H.ylori尿素酶B亚单位在保加利亚乳杆菌的表达与鉴定

目的构建表达幽门螺杆菌（Helicobacter pylori,H、pylori）尿素酶B亚单位（UreB）的基因工程乳杆菌,并对其进行初步的安全性评估。方法采用高保真PCR从H.pylori标准菌株NCTC 11637中扩增ureB基因,插入乳酸菌表达质粒pMG36e,将重组质粒电转入保加利亚乳杆菌L6032中,获得表达ureB的基因工程乳杆菌。在含乳糖的MRS培养基诱导目的蛋白表达,Western blot鉴定其免疫原性。连续传代培养60代,检测基因工程乳杆菌的稳定性、形态学与生理生化特性以进行初步的安全性评估。结果特异PCR、酶切和测序鉴定均证实ureB基因克隆入表达载体pMG36e,SDS-PAGE结果显示,重组质粒pMG36e-ureB电转入保加利亚乳杆菌所构建的基因工程乳杆菌能表达约64KD的蛋白,Western blot证明该蛋白能与抗H.priori ureB的兔血清反应。稳定性、形态学与生理生化特性检测结果表明,基因工程乳杆菌与原始菌株保加利亚乳杆菌完全一致。结论成功构建能表达H.pylori UreB的保加利亚乳杆菌L6032-UreB,该基因工程菌在形态与生理生化特性上未发生任何变异,从而为探索幽门螺杆菌感染的益生菌制剂调理疗法奠定了坚实的基础。     

幽门螺杆菌热休克蛋白A在乳酸乳球菌中的表达及免疫学活性分析

目的构建含幽门螺杆菌(H.pylori)热休克蛋白A编码基因的重组载体,并电转入乳酸乳球菌MG1363,表达目的蛋白并分析其免疫原性,为H.pylori基因工程口服疫苗的研究和开发奠定基础。方法以H.py-loriNCTC 11637株基因组DNA为模板,PCR扩增hspA基因,并克隆至乳酸乳球菌表达载体pMG36e中。将重组质粒转化E.coliDH5α,经鉴定的阳性重组质粒命名为pMG36e/hspA。以电穿孔法将pMG36e/hspA转化乳酸乳球菌MG1363并用Western blot检测HspA蛋白的表达。结果克隆重组后得到pMG36e/hspA。将pMG36e/hspA电转化MG1363后,收集菌体蛋白进行Western blot分析,在HspA的相对分子质量(Mr≈13 kDa)处出现特异性条带。结论首次成功构建了表达H.pyloriHspA的重组乳酸乳球菌MG1363,为进一步口服疫苗的相关研究奠定了基础。     

UreB酸奶的研制及其抗幽门螺杆菌感染的动物实验研究

目的 利用UreB基因工程乳杆菌(UreB-L6032)研制一种预防幽门螺杆菌(Helicobacter pylori,H.pylori)感染的UreB活性酸奶,并对其抗H.pylori感染的能力进行动物实验研究.方法 采用发酵奶制备技术制成酸奶,并对其pH、活菌含量和质粒稳定性进行检测.小鼠在喂食UreB活性酸奶1个月后再感染H.pylori,并设普通酸奶组,阴性和阳性感染组,2个月后测定IL-10(interleukin-10)、IFN-γ(interferon-γ)、sIgA和特异性IgG的水平,并利用快速脲酶法和细菌培养法对小鼠胃部H.pylori进行检测.结果 成功的研制出了色香味颇佳,含UreB-L6032活菌较高,质粒能稳定存在的UreB活性酸奶.与一般酸奶相比,其能诱导小鼠体内特异性IgG的产生,显著增加IL-10的表达,降低小鼠H.pylori感染率.结论 UreB活性酸奶比一般酸奶能更加有效预防H.pylori感染.     

作为宿主系统的几株乳酸菌的表型特征

目的：研究乳酸菌载体－宿主系统。方法：采用涂片染色和形态特征观察,用鉴别生化实验如过氧化氢酶实验,碳水化合物发酵产酸实验,精氨酸水解实验及抗生素抗性实验等对含有pMG36e质粒的乳酸菌MG1363,乳球菌IL1403和乳杆菌ATCC4356进行研究鉴定。结果：乳酸乳球菌乳脂亚种MG1363,乳酸乳球菌乳酸亚种IL1403含有质粒pMG36e的MG1363致及嗜酸乳杆菌ATCC4356其表型特征分别与伯杰氏手册中相应细菌特征一致,质粒pMG36e含有红霉素抗性基因,结论：此乳酸菌宿主一载体系统可用载体来源的红霉素抗性进行筛选,用于外源基因在乳酸菌中克隆和表达的研究。     

乳酸菌载体pMG36e的应用现状

乳酸乳球菌通用表达质粒pMG36e是一个经典的人工构建的组成型表达载体,是以乳酸乳球菌乳脂亚种蛋白酶基因的转录和翻译信号为基础构建而成的。它包含一个强启动子,能够在多种细菌中表达外源蛋白。已用于研究细菌素作用机制,乳酸菌基因工程菌株的改造以及口服疫苗的开发等,应用领域十分广泛,已成为乳酸菌基因工程研究的重要工具质粒之一。本文主要从载体构成、基因表达与食品级载体改造等三方面的应用对其进行综述,旨在为该质粒今后研究提供资料。     

幽门螺杆菌napA基因在乳酸菌中的表达及免疫原性分析

为在乳酸菌中表达幽门螺杆菌（Helicobacter pylori,H.pylori）中性粒细胞激活蛋白(NAP),口服免疫小鼠后检测其免疫原性。在实验中利用了分子克隆技术构建携带nap基因的重组原核表达质粒pNICE:secnap,将重组质粒转化乳酸菌NZ9000株,筛选鉴定阳性菌落,诱导表达的NAP蛋白用SDSPAGE和Western blot进行鉴定。将雌性ICR（CV级）小鼠随机分为4组,分别用PBS、携带空质粒的乳酸菌、携带pNICE:secnap的乳酸菌、灭活的H.pylori 灌胃。免疫7次后检测其特异性IgG和IgA的产生。成功扩增了nap基因并构建了重组原核表达质粒pNICE:secnap,转化乳酸菌NZ9000后经nisin诱导可表达Mr约17kDa的重组蛋白,表达量约占菌体总蛋白量的9.5%,表达的蛋白能与兔抗H.pylori 血清特异性反应,具有良好的免疫原性。携带pNICE:secnap质粒的乳酸菌刺激产生的IgG水平明显高于携带空质粒组,与灭活H.pylori组没有明显的差异,但其刺激产生的IgA水平明显高于其他组。以上结果说明表达NAP蛋白的乳酸菌口服免疫小鼠后,能够刺激小鼠产生特异的IgG和IgA,对幽门螺杆菌疫苗的研究开发具有理论意义。为乳酸菌作为抗原递送载体的研究和H.pylori口服疫苗的开发提供了一定的实验基础。     

幽门螺杆菌napA基因在乳酸菌中的表达及免疫原性分析

本实验目的是在乳酸菌中表达幽门螺杆菌（Helicobacter pylori,H.pylori） 中性粒细胞激活蛋白(NAP),口服免疫小鼠后检测其免疫原性。在实验中利用了分子克隆技术构建携带nap基因的重组原核表达质粒pNICE:sec-nap,将重组质粒转化乳酸菌NZ9000株,筛选鉴定阳性菌落,诱导表达的NAP蛋白用SDS-PAGE和Western blot进行鉴定。将雌性ICR（CV级）小鼠随机分为4组,分别用PBS、携带空质粒的乳酸菌、携带pNICE:sec-nap的乳酸菌、灭活的H.pylori 灌胃。免疫7次后检测其特异性IgG和IgA的产生。成功扩增了nap基因并构建了重组原核表达质粒pNICE:sec-nap,转化乳酸菌NZ9000后经nisin诱导可表达Mr约17kDa的重组蛋白,表达量约占菌体总蛋白量的9.5%,表达的蛋白能与兔抗H.pylori 血清特异性反应,具有良好的免疫原性。携带pNICE:sec-nap质粒的乳酸菌刺激产生的IgG水平明显高于携带空质粒组,与灭活H.pylori组没有明显的差异,但其刺激产生的IgA水平明显高于其他组。以上结果说明表达NAP蛋白的乳酸菌口服免疫小鼠后,能够刺激小鼠产生特异的IgG和IgA,对幽门螺杆菌疫苗的研究开发具有理论的意义。为乳酸菌作为抗原递送载体的研究和H.pylori口服疫苗的开发提供一定的实验基础。     

表达增强绿色荧光蛋白重组乳酸菌的构建

以乳酸乳球菌通用表达载体pMG36e为基本骨架,将增强型绿色荧光蛋白基因(eGFP)插入该载体组成型表达强启动子p32的下游,构建了eGFP标记的乳酸菌表达载体pMG36e-eGFP。重组质粒电击转化一株乳酸乳球菌BLCC02-0018。筛选阳性转化子,通过连续荧光强度检测,验证eGFP基因能否在重组菌中稳定表达。重组表达质粒pMG36e-eGFP酶切及测序结果与预期片段大小相符,表明成功构建了重组乳酸菌表达载体pMG36e-eGFP;对阳性转化子进行蓝光激发,可见明显的绿色荧光,且荧光稳定性强,连续传代30代仍可见荧光,结果表明已经成功构建表达增强型绿色荧光蛋白的重组乳酸乳球菌,为下一步研究乳酸菌在动物体内的分布定殖规律打下了基础。     

幽门螺杆菌尿素酶B亚单位基因工程菌发酵工艺的研究

通过不同培养基、不同葡萄糖浓度、不同溶氧条件、补料与非补料对幽门螺杆菌尿素酶B亚单位(UreB)基因工程菌的菌体生长与外源蛋白表达量的影响的比较 ,建立了稳定、适宜的幽门螺杆菌尿素酶B亚单位基因工程菌发酵工艺。多批实验结果证明 ,菌体单产可达 86 g/L ,目的蛋白的表达率为 38.2 %。     

人铜锌超氧化物歧化酶基因的克隆和在乳酸乳球菌中的表达

采用RT-PCR技术从人肝总RNA中分离扩增了0.45kb的人铜锌超氧化物歧化酶(Cu/Zn SOD)基因的cDNA序列,首先克隆至大肠杆菌表达质粒pET23b,进行了序列测定和超高表达。将Cu/Zn SOD cDNA亚克隆至乳酸乳球菌表达载体pMG36e,用电穿孔法将重组质粒pMG36esod转化到乳酸乳球菌中,获得Cu/Zn SOD的组成型表达,其表达量约占乳酸乳球菌可溶性蛋白的5%以上,活性染色表明该工程菌表达的Cu/Zn SOD具有较好的酶活性。     

德氏乳杆菌保加利亚亚种电转化平台的构建和优化

德氏乳杆菌保加利亚亚种(Lactobacillus delbrueckiisub sp.bulgaricus)是最具经济价值的乳酸菌之一,在世界上广泛应用于酸奶和其它发酵乳生产。当前对该菌的代谢机制研究甚少。外源基因的转化效率是制约其分子代谢机制研究的重要因素。本研究以pMG36c为材料,对L.delbrueckiisub sp.bulgaricus CH3进行电转化条件研究。结果表明,在电转化过程中,电场强度、质粒的浓度、细胞生长状态均对转化效率有明显影响,得到了该菌株的最适电转化条件为:对数初期的细胞,质粒浓度为100 ng/50μl菌液,在10 kV/cm电场强度下电转化,转化后细胞在复壮培养液中培养3 h后涂布选择性培养基,转化率可达2.6×103CFU/μg DNA。以甘氨酸、醋酸锂、二硫苏糖醇处理细胞壁,发现醋酸锂和二硫苏糖醇共同处理对转化效率有明显改善,可提高转化效率。     

Regulation and Adaptive Evolution of Lactose Operon Expression in Lactobacillus delbrueckii

Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis are both used in the dairy industry as homofermentative lactic acid bacteria in the production of fermented milk products. After selective pressure for the fast fermentation of milk in the manufacture of yogurts, L. delbrueckii subsp. bulgaricus loses its ability to regulate lac operon expression. A series of mutations led to the constitutive expression of the lac genes. A complex of insertion sequence (IS) elements (ISL4 inside ISL5), inserted at the border of the lac promoter, induced the loss of the palindromic structure of one of the operators likely involved in the binding of regulatory factors. A lac repressor gene was discovered downstream of the beta-galactosidase gene of L. delbrueckii subsp. lactis and was shown to be inactivated by several mutations in L. delbrueckii subsp. bulgaricus. Regulatory mechanisms of the lac gene expression of L. delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis were compared by heterologous expression in Lactococcus lactis of the two lac promoters in front of a reporter gene (beta-glucuronidase) in the presence or absence of the lac repressor gene. Insertion of the complex of IS elements in the lac promoter of L. delbrueckii subsp. bulgaricus increased the promoter's activity but did not prevent repressor binding; rather, it increased the affinity of the repressor for the promoter. Inactivation of the lac repressor by mutations was then necessary to induce the constitutive expression of the lac genes in L. delbrueckii subsp. bulgaricus.     

Protective effect of vaccination with DNA of the H. Pylori genomic library in experimentally infected mice

Immunologically mediated protection against H. pylori infection is an attractive alternative to antibiotic treatment. We compared the efficacy of conventional protein vaccination with that of genetic vaccination against experimental infection with H. pylori in mice. For oral immunization, we used the recombinant peptide of an antigenic fragment of UreB (rUreB) or H. pylori-whole cell lysate antigens, and for genetic immunization, we used recombinant pcDNA and pSec plasmids inserted with the fragment of ureB or DNA of the H. pylori genomic library. Mice were challenged with the mouse stomach-adapted H. pylori Sidney Strain. The detection of gastric bacterial colonization was performed by real-time PCR of a 26-kDa Helicobacter-specific gene, and the presence of serum H. pylori-specific antibodies was determined using direct ELISA assay. The most effective treatment appeared to be oral vaccination with rUreB and either intramuscular or intradermal vaccination with DNA of the H. pylori genomic library. Intradermal genetic vaccination with genomic library DNA significantly increased the IgG antibody response. Our study revealed acceptable efficacies of genetic vaccination with DNA of the H. pylori genomic library.     

Evolution of the bacterial species Lactobacillus delbrueckii: a partial genomic study with reflections on prokaryotic species concept

The species Lactobacillus delbrueckii consists at present of three subspecies, delbrueckii, lactis and bulgaricus, showing a high level of DNA-DNA hybridization similarity but presenting markedly different traits related to distinct ecological adaptation. The internal genetic heterogeneity of the bacterial species L. delbrueckii was analyzed. Phenotypic and several genetic traits were investigated for 61 strains belonging to this species. These included 16S rDNA sequence mutations, expression of beta-galactosidase and of the cell wall-anchored protease, the characterization of the lactose operon locus and of the sequence of lacR gene, galactose metabolism, and the distribution of insertion sequences. The high genetic heterogeneity of taxa was confirmed by every trait investigated: the lac operon was completely deleted in the subsp. delbrueckii, different mutation events in the repressor gene of the operon led to a constitutive expression of lacZ in the subsp. bulgaricus. Structural differences in the same genetic locus were probably due to the presence of different IS elements in the flanking regions. The different expression of the cell wall-anchored protease, constitutive in the subsp. bulgaricus, inducible in the subsp. lactis, and absent in the subsp. delbrueckii was also a consequence of mutations at the gene level. The galT gene for galactose metabolism was found only in the subsp. lactis, while no specific amplification product was detected in the other two subspecies. All these data, together with the absence of a specific IS element, ISL6, from the major number of strains belonging to the subsp. bulgaricus, confirmed a deep internal heterogeneity among the three subspecies. Moreover, this evidence and the directional mutations found in the 16S rDNA sequences suggested that, of the three subspecies, L. delbrueckii subsp. lactis is the taxon closer to the ancestor. Limitations of the current prokaryotic species definition were also discussed, based on presented evidences. Our results indicate the need for an accurate investigation of internal heterogeneity of bacterial species. This study has consequences on the prokaryotic species concept, since genomic flexibility of prokaryotes collides with a stable classification, necessary from a scientific and applied point of view.     

UreA2B2: a second urease system in the gastric pathogen Helicobacter felis

Urease activity is vital for gastric colonization by Helicobacter species, such as the animal pathogen Helicobacter felis. Here it is demonstrated that H. felis expresses two independent, and distinct urease systems. H. felis isolate CS1 expressed two proteins of 67 and 70 kDa reacting with antibodies to H. pylori urease. The 67-kDa protein was identified as the UreB urease subunit, whereas the N-terminal amino acid sequence of the 70-kDa protein displayed 58% identity with the UreB protein and was tentatively named UreB2. The gene encoding the UreB2 protein was identified and located in a gene cluster named ureA2B2. Inactivation of ureB led to complete absence of urease activity, whereas inactivation of ureB2 resulted in decreased urease activity. Although the exact function of the UreA2B2 system is still unknown, it is conceivable that UreA2B2 may contribute to pathogenesis of H. felis infection through a yet unknown mechanism.     

Deoxyribonuclease activities in Lactobacillus delbrueckii

DNase activity was examined in the extracellular and subcellular fractions of six non-transformable strains belonging to Lactobacillus delbrueckii subsp. lactis (L. lactis) and Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) and compared with the activity present in Lactobacillus johnsonii NCK 65, a transformable strain of Lactobacillus. In the extracellular fraction of the L. delbrueckii strains, a common protein band of 36 kDa was detected, while a band of 29 kDa was found in the same fraction of L. johnsonii. No nuclease activity was detected in the cytoplasmic fraction of this strain, indicating that the localization of the DNase activity could be a key factor in the uptake of foreign DNA.     

Organization and nucleotide sequence of the glutamine synthetase (glnA) gene from Lactobacillus delbrueckii subsp. bulgaricus.

A 3.3-kb BamHI fragment of Lactobacillus delbrueckii subsp. bulgaricus DNA was cloned and sequenced. It complements an Escherichia coli glnA deletion strain and hybridizes strongly to a DNA containing the Bacillus subtilis glnA gene. DNA sequence analysis of the L. delbrueckii subsp. bulgaricus DNA showed it to contain the glnA gene encoding class I glutamine synthetase, as judged by extensive homology with other prokaryotic glnA genes. The sequence suggests that the enzyme encoded in this gene is not controlled by adenylylation. Based on a comparison of glutamine synthetase sequences, L. delbrueckii subsp. bulgaricus is much closer to gram-positive eubacteria, especially Clostridium acetobutylicum, than to gram-negative eubacteria and archaebacteria. The fragment contains another open reading frame encoding a protein of unknown function consisting of 306 amino acids (ORF306), which is also present upstream of glnA of Bacillus cereus. In B. cereus, a repressor gene, glnR, is found between the open reading frame and glnA. Two proteins encoded by the L. delbrueckii subsp. bulgaricus gene were identified by the maxicell method; the sizes of these proteins are consistent with those of the open reading frames of ORF306 and glnA. The lack of a glnR gene in the L. delbrueckii subsp. bulgaricus DNA in this position may indicate a gene rearrangement or a different mechanism of glnA gene expression.     

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。     

Oral vaccination of mice against Helicobacter pylori with recombinant Lactococcus lactis expressing urease subunit B

To determine whether a protective immune response could be elicited by oral delivery of a recombinant live bacterial vaccine, Helicobacter pylori urease subunit B (UreB) was expressed for extracellular expression in food-grade bacterium Lactococcus lactis . The UreB-producing strains were then administered orally to mice, and the immune response to UreB was examined. Orally vaccinated mice produced a significant UreB-specific serum immunoglobulin G (IgG) response. Specific anti-UreB IgA responses could be detected in the feces of mice immunized with the secreting lactococcal strain. Mice vaccinated orally were significantly protected against gastric Helicobacter infection following a challenge with H. pylori strain SS1. In conclusion, mucosal vaccination with L. lactis expressing UreB produced serum IgG and UreB-specific fecal IgA, and prevented gastric infection with H. pylori .