKGROUND: The gastric pathogen Helicobacter pylori produces urease in amounts up to 10% of its cell protein. This enzyme, which catalyzes the hydrolysis of urea to ammonia and carbon dioxide, protects the bacterium from gastric acid. Urease, a nickel metalloenzyme, requires active uptake of nickel ions from the environment to maintain its activity. NixA is a nickel transport protein that resides in the cytoplasmic membrane. Mutation of nixA significantly reduces but does not abolish urease activity, strongly suggesting the presence of a second transporter. We postulated that the dipeptide permease (dpp) genes that are homologous to the nik operon of Escherichia coli could be a second nickel transporter. The predicted Dpp polypeptides DppA, DppC, and DppD of H. pylori share approximately 40%, 53%, and 56% amino acid sequence identity with their respective E. coli homologs. METHODS: A mutation in dppA, constructed by insertional inactivation with a chloramphenicol resistance cassette, was introduced by allelic exchange into H. pylori strain 26695. RESULTS: When compared to the parental strain, urease activity was not decreased in a dppA mutant. CONCLUSIONS: DppA does not contribute to the synthesis of catalytically active urease in H. pylori 26695 and is likely not a nickel importer in H. pylori.     

Structure-based mutational analysis of the active site residues of -hydantoinase

We previously proposed the hydrophobic and bulky residues of the three loops, designated stereochemistry gate loops (SGLs), to constitute a hydrophobic substrate binding pocket of -hydantoinase from Bacillus stearothermophilus SD1. Simulation of substrate binding in the active site of -hydantoinase and sequence alignment of various -hydantoinases revealed the critical hydrophobic residues closely located around the exocyclic substituent of substrate. To evaluate the roles of these residues in substrate binding pocket, site-directed mutagenesis was performed specifically for Leu 65, Tyr 155, and Phe 159. When Tyr 155 was mutated to Phe and Glu, both mutants Y155F and Y155E were totally inactive for nonsubstituted hydantoin and -5-hydroxyphenyl hydantoin (HPH), which indicates that Tyr 155 is involved in substrate binding via a hydrogen bond with the hydantoinic ring. Furthermore, replacement of the hydrophobic residues Leu 65 and Phe 159 with Glu, a charged amino acid, resulted in a significant decrease in activity for nonsubstituted hydantoin, but not for HPH. The K_cat values of both mutants for nonsubstituted hydantoin also severely decreased, but a slight change in the K_cat values was observed towards HPH. These results suggest that the hydrophobic residues in SGLs play an essential role in substrate binding, and differentially interact according to the property of the exocyclic substituent.     

Mechanism of action of low recurrence of gastritis caused by Helicobacter pylori with the type II urease B gene

Background. Low recurrence of gastritis is seen in patients infected with Helicobacter pylori carrying the type II urease B gene, compared with H. pylori carrying types I and III. The underlying mechanism has been studied in terms of the urease activity and interleukin (IL)‐8 production capacity of different strains of H. pylori. Materials and Methods. Forty‐five patients infected with different strains of H. pylori (type I; 15, type II; 15 and type III; 15) were enrolled in the study. H. pylori was isolated from gastric mucosa and cultured in the presence of urea at pH 5.5 to evaluate urease activity. The capacity of different strains of H. pylori to induce IL‐8 mRNA and IL‐8 from a human gastric cancer cell line and human peripheral blood mononuclear cells was evaluated. Results. The urease activity of type II H. pylori[523 ± 228 µg of ammonia/dl/10⁸ colony‐forming units (CFU)/ml] was significantly lower than that of type I (1355 ± 1369 µg of ammonia/dl/10⁸ CFU/ml) and type III (1442 ± 2229 µg of ammonia/dl/10⁸ CFU/ml) (p < .05). Gastric cancer cells cocultured with type II H. pylori produced lower levels of IL‐8 mRNA compared with type I and type III H. pylori. The levels of IL‐8 were also significantly lower in cultures induced by type II H. pylori compared with those induced by type I and type III H. pylori. Peripheral blood mononuclear cells also produced lower levels of IL‐8 when cocultured with type II compared with type I H. pylori. Conclusions. These results indicate that both the lower level of urease activity and the low IL‐8‐inducing capacity of type II H. pylori might underlie the lower recurrence rate of gastritis caused by type II H. pylori.     

幽门螺杆菌尿素酶的纯化及其特性

本文利用Ｓｅｐｈａｃｒｙ１ Ｓ－２００和Ｑ－Ｓｅｐｈａｒｏｓｅ两步层析,从ＨＰ蒸馏水浸液中提取纯化尿素酶,并对其特性进行了测定,证明ＨＰ尿素酶各含一个６６ｋＤ和２９．５ｋＤ的亚单位,并以６个分子聚合成６２５ｋＤ大分子蛋白,有很好的抗原性,并显示特异的血清学反应。用ＨＰ超声浸液抗原和尿素酶,加入２μｇ霍乱毒素粘膜佐剂,给ＳＰＦ ＢＡＬＢ／Ｃ小鼠口服免疫,可使８０％小鼠抵抗ＨＰ活菌的攻击,证明尿素酶是一种抗ＨＰ的保护性抗原。