乳酸杆菌S-层蛋白对鼠伤寒沙门氏菌黏附及入侵Caco-2细胞的拮抗作用

【目的】对嗜酸乳杆菌的S-层蛋白(S-layer protein)进行提纯,研究嗜酸乳酸杆菌和S-层蛋白对鼠伤寒沙门氏菌黏附和入侵的拮抗作用。【方法】应用阴离子交换柱(DE52)对嗜酸乳酸杆菌的S-层蛋白进行提纯,然后分别研究了嗜酸乳酸杆菌和S-层蛋白对鼠伤寒沙门氏菌黏附及入侵Caco-2细胞的作用。【结果】S-层蛋白能显著地抑制鼠伤寒沙门氏菌的黏附及入侵;在竞争、排斥、置换3种黏附试验中,S-层蛋白可显著降低鼠伤寒沙门氏菌的黏附,其相对黏附力分别为1.17%±5.97%、8.71%±1.36%、10.56%±0.92%,差异极显著(p0.01),其中竞争试验效果最好;并且S-层蛋白对鼠伤寒沙门氏菌黏附抑制作用极显著高于嗜酸乳酸杆菌(p0.01);此外,S-层蛋白也能显著抑制鼠伤寒沙门氏菌入侵。【结论】乳酸杆菌S-层蛋白对鼠伤寒沙门氏菌可产生显著的拮抗作用,这可能与S-层蛋白和鼠伤寒沙门氏菌的宿主黏附受体存在竞争作用有关;提示乳酸杆菌S-层蛋白可用于预防和治疗鼠伤寒沙门氏菌感染,并有望成为抗生素的替代品。     

金黄色葡萄球菌重组GapC蛋白的GAPDH活性及免疫原性分析

为研究金黄色葡萄球菌(Staphylococcus aureus)表面GapC蛋白的GAPDH活性、免疫原性及免疫保护作用, 应用PCR方法扩增出S. aureus的gapC基因, 插入到pQE-30载体相应位点, 构建重组质粒pQE/gapC。将其导入宿主菌E.coli M15(pREP4)后, IPTG诱导表达。重组蛋白纯化后进行GAPDH活性检测, 并与灭活全菌体分别免疫健康家兔。然后, 应用ELISA方法检测血清中IgG抗体水平及IFN-g、IL-4细胞因子浓度, 并用1.0×108CFU/mL S. aureus菌株Wood46对免疫家兔攻毒。SDS-PAGE结果显示, GapC蛋白在E. coli M15(pREP4)中获得表达; 经GAPDH活性检测及Western Blot检测, 重组蛋白具有较高的GAPDH活性和抗原特异性; 经ELISA检测, GapC蛋白及全菌体组兔血清中IgG抗体水平迅速升高, 并在加强免疫后第28天达到最高(1:64 000), 加强免疫后第14 d, 血清中细胞因子IFN-g和IL-4浓度与对照组相比, 显著升高(P<0.05), 而全菌体免疫组升高不明显(P>0.05); 攻毒结果为蛋白免疫组家兔获得一定的免疫保护(4/5)。以上结果表明, 表达的重组GapC蛋白具有GAPDH活性、较好的免疫原性及免疫保护力, 可作为深入研究S. aureus基因工程疫苗的良好靶向。     

金黄色葡萄球菌重组GapC蛋白的GAPDH活性及免疫原性分析

为研究金黄色葡萄球菌(Staphylococcus aureus)表面GapC蛋白的GAPDH活性、免疫原性及免疫保护作用, 应用PCR方法扩增出S. aureus的gapC基因, 插入到pQE-30载体相应位点, 构建重组质粒pQE/gapC。将其导入宿主菌E.coli M15(pREP4)后, IPTG诱导表达。重组蛋白纯化后进行GAPDH活性检测, 并与灭活全菌体分别免疫健康家兔。然后, 应用ELISA方法检测血清中IgG抗体水平及IFN-g、IL-4细胞因子浓度, 并用1.0×108CFU/mL S. aureus菌株Wood46对免疫家兔攻毒。SDS-PAGE结果显示, GapC蛋白在E. coli M15(pREP4)中获得表达; 经GAPDH活性检测及Western Blot检测, 重组蛋白具有较高的GAPDH活性和抗原特异性; 经ELISA检测, GapC蛋白及全菌体组兔血清中IgG抗体水平迅速升高, 并在加强免疫后第28天达到最高(1:64 000), 加强免疫后第14 d, 血清中细胞因子IFN-g和IL-4浓度与对照组相比, 显著升高(P<0.05), 而全菌体免疫组升高不明显(P>0.05); 攻毒结果为蛋白免疫组家兔获得一定的免疫保护(4/5)。以上结果表明, 表达的重组GapC蛋白具有GAPDH活性、较好的免疫原性及免疫保护力, 可作为深入研究S. aureus基因工程疫苗的良好靶向。     

白喉毒素突变体CRM197在白喉杆菌中的表达纯化

CRM197是一种白喉毒素突变体,第52位的甘氨酸突变为谷氨酸,作为载体蛋白广泛用于疫苗开发。将阐述一种新的生产CRM197方法。将合成的CRM197基因片段克隆到表达载体pCKM4.1中,表达质粒pCKM5.1电转化至大肠杆菌E.coli S17-1中,通过结合转移转化至白喉杆菌(ATCC?27010TM的白喉杆菌)中,载体上的导肽序列可以使得CRM197作为可溶性蛋白分泌到胞外表达,CRM197蛋白可占到菌体总蛋白的70%。增强对铁的调控,进一步优化培养基及发酵条件以提高产量。经过Q膜、硫酸铵沉淀、阴离子交换纯化步骤获得纯度达到95%的CRM197样品,提高了蛋白得率,节约了纯化时间和成本。     

昂立植物乳杆菌及其抑菌物质的特性研究

该文研究了昂立植物乳杆菌(LP-Onlly)菌体及其代谢产物的抑菌性能,并对其代谢产物中的抑菌物质进行了部分理化特性的考察,发现LP-Onlly菌体对部分肠道有害菌有抑制作用,代谢产物中的抑菌物质对常见的肠道致病菌和食品腐败微生物具有广谱抑菌作用,对嗜酸乳杆菌及双歧杆菌等益生菌无抑制作用.该物质具有热稳定性,但抑菌活性受pH值的影响较大.     

血管生成抑制素基因工程大肠杆菌的高密度发酵研究

用5L发酵罐研究了E.coli TG1/pBVA2和E.coli TG1/pBVK13的高密度培养工艺,确定了诱导及补料策略,在不降低外源基因表达量的前提下,工程菌TG1/pBVA2高密度发酵菌体干重为16.8g/L,hAGN(K1-4)的表达量为菌体总蛋白的24.1%,相当于1.39 g/L;同样的方法, 工程菌TG1/pBVK13菌体干重可达16g/L, hAGN(K1-3)占菌体总蛋白25.8%,相当于1.45 g/L。     

以thyA基因为选择压力非抗性质粒载体的构建

以干酷乳杆菌L.casei34103染色体DNA为模板,利用PCR技术扩增胸苷酸合成酶（Thymidylatesynthase,thyA)基因,回收纯化,选择以红霉素抗性为选择压力的可以在大肠杆菌和乳酸菌中穿梭表达的质粒pW425e为基本质粒,以thyA基因取代红霉素基因,获得重组载体并鉴定,此重组载体可以对thyA基因缺陷的大肠杆菌E.coli X51和嗜酸乳杆菌DOMLaS 107进行功能弥补,进而构建了以thyA基因为地选择压力的非抗生素抗性穿梭表达载体,其大小为3716bp,并命名为pW425t。     

苏云金芽胞杆菌S-层蛋白基因的异源表达及分离纯化

S-层(S-layer)普遍存在于古菌、G~+、G~-菌中,由S-层蛋白所构成细菌S-层结构的生物体中,其功能引起了科学家的广泛关注,但目前S-层的功能大都处于推测阶段。究其原因,是因为外源S-层基因可以造成宿主大肠杆菌的致死效应。本研究将一株苏云金芽胞杆菌的两个S-层蛋白基因(GenBank登录号为AJ012290和AY460125)的3’端序列在大肠杆菌BL21(DE3)中成功进行异源表达,在0.8mmol/LIPTG诱导培养6h时,菌体中包涵体表达量达到最高;并对表达蛋白进行了初步分离纯化,以期为后期利用该纯化蛋白进行抗血清制备,进而对该菌的S-层蛋白在宿主菌中的定位、功能分析打下基础。     

优化的绿色荧光蛋白在超嗜热嗜酸古菌冰岛硫化叶菌中的表达与应用

【目的】开发可用于在极端嗜热嗜酸模式泉古菌冰岛硫化叶菌(Sulfolobus islandicus)中进行高效表达的eCGP123(enhanced consensus green protein variant 123)荧光蛋白,并用作S.islandicus的细胞内蛋白定位工具。【方法】绿色荧光蛋白突变体eCGP123具有极高的热稳定性、耐酸性和可逆的荧光特性等。本研究主要对eCGP123的基因根据S.islandicus密码子偏好性进行优化与合成,在大肠杆菌(Escherichia coli)中表达并研究其蛋白性质;通过在eCGP123的C末端分别融合具有不同细胞内定位的蛋白(包括E.coli来源的Fts Z和S.islandicus来源的Ups E、PCNA1和SiRe_1200等),构建eCGP123及其融合蛋白的表达菌株,用激光共聚焦显微镜分析eCGP123及其融合蛋白在E.coli和S.islandicus活细胞中的亚细胞定位。【结果】我们确认了在E.coli中表达并纯化密码子优化后的e CGP123具有与野生型绿色荧光蛋白相同的吸光值和较高的热稳定性。细胞学分析显示细胞分裂相关蛋白FtsZ和Si Re_1200分别主要定位于E.coli和S.islandicus分裂细胞的中间;鞭毛组分蛋白Ups E呈点状均匀分布,可能定位于细胞膜上;DNA复制滑动夹亚基PCNA1呈区域性点状分布,暗示了DNA复制区域的位置。蛋白的亚细胞定位与预期结果基本吻合。【结论】绿色荧光蛋白e CGP123可以作为报告蛋白,应用于S.islandicus细胞的蛋白定位分析中,可作为该模式菌株中功能基因研究的重要工具,但需要进一步优化条件。     

破菌时可自动降解宿主核酸的大肠杆菌BL21(DE3)的lpxM突变株构建

研究利用Red同源重组技术对常用大肠杆菌表达宿主菌BL21(DE3)进行改良, 构建破菌时可自动降解宿主核酸的大肠杆菌表达宿主菌, 该菌株可望有助于解决因破菌时宿主菌染色体核酸释放给后续纯化重组蛋白工作带来的困难。将N端连有OmpA的信号肽的S. aureus nucleaseB(nucB)表达框整合至E. coli BL21(DE3)的lpxM位点, 改造后菌株(称为BLN)经诱导能表达nucB、并分泌至周质空间, 这样可使宿主核酸免受该酶“毒性”影响, 菌体裂解后, nucB释放,能自动降解宿主核酸。BLN菌体生长状态以及表达外源重组蛋白的能力与出发菌基本一致。     

Bactericidal activity of human peritoneal macrophages from patients undergoing peritoneal dialysis

Peritoneal macrophages (PM) play an essential role in the pathogenesis of bacterial peritonitis, the main complication of peritoneal dialysis (PD). We determined the antibacterial activity of PM from 31 PD patients using gram-positive (Staphylococcus aureus, Staphylococcus epidermidis) and gram-negative (Escherichia coli, Pseudomonas aeruginosa) test organisms. In an 8-hour test assay, PM revealed the highest antibacterial activity against E. coli [median bactericidal index (Bi) = 5.46 representing 0.74 log growth inhibition compared to controls] and the lowest against P. aeruginosa (Bi = 1.63, 0.21 log growth inhibition, p less than 0.05). The antibacterial activity against S. aureus (Bi = 1.99, 0.3 log growth inhibition) and S. epidermidis (Bi = 2.0, 0.31 log growth inhibition) was within this range. When compared to peripheral blood polymorphonuclear leukocytes, PM reached only 4% (S. aureus) and 8.1% (E. coli) of their antibacterial activity (p less than 0.05). Using E. coli as a test organism, PM isolated after a 4-hour dialysis period revealed the highest antibacterial activity when compared to PM isolated after longer dialysis periods (p less than 0.05). Increasing the duration of PD to 6 and 8 h subsequently decreased the antibacterial activity of PM, suggesting that unphysiologic concentrations of toxic metabolites in the peritoneal effluent might have a harmful influence on PM functions.     

莳萝蒿精油成分分析及抑菌活性机理探究

为了确定莳萝蒿精油的化学成分,并探究其抑菌活性及抑菌机理。该研究采用水蒸气蒸馏法提取莳萝蒿精油,并通过气相色谱-质谱联用法测定其化学成分。采用抑菌圈法、二倍稀释法和生长曲线法测定精油的抑菌活性,采用电导率法和扫描电镜法探究精油的抑菌机理。结果表明:(1)莳萝蒿精油的主要化学成分包括醇类(47.12%)和萜烯类(19.90%),在所有成分中桉油精(12.39%)含量最高,其次为松油醇(8.70%)。(2)精油对金黄色葡萄球菌和大肠杆菌的抑菌圈直径分别为(22.57±1.68)mm和(15.36±0.71)mm。(3)精油对金黄色葡萄球菌和大肠杆菌的最小抑菌浓度分别为3.25和7.5μL/mL,最小杀菌浓度分别为7.5和15μL/mL。(4)当精油浓度为1.625和3.25μL/mL时,其分别能够延缓金黄色葡萄球菌和大肠杆菌的生长;当精油浓度为3.25和7.5μL/mL时,其能够完全抑制金黄色葡萄球菌的生长;当精油浓度为7.5和15μL/mL时,其能够完全抑制大肠杆菌的生长。(5)经精油处理之后的细菌,其相对电导率明显增大,且随精油浓度的增加而增大,同时其细胞膜发生了萎缩和破裂的现象。研究发现,莳萝蒿精油富含醇类和萜烯类等多种活性物质,对金黄色葡萄球菌和大肠杆菌具有良好的抑菌活性,且莳萝蒿精油能够改变细胞的膜结构,导致细菌中的内溶物发生泄漏,从而抑制细菌生长。     

Importance of S-layer proteins in probiotic activity of Lactobacillus acidophilus M92

AIMS: To investigate the functional role of surface layer proteins (S-layer) in probiotic strain Lactobacillus acidophilus M92, especially its influence on adhesiveness to mouse ileal epithelial cells. METHODS AND RESULTS: Sodium dodecyl sulphate polyacrylamide gel electrophoresis of cell surface proteins revealed the presence of potential surface layer (S-layer) proteins, ca at 45 kDa in L. acidophilus M92. Southern blot with pBK1 plasmid, containing slpA gene, gave a positive signal, suggesting that L. acidophilus M92 has a slpA gene coding for the S-layer proteins. S-layer proteins of this strain are present during all phases of growth. The S-layer proteins appeared when cells treated with 5 mol l(-1) LiCl were allowed to grow again. Removal of the S-layer proteins reduced adhesion of L. acidophilus M92 to mouse ileal epithelial cells. Furthermore, the viability of cells without S-layer were reduced in simulated gastric juice at low pH range (2, 2.5, 3) and simulated pancreatic juice with bile salts (1.5 and 3 g l(-1)). S-layer proteins of L. acidophilus M92 were resistant to pepsin and pancreatin, in contrast, the treatment with proteinase K led to a significant proteolysis of the S-layer proteins. CONCLUSIONS: These results demonstrated functional role of S-layer; it is responsible for adhesiveness of Lactobacillus acidophilus M92 to mouse ileal epithelial cells and has a protective role for this strain. SIGNIFICANCE AND IMPACT OF THE STUDY: S-layer proteins have an important role in the establishment of probiotic strain Lactobacillus acidophilus M92 in the gastrointestinal tract.     

Studies on the antibacterial activity of phanquone: chelating properties in relation to mode of action against Escherichia coli and Staphylococcus aureus

Phanquone is active against a wide range of Gram-positive and Gram-negative organisms. Its activity is affected by the nature of the suspending fluid, pH and anaerobic growth conditions. Its ability to chelate metal ions was examined and found to be related to its antibacterial activity, which was reduced by the presence of added metal ions, e.g. Co (II), Cu(II), Fe(II) and Fe(III) in nutrient media for both E. coli and S. aureus. When antibacterial activity was examined in dis-nutrient media for both E. coli and S. aureus. When antibacterial activity was examined in distilled water, then certain added metal ions, whilst antagonizing activity was examined in distilled water, then certain added metal ions, whilst antagonizing the activity of Phanquone against E. coli, exerted a co-operative effect in the case of S. aureus. The addition of EDTA and NTA lowered the activity of Phanquone against S. aureus, but not E. coli, while the addition of thiol-containing compounds lowered its activity against E. coli but not S. aureus. concentration quenching was observed for S. aureus but not for E. coli, while overnight pre-incubation at 4 degrees C resulted in the appearance of a growth zone inside the zone of inhibition in the case of S. aureus but not E. coli. Phanquone may have a different mode of action against the two organisms.     

A new route for chitosan immobilization onto polyethylene surface

Low-density polyethylene (LDPE) belongs to commodity polymer materials applied in biomedical applications due to its favorable mechanical and chemical properties. The main disadvantage of LDPE in biomedical applications is low resistance to bacterial infections. An antibacterial modification of LDPE appears to be a solution to this problem. In this paper, the chitosan and chitosan/pectin multilayer was immobilized via polyacrylic acid (PAA) brushes grafted on the LDPE surface. The grafting was initiated by a low-temperature plasma treatment of the LDPE surface. Surface and adhesive properties of the samples prepared were investigated by surface analysis techniques. An antibacterial effect was confirmed by inhibition zone measurements of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). The chitosan treatment of LDPE led to the highest and most clear inhibition zones (35mm(2) for E. coli and 275mm(2) for S. aureus).     

细菌对肉鸡肠粘液的粘附作用

研究两歧双歧杆菌、嗜酸乳杆菌、禽大肠杆菌O78、大肠杆菌 ATCC 25922、鸡白痢沙门氏菌和鼠伤寒沙门氏菌与肉鸡不同部位肠粘液糖蛋白的粘附性能,探讨两歧双歧杆菌和嗜酸乳杆菌对所试病原菌的抗粘附作用。结果表明：在不同的肠道部位,两歧双歧杆菌、嗜酸乳杆菌、鸡白痢沙门氏菌和鼠伤寒沙门氏菌与肠粘液糖蛋白均有不同的粘附作用,而禽大肠杆菌O78、大肠杆菌 ATCC 25922在各肠段粘液上的粘附性能则相近;在相同的肠道部位,所试益生菌的粘附能力大于病原菌;两歧双歧杆菌和嗜酸乳杆菌对所试病原菌的粘附有不同的阻断作用,同时二者有时还存在互补抗粘附作用。     

Antibacterial action of a heat-stable form of L-amino acid oxidase isolated from king cobra (Ophiophagus hannah) venom

The major l-amino acid oxidase (LAAO, EC 1.4.3.2) of king cobra (Ophiophagus hannah) venom is known to be an unusual form of snake venom LAAO as it possesses unique structural features and unusual thermal stability. The antibacterial effects of king cobra venom LAAO were tested against several strains of clinical isolates including Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli using broth microdilution assay. For comparison, the antibacterial effects of several antibiotics (cefotaxime, kanamycin, tetracycline, vancomycin and penicillin) were also examined using the same conditions. King cobra venom LAAO was very effective in inhibiting the two Gram-positive bacteria (S. aureus and S. epidermidis) tested, with minimum inhibitory concentration (MIC) of 0.78μg/mL (0.006μM) and 1.56μg/mL (0.012μM) against S. aureus and S. epidermidis, respectively. The MICs are comparable to the MICs of the antibiotics tested, on a weight basis. However, the LAAO was only moderately effective against three Gram-negative bacteria tested (P. aeruginosa, K. pneumoniae and E. coli), with MIC ranges from 25 to 50μg/mL (0.2-0.4μM). Catalase at the concentration of 1mg/mL abolished the antibacterial effect of LAAO, indicating that the antibacterial effect of the enzyme involves generation of hydrogen peroxide. Binding studies indicated that king cobra venom LAAO binds strongly to the Gram-positive S. aureus and S. epidermidis, but less strongly to the Gram-negative E. coli and P. aeruginosa, indicating that specific binding to bacteria is important for the potent antibacterial activity of the enzyme.     

In vitro growth control of selected pathogens by Lactobacillus acidophilus- and Lactobacillus casei-fermented milk

AIMS: Food-borne pathogen inhibition was tested in the presence of a mixture of Lactobacillus acidophilus and Lactobacillus casei during fermentation under controlled pH conditions. METHODS AND RESULTS: The growth of Escherichia coli O157:H7, Salmonella serotype Typhimurium, Staphylococcus aureus, Listeria innocua, Enterococcus faecium and Enterococcus faecalis was evaluated for 48 h at 37 degrees C. In the presence of the lactic acid bacteria (LAB), an increase of the generation time was observed for all the gram-positive bacteria evaluated. Staphylococcus aureus was the most sensitive strain showing an increase of the generation time by 210%. However, for all the gram-negative bacteria evaluated, no inhibition occurred after 8 h of fermentation. The soluble portion of Lact. acidophilus- and Lact. casei-fermented milk was recuperated and tested for its antimicrobial activity. Listeria innocua and Staph. aureus were the most sensitive to the presence of fermented milk supernatant showing an inhibition of 85.9% and 84.7%, respectively. This soluble fraction was neutralized to eliminate the antimicrobial effect of the organic acids produced; the most sensitive strains were L. innocua and E. coli O157:H7 showing an inhibition of 65.9% and 61.9%, respectively. Finally, the soluble fraction was neutralized and irradiated at 45 kGy using a (60)Co source to eliminate the possible antimicrobial effect of both organic acids and bacteriocin-like substances. Enterococcus faecalis, E. coli O157:H7 and Staph. aureus were the most affected bacteria by this fraction, showing 39.1, 32 and 31.2% inhibition, respectively. CONCLUSIONS: The results obtained in this study suggest the implication of both organic acids and bacteriocin-like inhibitory substances in the antimicrobial activity observed in the soluble fraction of the probiotic preparation. SIGNIFICANCE AND IMPACT OF THE STUDY: This study revealed the antimicrobial mechanisms of action of Lact. acidophilus- and Lact. casei-fermented milk used to prevent antibiotic-associated diarrhoea.     

载银纳米抗菌复合骨填充材料体外抗菌性能研究

目的探讨新型载银纳米抗菌复合骨填充材料(TiO2-Ag-nHA/PA66)的体外抗菌性能。方法采用抑菌环试验及菌落总数测定法检测不同纳米抗菌复合骨填充材料(A1、A2、A3)对金黄色葡萄球菌及大肠埃希菌的体外抗菌效果;扫描电镜观察其对细菌的抗粘附作用。结果抑菌环试验显示,不同载银纳米抗菌复合骨填充材料对金黄色葡萄球菌和大肠埃希菌均形成明显的抑菌环,以作用24 h抑菌环直径最大,并随作用时间延长,抑菌环直径逐渐缩小。其中银含量为0.64%(质量比)的材料A3的抗菌作用最明显,持续时间最长,其对金黄色葡萄球菌和大肠埃希菌的抑菌作用持续时间分别达到33 d和24 d;菌落总数测定法显示细菌与材料A3接触24 h后,对金黄色葡萄球菌和大肠埃希菌的抗菌率分别为94.18%和85.96%;扫描电镜发现载银材料能够明显减少细菌在材料表面的粘附。结论载银纳米抗菌复合骨填充材料体外对金黄色葡萄球菌及大肠埃希菌有明显抗菌作用,为其应用于慢性骨髓炎术后骨缺损修复提供理论依据。     

青海湖M1菌发酵液体外抗菌活性及稳定性的初步研究

从青海湖中分离得到一株霉菌M1,采用平板抑菌法进行体外抗菌作用以及pH值、温度因素对其抗菌活性的影响。结果表明：该菌株发酵液对大肠杆菌、枯草芽孢杆菌及金黄色葡萄球菌都有明显的抑菌效果。M1菌株发酵液的抑菌活性对热稳定性较差,在pH值3～7的条件下抑菌效果最佳,遗传稳定性也不高。