PCR Detection of Virulence Genes Colv,Stxs and HlyE of Escherichia coli

[Objective] This study aimed to explore the presence of three causative genes Colv,Stxs and HlyE of the pathogenic E.coli from chickens,pigs and food.[Method] By using 44 E.coli strains from chickens,24 from pigs and 26 from food as the experimental materials,virulence genes Colv,Stxs（stx2,stx2e） and HlyE were detected with polymerase chain reaction（PCR） method.[Result] Among all the E.coli strains,the detection rate of Colv was 25% from chickens,4.2% from pigs,and 0 from food;the detection rate of Stx2（Stx2e） from all E.coli strains was 0;the detection rate of HlyE was 2.27% from chickens,0 from pigs,and 11.5% from food.[Conclusion] Virulence gene Colv shows relatively high carrying rate in E.coli from chickens and pigs;HlyE also shows a certain degree of presence in E.coli from chickens and food.

动物源E.coli O157:H7 stx基因的检测与系统进化分析

为了解2009-2010年间在河南、甘肃地区分离鉴定的5株大肠埃希菌O157(E.coli O157)携带stx的情况及不同分离株间stx分子进化与变迁的情况,本研究利用PCR方法对分离株进行了stx基因检测,并完成了序列测定与系统演化分析。结果表明,5株不同动物源的分离株均含有stx1及stx2基因。序列分析结果显示5株分离株间stx1、stx2的核苷酸及氨基酸同源性均较高;stx1基因均与参考株中的山羊源和食品源E.coli O157菌株的同源性较高,进化树中遗传距离最近;分离株的stx2基因与多株牛源及少数人源参考株也具有较高的同源性,进化树中虽然5株分离菌均在一个大主干分支中,但分离株27与其他各分离株及参照株遗传距离最远,独自处于一次级分支中;分离株L37与W、12与50分别分布于牛源、人源E.coli O157小次级分支中;由此可推测,分离株所携带的stx1很有可能是经食品源或羊源E.coli O157传递而来;分离株L37与W、分离株12与50的stx2可能是由牛源、人源E.coli O157菌株传递而来,分离株27的stx2来源不清楚。研究结果表明,5株E.coli O157分离株均含有stx1、stx2基因,但两个基因的起源存在差异。

突变和修饰IFN-α2b基因的5’和3’端对其在E.coli表达的调控作用

目的对人 IFN- α2 b基因进行突变和修饰 ,从翻译水平上调控 IFN- α2 b基因在大肠杆菌中的表达。方法采用PCR技术 ,人工合成寡核苷酸引物 ,对人 IFN- α2 b基因进行了改造 :在不改变氨基酸的前提下 ,将 5’端编码区的四个 G、C位点突变为 A、T;去除 3’端非编码区。将突变和修饰后的基因片段分别克隆入原核表达载体 p BV32 1,在大肠杆菌中进行表达。对表达产物进行活性效价测定。结果 3’端删除非编码区 ,表达水平比删除前提高 4倍 ;5’端四个位点突变 ,表达水平比突变前降低 2倍。结论 IFN-α2 b基因的 3’端非编码区抑制其在原核系统的表达 。

工程菌E.coli MM2的固定化细胞制备

固定化细胞技术是七十年代兴起的一门应用技术。固定化细胞具有细胞密度高、易进行连续生产且稀释率高、不易被污染、产物易分离、可降低设备规模等优点,已成为生物合成、生物转化的有效工具,得到广泛应用。近十年来,人们把固定化细胞技术应用到工程菌的培养中取得了一些有价值的结果。用工程菌制得的固定化细胞裂解青霉素生产6-APA,已达工业化生产水平。据文献报道,工程菌固定化后可大大提高重组质粒的稳定性,并且外源基因能稳定地表达。

高密度CO_2处理对E.coli细胞膜渗透性的影响

以E.coli菌悬液为研究对象,通过测定高密度CO2处理(DPCD)后E.coli上清液中蛋白质、核酸、Mg2+、K+离子和丙二醛的含量,辅助透射电镜观察,研究DPCD对E.coli细胞膜渗透性的影响。在7MPa、37℃条件下,E.coli经高密度CO2处理10min后,99%以上的E.coli失活,同时研究发现蛋白质、核酸及Mg2+、K+离子等胞内物质均发生了不同程度的泄漏,丙二醛含量增加,E.coli胞内物质密度降低。密度CO2处理造成E.coli细胞膜渗透性的增加,这也是导致E.coli死亡的原因之一。

Acetylsalicylic acid supplementation improves protein utilization efficiency while vitamin E supplementation reduces markers of the inflammatory response in weaned pigs challenged with enterotoxigenic E.coli

Background： This experiment was conducted to test the hypothesis that vitamin E（Vit E） and acetylsalicylic acid（ASA）, a cyclooxygenase-2（COX-2） inhibitor, will additively reduce the production of the immunosuppressive molecule prostaglandin E_2（PGE_2） and hence reduce inflammatory responses in weaner pigs experimentally infected with an enterotoxigenic strain of E. coli.Methods： The experiment was conducted in a research facility with 192 individually-housed male weaner pigs（Landrace × Large White） weighing 6.6 ± 0.04 kg（mean ± SEM）. The pigs were experimentally infected with an enterotoxigenic strain of E. coli and were allocated to a 2 × 3 factorial design with the respective factors being without and with 125 ppm ASA and three levels of Vit E supplementation（50, 100 or 200 IU/kg diet, dl-α-tocopheryl acetate）.Results： Acetylsalicylic acid supplementation improved average daily gain（P 〈 0.05） and tended to improve feed：gain ratio（P 〈 0.10） during the first 14 d after weaning. Acetylsalicylic acid supplementation also improved（P 〈 0.001） amino acid utilization efficiency（as assessed by plasma urea level） and tended to decrease（P 〈 0.10） PGE2 production in the liver without affecting smal intestinal histology and tight junction protein mR NA expression in the jejunal epithelium. Vitamin E supplementation greater than 100 IU/kg diet sustained both the plasma Vit E concentration（P 〈 0.001） and plasma haptoglobin content（P 〈 0.001） after weaning. However, there was no additive effects of the combined supplementation of ASA and Vit E on performance, intestinal barrier function and inflammatory responses of weaned pigs.Conclusions： Although ASA and vitamin E improved amino acid utilization efficiency and reduced acute inflammatory responses, ASA and vitamin E did not additively reduce production of PGE2 and inflammatory responses in weaner pigs experimental y infected with an enterotoxigenic strain of E. coli.

Biological Effect Studies of Norfloxacin Complexes on Tetrahymena and Escherichia Coli by Microcalorimetry

Norfloxacin complexes Co（nor）2.8H2O （nor=norfloxaein） and [Co（nor）（phen）]NO3.2H2O （phen=1,10-phenanthroline） were synthesized and their biological activity on Tetrahymena and Escherichia coli （E.coli） was assayed by means of microcalorimetry. Massive experimental parameters such as the growth constant k, inhibitory ratio I, half inhibition concentration IC5o and generation time TG were obtained. Data showed that with the increase of exposing complex concentration, both complexes exhibited strong inhibition during the growth of organisms. While toxic degrees were quite different on Tetrahymena and E.coli, due to various toxic mechanisms. And complex molecular volume and the ability of the complex penetrating into cells may be the keys.

Differential expression of bcl-2 and bax genes during the E.coli-induced apoptosis of human U937 cells

To explore the role of bcl-2 and bax genes in the apoptosis of human U937 cells induced by E.coli, flow cytometry assay with annexinⅤ-FITC/PI double staining was used to determine the condition of apoptosis, and the expressions of mRNA of bcl-2 and bax genes were assayed with RT-PCR. It was found that the apoptosis of human U937 cells could be induced by E.coli at various concentration ratios between cells and bacteria for 30 min in a dose-dependent manner. The apoptotic rates at cell/bacteria ratios of 0, 1∶5, 1∶10, 1∶20, 1∶50 and 1∶100 were 3.16%±0.90%, 9.46%±0.84%, 17.90%±1.41%, 35.59%±3.76%, 38.35%±7.12% and 55.07%±5.82% respectively. Also, there was a tendency of alterations in the expression levels of bcl-2 and bax genes with an increased expression level of bax gene and a reduced expression level of bcl-2 gene. It is concluded that E.coli can induce apoptosis in human U937 cells with a down-regulated expression of Bcl-2 and an up-regulated expression of Bax, and this might be related to the induction of apoptosis of the infected cell.

Core 2和Core 4 O-型糖链参与大肠杆菌对肠上皮细胞的黏附与侵袭

目的探讨大肠杆菌对Core 2和Core 4 O-型糖链合成障碍的肠上皮细胞的黏附和侵袭的影响。方法采用针对C2GnT-2的干扰质粒和对照质粒对结肠癌HT-29细胞进行转染,通过G418筛选建立稳定转染的细胞系,采用Realtime PCR和Western blot方法检测干扰效率,选取干扰最有效的稳定细胞系与肠致病性大肠杆菌(enteropathogenic Escherichia coli,EPEC)和肠出血性大肠杆菌(EHEC O157∶H7)37℃共培养。最后采用系列稀释克隆计数法观察干扰Core 2和Core 4 O-型糖链合成的C2GnT-2的HT-29细胞对细菌黏附及侵袭的影响。结果成功筛选获得shRNA-C2GnT-2及shRNA-Ctr稳定转染的HT-29细胞。Real-time PCR和Western blot检测证实干扰质粒能够有效地抑制HT-29细胞内C2GnT-2的mRNA及蛋白水平表达(P<0.01),黏附于shRNA-C2GnT-2/HT-29细胞表面的EPEC或EHEC O157∶H7的数量较对照细胞显著减少(P<0.01,P<0.05),而侵袭入shRNA-C2GnT-2/HT-29细胞内的EPEC或EHEC O157∶H7的数量较对照细胞显著增加(P<0.01,P<0.05)。结论 Core 2和Core 4 O-型糖链参与了肠上皮细胞细菌的黏附与侵袭。

PEC HPI irp2基因缺失株的构建及蛋白结构功能预测

为了探讨致病性大肠杆菌内HMWP2蛋白的生物学结构和功能,采用Red同源重组技术,对致病性E.coli的HPI irp2基因进行敲除,成功构建了致病性E.coli irp2基因缺失株,并利用测序结果对HMWP2蛋白进行了多重预测分析,以期为致病性大肠杆菌相关疾病的防治和发生机理研究提供理论基础。

猪2型圆环病毒间接ELISA检测方法研究

应用PCR扩增得到猪2型圆环病毒ORF2基因的3′端约600 bp,该PCV2 ORF2基因已去除终止子,并分别在其上下游添加Kpn Ⅰ和Hind Ⅲ酶切位点。PCR产物用Kpn Ⅰ和 Hind Ⅲ酶切后,与经同样处理过的 pBAD/g Ⅲ B Vector连接,转化TOP10细胞后挑取阳性克隆,经酶切鉴定和测序验证后用L-arabinose诱导进行融合表达,表达的蛋白主要以包涵体存在。经过镍离子亲和层析柱纯化,SDS-PAGE显示1条约28 ku的目的条带, 纯度达到85％以上。以该28 ku的多肽包被酶标板,建立了PCV2的间接ELISA检测方法。

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。

Zinc phosphate-based nanoparticles as alternatives to zinc oxide in diet of weaned piglets

Background: The high doses of zinc oxide(Zn O) administered orally to piglets for the prevention of diarrhea and increase of growth rate can contaminate pig farms and the surrounding environment. Therefore, there is a need to find a replacement of high doses of dietary Zn O with an equally effective alternative. In the present study, the effect of two formulations of zinc phosphate-based nanoparticles(Zn A and Zn C NPs) on growth performance,intestinal microbiota, antioxidant status, and intestinal and liver morphology was evaluated. A total of 100 weaned piglets were randomly divided into 10 equal groups with the base diet(control) or the base diet supplemented with Zn A, Zn C, or Zn O at concentrations 500, 1000, and 2000 mg Zn per kilogram of diet. Supplements were given to animals for 10 days. Fecal samples were collected on day 0, 5, 10 and 20. At the end of the treatment(day 10),three piglets from each group were sacrificed and analyzed.Results: Comparing to that of control, the significantly higher piglet weight gain was observed in all piglet groups fed with Zn A(P < 0.05). Differences in the total aerobic bacteria and coliform counts in piglet feces after NPs supplementation compared to that of control and Zn O groups were also found(P < 0.05). The majority of aerobic culturable bacteria from the feces represented Escherichia(28.57–47.62%), Enterococcus(3.85–35.71%), and Streptococcus(3.70–42.31%) spp. A total of 542 Escherichia coli isolates were screened for the virulence genes STa,STb, Stx2, F4, and F18. The substantial occurrence of E. coli virulence factors was found on day 5, mainly in fimbrillary antigen and thermostable toxins, except for piglets fed by Zn C. Zn treatment decreased Zn blood levels in piglets fed with Zn O and Zn A(500 mg/kg) and increased in Zn C(2000 mg/kg) compared to that of control(P < 0.05). The antioxidant status of piglets was affected only by Zn A. While some changes in the liver and the intestinal morphology of piglets with NPs were observed, none were serious as reflected by the normal health status and increased weigh gain performance.Conclusions: Our results indicate that Zn A NPs have a positive effect on the piglet growth performance even at the lowest concentration. The prevalence of E. coli virulence factors was lowest in pigs supplemented with Zn C.Zinc phosphate-based nanoparticles may be an effective alternative to Zn O.

Synthesis of Niobium Doped ZnO Nanoparticles by Electrochemical Method: Characterization, Photodegradation of Indigo Carmine Dye and Antibacterial Study

Niobium doped Zincoxide nanoparticles has been synthesized through electrochemical method and characterized by UV-Visible spectroscopy, IR Spectroscopy, SEM, XRD, ICPMS and EDAX data. The UV-Visible spectroscopy result reveals that the band gap energy of ZnO/Nb2O5 nanoparticles to be 3.8 eV. The XRD results show that the crystallite size is to be 31.9 nm. The ICPMS data indicate the presence of 3,3461,328 counts of 93 Nb and 577,906,390 counts of 66 Zn. An improvement in the photocatalytic degradation of Indigocarmine dye (IC) in comparison to commercially available pure ZnO was observed. The photodegradation efficiency for ZnO/Nb2O5 and ZnO were found to be 97.4% and 52.1% respectively. The enhancement in photocatalytic activity of ZnO/ Nb2O5 was ascribed to the extended light absorption range and suppression of electron hole pair recombination upon Nb loading. The antibacterial activity of ZnO/Nb2O5 nanoparticles was investigated. These particles were shown to have an effective bactericide.

Synthesis of Chromium(III) Oxide Nanoparticles by Electrochemical Method and Mukia Maderaspatana Plant Extract, Characterization, KMnO₄Decomposition and Antibacterial Study

Chromium oxide nanoparticles were synthesized by the reduction of potassium dichromate solution with Mukia Maderaspatana plant extract. In electrochemical methods, Cr2O3 nanoparticles were synthesized by two ways, using platinum (Pt) electrodes and K2Cr2O7 solution with H2SO4 as medium in the first case. And chromium doped platinum electrode (Pt/Cr) in presence of NaHCO3 solution in second case. The resulting Cr2O3 nanoparticles were characterized by X-ray diffraction (XRD), Scanning electron microscopy (SEM), UV-VIS absorption and Fourier-transform infrared (FTIR) spectroscopy. The enhancing influence of Cr2O3 nanoparticles as a catalyst for the decomposition of KMnO4 has been studied. The antibacterial effect of Cr2O3 nanoparticles against E. coli was investigated. These particles were shown to have an effective bactericide.

利用EDA融合标签高效表达猪圆环病毒2型壳蛋白(英文)

原核生物作为宿主细胞被广泛应用于异源蛋白质的重组表达,并且为生物活性蛋白质的制备提供了一种高效、经济的方法,因而在分子生物学中得到普遍的应用。然而,病毒蛋白在使用原核重组表达系统进行重组表达时,会出现病毒蛋白溶解性差和表达量低等问题。因此,通过使用各种融合标签以增加目的重组蛋白的表达量和溶解性成为有效的方法。本研究通过使用3种融合标签(EDA标签、MBP标签和GST标签)以获得表达量高的可溶性重组表达猪圆环病毒2型壳蛋白;并比较3种融合标签对该蛋白表达量、溶解性和稳定性的影响。研究结果表明,EDA标签可以显著提高重组表达的猪圆环病毒2型壳蛋白表达量,并且能够增强该蛋白的稳定性;MBP标签可增强重组表达的猪圆环病毒2型壳蛋白表达量,但是不能改善该蛋白的稳定性;GST标签能够增强该重组表达蛋白的表达量,但是不能增强该蛋白的溶解性和稳定性。本研究将EDA作为PCV2-CP蛋白的融合标签,显著提高PCV2-CP-EDA重组蛋白的表达量和增强该重组蛋白的稳定性,为病毒蛋白的可溶性重组表达提供了一种新的融合标签。

UV-TiO_2 photocatalytic disinfection and photoreactivation of pathogenic bacterium in municipal wastewater

The disinfected bacteria will be a photoreactivation under the irradiation of the sunlight,and the light intensity plays an important role in the bacteria resurrection.The effect of light intensity on photoreactivation of Escherichia coli(E.coli) and Enterococcus faecalis(E.faecalis) in secondary effluents which were disinfected respectively by pure UV and UV-TiO_2 was investigated.The results show that the disinfection efficiency of UV-TiO_2 is much higher than that of the pure UV disinfection.The photoreactivation rate of E.coli is much higher in pure UV disinfection than in UV-TiO_2 photocatalytic disinfection.Under high light intensity in UV-TiO_2 disinfection,high resurrection rate can be induced.However,a higher resurrection rate can be introduced even under low light intensity in pure UV disinfection alone.Meanwhile,UV-TiO_2 disinfection has a strong inhibition effect on E.faecalis photoreactivation.When the light intensity is lower than 21 μW/cm^2,nearly no resurrection of E.faecalis occurs after 72 h resurrection irradiation,and a little resurrection rate is observed only under a strong photoreactivating light intensity.

Bacterial UDP-Glucose Hydrolases and P2 Receptor-Mediated Responses to Infection: A Commentary

UDP-glucose hydrolases are a group of relatively little known membrane-bound or periplasmic enzymes found in Salmonella enterica and E. coli. UDP-glucose is an agonist for a specific P2 receptor (P2Y14) found on epithelial cells and cells associated with innate immunity. It is also recognised as a ‘danger signal’. Cells respond to mechanical damage by releasing UDP-glucose which activates P2Y14 to trigger an innate immune response;it is postulated that a similar response to bacterial infection may be protective against infection. However, the UDP-glucose hydrolases may constitute virulence factors able to abrogate this response by degradation of the released UDP-glucose.

人CIDE-3基因原核表达载体的构建及蛋白的初步表达

目的：构建人CIDE-3基因的原核表达载体，并在E．coli BL21（DE3）中表达．方法：提取人肝母细胞瘤细胞系HepG2细胞的总RNA，经RT—PCR扩增人CIDE-3基因片段，并将其克隆入原核表达载体pET28a（＋）中，构建重组质粒pET28a（＋）-CIDE-3.经限制性内切酶Bam H I、Xho I双酶切鉴定及序列测定后，转化E．coli BL21（DE3），经IPTG诱导表达组氨酸融合蛋白．

MUC2基因表达对益生菌调节肠屏障作用的影响

目的观察益生菌诱导的乳鼠结肠MUC2基因的表达及其对益生菌抑制E.coli K1株E44黏附侵袭肠屏障作用的影响。方法 SD乳鼠分别用益生菌、E44株和益生菌+E44株三组灌胃7 d后,RT-PCR法观察益生菌和E44株诱导结肠MUC2基因的表达;靶向MUC2基因的shRNA真核质粒表达载体(shRNA MUC2)和阴性对照shRNA NC转染人结肠癌Lovo细胞,荧光定量PCR法检测其表达水平,并通过竞争性排斥方法检测MUC2基因对益生菌拮抗致病菌E44粘附侵袭的影响。结果灌胃益生菌组SD乳鼠肠道MUC2基因明显上调,而E44组则显著降低。用shRNA MUC2转染Lovo细胞后,与阴性对照组和空白对照组相比,其表达水平明显降低,干扰率为66.7%;益生菌对E44株粘附侵袭抑制作用明显低于未处理组,与对照组相比,E44相对粘附率为56.64%,相对侵袭率为66.64%。结论益生菌诱导的MUC2基因表达上调可能成为拮抗致病菌易位的保护机制之一;MUC2基因沉默后,益生菌对E44粘附侵袭肠上皮细胞的抑制作用明显降低。