三聚氰胺脱氨酶基因克隆表达及活性测定

从载体pUC57-MDA中获得经过大肠杆菌密码子偏好性优化的三聚氰胺脱氨酶编码基因,将该基因连接至表达载体6HisT-pRSET中,构建了重组表达质粒6HisT-pRSET-MDA,将其转化大肠杆菌BL21(DE3),得到重组工程菌BL-MDA并进行诱导表达,利用SDS-PAGE对表达产物进行分析并测定酶活性。SDS-PAGE电泳分析结果显示,表达上清液在53 000处有明显的蛋白质条带,与理论相对分子质量的吻合,其脱氨酶活性达5.61 U。     

大肠杆菌链长控制基因ddsA原位替换及多基因共表达对辅酶Q_(10)合成能力的影响

目的强化大肠杆菌合成辅酶Q10(CoQ10)的能力。方法利用途径工程的基本原理,对大肠杆菌辅酶Q生物合成途径中链长控制基因ispB进行遗传操作,并强化表达该途径中多个功能基因。结果首次成功用来自Gluconobacter suboxydans的十聚异戊二烯焦磷酸合成酶基因ddsA原位替换大肠杆菌染色体上ispB基因,构建得到原位替换株JKL;同时强化表达CoQ生物合成途径的相关基因,发现在ubiCA和ddsA协同表达的重组菌pDCA/JKL中CoQ的合成能力比JKL提高了2.3倍,CoQ10合成量提高了2.5倍。检测该重组菌中ubiA的转录水平,发现其mRNA相对拷贝数是对照JKL的125倍。结论成功获得的重组大肠杆菌在降低内源性CoQ8合成能力的同时具备合成较长侧链CoQ10的能力,且通过强化表达相关基因使合成CoQ10的能力得到了提高。     

大肠杆菌链长控制基因ddsA原位替换及多基因共表达对辅酶Q_（10）合成能力的影响

目的强化大肠杆菌合成辅酶Q10（CoQ10）的能力。方法利用途径工程的基本原理,对大肠杆菌辅酶Q生物合成途径中链长控制基因ispB进行遗传操作,并强化表达该途径中多个功能基因。结果首次成功用来自Gluconobacter suboxydans的十聚异戊二烯焦磷酸合成酶基因ddsA原位替换大肠杆菌染色体上ispB基因,构建得到原位替换株JKL;同时强化表达CoQ生物合成途径的相关基因,发现在ubiCA和ddsA协同表达的重组菌pDCA/JKL中CoQ的合成能力比JKL提高了2.3倍,CoQ10合成量提高了2.5倍。检测该重组菌中ubiA的转录水平,发现其mRNA相对拷贝数是对照JKL的125倍。结论成功获得的重组大肠杆菌在降低内源性CoQ8合成能力的同时具备合成较长侧链CoQ10的能力,且通过强化表达相关基因使合成CoQ10的能力得到了提高。     

大肠杆菌链长控制基因ddsA原位替换及多基因共表达对辅酶Q10合成能力的影响

目的 强化大肠杆菌合成辅酶Q10(CoQ10)的能力.方法 利用途径工程的基本原理,对大肠杆菌辅酶Q生物合成途径中链长控制基因ispB进行遗传操作,并强化表达该途径中多个功能基因.结果 首次成功用来自Gluconobacter suboxydans的十聚异戊二烯焦磷酸合成酶基因ddsA原位替换大肠杆菌染色体上ispB基因,构建得到原位替换株JKL;同时强化表达CoQ生物合成途径的相关基因,发现在ubiCA和ddsA协同表达的重组菌pDCA/JKL中CoQ的合成能力比JKL提高了2.3倍,CoQ10合成量提高了2.5倍.检测该重组菌中ubiA的转录水平,发现其mRNA相对拷贝数是对照JKL的125倍.结论 成功获得的重组大肠杆菌在降低内源性CoQ8合成能力的同时具备合成较长侧链CoQ10的能力,且通过强化表达相关基因使合成CoQ10的能力得到了提高.     

人工合成的单链甜蛋白monellin基因在大肠杆菌中的高效表达

根据已报道的单链monellin甜蛋白的氨基酸序列,采用细菌偏爱密码子,人工合成了全长294bp的monellin基因。插入到大肠杆菌表达载体pET22b中,构建重组分泌型表达载体pETMO。经IPTG诱导pETMO所含有的甜蛋白基因可在大肠杆菌BL21(DE3)中高效表达,表达量占菌体可溶性蛋白的44.8%。且经纯化后测定其甜度是蔗糖的3000倍。     

假肠膜明串珠菌甘露醇脱氢酶基因的克隆及表达

利用聚合酶链式反应从假肠膜明串珠菌中扩增出甘露醇脱氢酶（mannitol dehydrogenase,MDA）基因的结构基因,克隆入表达载体pETDuet-1,构建了甘露醇脱氢酶表达质粒pETDuet-1-mdh,将其转化进入大肠杆菌BL21(DE3),经异丙基-β-D-硫代半乳糖苷诱导表达。假肠膜明串珠菌甘露醇脱氢酶结构基因长度为1 017 bp,重组甘露醇脱氢酶基因在大肠杆菌内成功表达,其蛋白质分子质量为36.0 kD;重组甘露醇脱氢酶活力为0.15 U/mg pro,高于假肠膜明串珠菌中甘露醇脱氢酶活力0.03 U/mg pro。     

泡菜中植物乳杆菌亚硝酸盐还原酶的克隆表达

目的:对植物乳杆菌亚硝酸盐还原酶基因(nirS)进行克隆及表达,检测重组酶表达情况及其酶活力。方法:以分离自传统泡菜植物乳杆菌的基因组DNA为模版进行PCR扩增nirS;重组构建TA克隆质粒pMD 19-T-nirS,并转化到大肠杆菌DH5a中保存;通过双酶切消化将nirS基因连接到pET-32a(+)上,获得重组表达质粒pET-32a(+)-nirS,并将其转入大肠杆菌BL21(DE3)中诱导表达;重组酶经纯化后进行SDS-PAGE电泳检测其表达情况;重组酶经复性后检测其酶活力。结果:扩增得到nirS基因并成功在大肠杆菌BL21(DE3)中表达,所得重组酶以包涵体形式存在,酶活力为2131.5U/mg。结论:采用基因工程技术获得亚硝酸盐还原酶具有一定的应用前景。     

碳源对manA基因突变大肠杆菌代谢的影响

ManA基因编码的甘露糖-6-磷酸异构酶在大肠杆菌中催化D-甘露糖和D-果糖的异构化,促进大肠杆菌对碳源的代谢吸收。本文通过研究manA基因突变大肠杆菌对碳源的利用和编码糖代谢基因情况,探讨甘露糖-6-磷酸异构酶对大肠杆菌糖代谢的影响。采用Ⅱ型内含子逆转录突变方法构建manA基因突变大肠杆菌,分析manA基因突变大肠杆菌对不同碳源的利用情况和manA基因突变对大肠杆菌糖代谢相关基因表达的影响,结果显示,大肠杆菌BL21(DE3)ΔmanA以甘露糖、果糖为碳源时,菌株生长受到显著抑制;以淀粉为碳源时,BL21(DE3)ΔmanA菌株的生长显著优于野生型大肠杆菌;以葡萄糖为碳源时,manA基因突变对大肠杆菌的生长无显著影响。通过基因表达分析,发现大肠杆菌BL21(DE3)ΔmanA中甘露糖代谢相关基因的表达显著性降低;果糖代谢途径中6-磷酸果糖激酶Ⅰ亚基的编码基因(pfkA)显著下调表达;水解淀粉的α-淀粉酶编码基因(malS)显著性上调表达。ManA基因突变影响大肠杆菌甘露糖、果糖和淀粉代谢途径中相关基因的表达,从而影响大肠杆菌对碳源的利用。     

植物乳杆菌细菌素基因plnEF的克隆与异源表达

目的:构建目的基因plnEF原核表达系统,诱导工程菌BL21-p ET28a-PL-plnEF表达目的融合蛋白并纯化,以期获得大量纯度较高的植物乳杆菌PlnEF细菌素,开发新的食品防腐剂。方法:构建含plnEF基因的重组质粒,将其转入大肠杆菌BL21中,经过IPTG诱导大量表达目标蛋白,融合蛋白PlnEF经纯化后进行分子量大小、抑菌活性等的检测。结果:重组质粒pET28a-PL-plnEF在大肠杆菌BL21中成功表达,合成PlnEF蛋白,其分子量为15.6 k Da,且该融合蛋白对大肠杆菌JM109具有良好的抑菌活性。结论:plnEF基因片段能够在原核细胞中正确表达且具有活性,本文为进一步研究开发该细菌素作为生物防腐剂奠定了基础。     

藤黄微球菌过氧化氢酶基因在大肠杆菌中的重组与表达

目的应用基因重组技术在大肠杆菌中高效表达藤黄微球菌过氧化氢酶(catalase,CAT)。方法从藤黄微球菌DNA中获得CAT编码基因,并应用pProEx-HTa质粒构建融合表达载体并表达和检测活性。结果通过融合表达获得可溶性带6×His标签重组蛋白,该蛋白经过Ni-NTA纯化后可获得活性物质。结论从大肠杆菌表达体系中表达了具有生物活性的CAT。     

多重耐药与药物敏感大肠杆菌菌株在体外模拟胃肠道环境的生存力研究

目的 大肠杆菌是人和温血动物肠道重要的兼性厌氧寄居菌群, 也是水源和食品粪便污染指示菌。研究多重耐药型大肠杆菌和药物敏感型大肠杆菌菌株在体外模拟胃肠道环境下的生存能力, 本文数据为控制有害大肠杆菌提供理论依据。方法 从奎屯市超市采集30份零售食品, 分离出8份大肠杆菌阳性样品, 污染率为27%。利用纸片扩散法对分离的8株大肠杆菌进行耐药实验, 从8株菌中选出1株具有多重耐药的E27号菌和1株药物敏感型菌株E25号菌进行人工胃液和人工肠液耐受试验。结果 所测定的8株大肠杆菌对大多数耐药纸片敏感, 大肠杆菌对氨苄西林和四环素耐药率较高。8株菌中有4株耐药菌和4株敏感菌。结论 所选出的两株菌, E25在人工胃肠液中的耐受力较好, 菌株E27在人工胃肠液中耐受力较差, 敏感菌株E25要比多重耐药菌株E27菌数减少的缓慢。     

The fate of Escherichia coli O157 in soil and its potential to contaminate drinking water.

The survival and transport of Escherichia coli and E. coli O157 after cattle slurry application were studied on drained plots in both grassland and arable stubble at three sites in Scotland. Leaching losses were between 0.2% and 10% of total E. coli and were dependent on rainfall. Recovery of E. coli in grass and soil declined with approximately first order kinetics. Residual numbers, in excess of background declined more slowly. The pattern was similar for both grass and arable plots. Laboratory incubations of soil cores, with applied slurry containing E. coli and E. coli O157 were performed in soils with different moisture contents at two temperatures for clay loam and sandy loam soils. Both E. coli populations were measured over a 4-week period. Using a dual population approach, the die off of the susceptible pool was linear with a half-life of 3-4 days, and was faster at the higher temperature and lowest moisture content. The resistant pool was not strongly affected by temperature or moisture and had a half-life for die off of between 18 and 24 days. After a 4-week period, < 100 cfu g/soil of E. coli and E. coli O157 remained. The die off rate of E. coli O157 was the same or slightly faster than that of the commensal E. coli population, indicating that the field behaviour of E. coli O157 can be studied by monitoring the total population of E. coli applied with slurry. The risk of significant pollution of water by E. coli is highest immediately after application of slurry, and the first increments of drainflow carry significant concentrations. Thereafter, the risk of pollution is very low. If weather conditions are dry after application on well-drained sandy soils, it is unlikely that any significant losses of organisms to drains will occur. Such data can be used to control and minimise the risk of E. coli O157 contaminating drinking water.     

核桃青皮中胡桃醌对大肠杆菌的抗菌作用及机制

为研究胡桃醌对大肠杆菌的抗菌活性及其作用机理,用不同质量浓度胡桃醌处理大肠杆菌,分别对最小抑菌质量浓度（minimal inhibitory concentration,MIC）、生长曲线、细胞膜相对电导率、荧光发射光谱等进行测定,并进行生物膜形成和细胞活性分析、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（sodium dodecyl sulphate-polyacrylamide gel electrophoresis,SDS-PAGE）以及大肠杆菌基因组合成分析。结果表明,胡桃醌对大肠杆菌具有有效的抗菌活性,MIC为0.062 5 mg/mL。经不同质量浓度胡桃醌处理后大肠杆菌细胞膜相对电导率升高,表明胡桃醌破坏了大肠杆菌膜的完整性,增加了细胞膜的通透性。荧光发射光谱分析结果表明,胡桃醌能够与膜蛋白相互作用从而改变大肠杆菌细胞膜结构。结晶紫和刃天青染色实验结果表明胡桃醌能够通过抑制大肠杆菌生物膜的形成减弱大肠杆菌的呼吸作用,进而抑制其活性。SDS-PAGE和大肠杆菌基因组合成分析结果显示胡桃醌可抑制大肠杆菌中蛋白质、DNA和RNA的表达。通过分子对接实验可知,胡桃醌可以结合到基因组DNA的...     

Development of a medium for differentiation between Escherichia coli and Escherichia coli O157:H7.

A new medium (Escherichia coli O157:H7 medium: EOH) was developed for differentiation between E. coli and E. coli O157:H7. The EOH medium was compared with sorbitol MacConkey agar (SMAC), which is the most popular medium to enumerate E. coli O157:H7. Several combinations of 35 dyes were evaluated to develop the new medium. Indigo carmine (0.03) g/liter) and phenol red (0.036 g/liter) were found as the best combination for differentiation between E. coli O157:H7 and E. coli and added to the basal agar medium (SMAC medium excluding neutral red and crystal violet) for EOH medium. On the dark blue EOH medium, E. coli produced a yellow color with clear zone, whereas E. coli O157:H7 produced a red color without clear zone. For differentiation between E. coli and E. coli O157:H7, EOH has much better potential than SMAC. Furthermore. the red color produced by normal E. coli in SMAC may mask the light gray color produced by E. coli O157: H7, whereas the yellow color with clear zone did not mask the red color without clear zone in the EOH medium. The recovery numbers of E. coli O157:H7 from inoculated ground beef, pork, and turkey were not significantly different between SMAC and EOH media (P > 0.05). The recovery rates of heat- and cold-injured E. coli O157:H7 also were not significantly different (P > 0.05).     

Polymerase chain reaction assay for detection of Escherichia coli O157: H7 and Escherichia coli O157: H-.

The DNA band patterns generated by polymerase chain reaction (PCR) using the du2 primer and template DNAs from various strains of Escherichia coli and non-E. coli bacteria were compared. Among three to five prominent bands produced, the three bands at about 1.8, 2.7, and 5.0 kb were detected in all of the E. coli O157 strains tested. Some nonpathogenic E. coli and all pathogenic E. coli except E. coli O157 showed bands at 1.8 and 5.0 kb. It seems that the band at 2.7 kb is specific to E. coli O157. Sequence analysis of the 2.7-kb PCR product revealed the presence of a DNA sequence specific to E. coli O157:H- and E. coli O157:H7. Since the DNA sequence from base 15 to base 1,008 of the PCR product seems to be specific to E. coli O157, a PCR assay was carried out with various bacterial genomic DNAs and O157-FHC1 and O157-FHC2 primers that amplified the region between base 23 and base 994 of the 2.7-kb PCR product. A single band at 970 bp was clearly detected in all of the strains of E. coli O157:H- and E. coli O157:H7 tested. However, no band was amplified from template DNAs from other bacteria, including both nonpathogenic and pathogenic E. coli except E. coli O157. All raw meats inoculated with E. coli O157:H7 at 3 x 10(0) to 3.5 x 10(2) CFU/25 g were positive both for our PCR assay after cultivation in mEC-N broth at 42 degrees C for 18 h and for the conventional cultural method.     

MALDI-TOF MS方法快速鉴定大肠杆菌及ESBLs菌株检测探索研究

目的研以基质辅助激光解析电离飞行时间质谱(matrix assisted laser desorption ionization-time of flight mass spectrometry,MALDI-TOF MS)方法检测大肠杆菌,并利用MALDI-TOF MS方法鉴别ESBLs菌株及聚类分析法区分不同分型的ESBLs菌株,探索MALDI-TOF MS方法鉴定大肠杆菌及ESBLs菌株的可行性。方法利用MALDI-TOF MS采集47株大肠杆菌分离株的质谱数据,生成肽指纹图谱,并利用biotyper软件进行菌种鉴定,同时研究其耐药性鉴定结果,并利用聚类分析法研究大肠杆菌ESBLs菌株,最终确定MALDI-TOF MS对大肠杆菌菌种鉴定及ESBLs菌株鉴别的可行性。结果 MALDI-TOF MS方法鉴定大肠杆菌的结果与传统方法及PCR方法检测结果均符合,经MALDI-TOF MS鉴定方法提示为ESBLs菌株的10株大肠杆菌中有9株为ESBLs菌株,且聚类分析法对OXA型ESBLs大肠杆菌具有较好的鉴别能力。结论MALDI-TOF MS方法可以快速、准确地鉴定大肠杆菌,对ESBLs菌株具有一定的筛选鉴别能力。     

Ability of Lactobacillus gasseri K 7 to inhibit Escherichia coli adhesion in vitro on Caco-2 cells and ex vivo on pigs' jejunal tissue

The ability of Lactobacillus (Lb.) gasseri K 7 to inhibit adhesion of Escherichia coli O8:K88 to intestinal mucosa was studied on cultured Caco-2 cells and ex vivo on pigs' small intestinal tissue. Lactobacilli were added simultaneously with E. coli, before E. coli and after E. coli for competition, exclusion and displacement assays. The concentration of lactobacilli on fully differentiated Caco-2 cells was 4.5+/-0.3 x 10(8) cfu/well, while the concentration of E. coli varied from 1.5 x 10(6) to 4.3 x 10(8) cfu/well. The number of E. coli adhered to Caco-2 monolayer (cfu/well) was lineary correlated (R(2)=0.97) to the concentration of added cells. In the assay simulating exclusion, E. coli adhesion was reduced by Lb. gasseri K 7 strain by 0.1 to 0.6 log cfu/well. The binding of E. coli was inhibited even more when incubated simultaneously with lactobacilli, particularly at the lowest concentration of E. coli (ratio E. coli/lactobacilli 1:248), where five-times reduction (or 0.7 log) was observed. When adhesion to tissue derived from pigs' jejunum was tested, concentration of E. coli was constant (6.9+/-0.14 x 10(7) cfu/ml), while the concentration of Lb. gasseri K 7 was 5.9 x 10(7) and 1.3 x 10(7) cfu/ml in two independent experiments, respectively. The adhesion of E. coli and Lb. gasseri K 7 cells to jejunal mucosa was similar (1.0+/-0.17 x 10(6) and 1.54+/-0.10 x 10(6) cfu/cm(2)) when the concentrations of single strains in suspensions were approximately the same. No significant competition, exclusion or displacement of E. coli by lactobacilli was observed on jejunal tissue. In conclusion, Lb. gasseri K 7 was found to be effective in reducing E. coli adhesion to Caco-2 enterocytes, but it was not able to do so in ex vivo conditions tested for pig jejunal tissue.     

Understanding the role of agricultural practices in the potential colonization and contamination by Escherichia coli in the rhizospheres of fresh produce

To better protect consumers from exposure to produce contaminated with Escherichia coli, the potential transfer of E. coli from manure or irrigation water to plants must be better understood. We used E. coli strains expressing bioluminescence (E. coli O157:H7 lux) or multiantibiotic resistance (E. coli2(+)) in this study. These marked strains enabled us to visualize in situ rhizosphere colonization and metabolic activity and to track the occurrence and survival of E. coli in soil, rhizosphere, and phyllosphere. When radish and lettuce seeds were treated with E. coli O157:H7 lux and grown in an agar-based growth system, rapid bacterial colonization of the germinating seedlings and high levels of microbial activity were seen. Introduction of E. coli2(+) to soil via manure or via manure in irrigation water showed that E. coli could establish itself in the lettuce rhizosphere. Regardless of introduction method, 15 days subsequent to its establishment in the rhizosphere, E. coli2(+) was detected on the phyllosphere of lettuce at an average number of 2.5 log CFU/g. When E. coli2(+) was introduced 17 and 32 days postseeding to untreated soil (rather than the plant surface) via irrigation, it was detected at low levels (1.4 log CFU/g) on the lettuce phyllosphere 10 days later. While E. coli2(+) persisted in the bulk and rhizosphere soil throughout the study period (day 41), it was not detected on the external portions of the phyllosphere after 27 days. Overall, we find that E. coli is mobile in the plant system and responds to the rhizosphere like other bacteria.     

Development of a rapid immunochromatographic strip for detection of Escherichia coli O157

We developed an immunochromatographic (IC) strip for the rapid detection of Escherichia coli O157 in enriched samples. Murine monoclonal antibody to E. coli O157:H7 lipopolysaccharide was conjugated with 40 nm of colloidal gold particles by the citrate method. The specificity of the IC strip was determined using 48 pure-cultured bacteria, including 32 E. coli strains and 16 non-E. coli strains. Regardless of H serotype, E. coli O157 strains produced a positive signal, whereas the others, representing 29 E. coli serotypes, did not. Among 16 non-E. coli strains, only Citrobacter amalonaticus yielded a positive signal. The sensitivity of the IC strip was determined using 10-fold diluted E. coli O157:H7, with a range of 1.8 X 10(7) to 1.8 CFU/ml in enriched raw beef. E. coli O157 could be detected at a minimum of 1.8 x 10(5) CFU/ml without enrichment and 1.8 CFU/ml after enrichment. Various samples were enriched to detect E. coli O157 using the IC strip and to isolate E. coli O157:H7 using traditional culture procedures. The IC strip test results exhibited 100% agreement with traditional methods after selective enrichment, since E. coli O157:H7 was also isolated from all the samples with positive strip test results. However, the specificity of the strip was somewhat higher with pork (98.8%) than with bovine feces (87.9%) and swine feces (93.4%). These results indicated that the IC strip exhibits high specificity and sensitivity in the detection of E. coli O157, and this assay is rapid, economical, and simple, without requirement of complicated equipment.     

Construction of a novel hydroxyproline-producing recombinant Escherichia coli by introducing a proline 4-hydroxylase gene

An Escherichia coli recombinant strain producing trans-4-hydroxy-L-proline (Hyp) was constructed by introducing a proline 4-hydroxylase gene into an L-proline-producing E. coli. Plasmid pPF1, which contains a gene encoding feedback resistant gamma-glutamyl kinase (proB74), was constructed and introduced into E. coli W1485 putA. The recombinant E. coli W1485 putA/pPF1 strain produced L-proline (1.2 g/l). The proline production by W1485 putA/pPF1 was converted to Hyp production by introducing pWFH1 which contains a proline 4-hydroxylase gene. E. coli W1485 putA which harbors pWFP1 carrying the proline 4-hydroxylase gene, proB74, and proA produced 25 g/l of Hyp in 96 h. A novel biosynthetic pathway of Hyp, which has not previously been produced in E. coli, was constructed in E. coli.