Detection and frequency of Stx2 gene in Escherichia coli O157 and O157:H7 strains isolated from sheep carcasses in Shiraz-Iran

Enterohaemorrhagic Escherichia coli constitute a subset of serotypes (E. coli O157 and some other serogroups) of Shiga toxin-producing E. coli firmly associated with severe human illnesses like bloody diarrhoea and haemolytic uraemic syndrome. Escherichia coli O157:H7 is a zoonotic pathogen. They rarely cause disease in animals, live in the intestines of healthy sheep and ruminants are recognized as their main natural reservoir, so they can contaminate meat during slaughtering practices. The purpose of this study was epidemiological survey on the occurrence of E. coli O157:H7 in healthy sheep in Shiraz-Iran. Polymerase Chain Reaction (PCR) assay was developed to detect the Stx2 gene the only bacterial factor that has been associated with more severe disease. During a period of 7 months (December 2005 to June 2006), 153 slaughtered sheep at Shiraz slaughterhouse, were randomly selected and examined for surface carriage of E. coli O157:H7 by conventional plating and Stx2 gene detection by PCR technique. E. coli O157:H7 was found in 6(3.92%) of 153 sheep. The bacteria were isolated from 5(3.34%) of 114 and 1(2.63%) of 38 sheep two or under two and more than 2 years old, respectively (p = 0.5). The contamination rate might vary depending on season, age and infection time. The higher frequency for younger animals may be due to differences in the composition of the gastrointestinal flora resulting from differences in diet. This is the first report of the presence of E. coli O157:H7 in sheep from Iran.     

Chitosan-Based Intranasal Vaccine against Escherichia coli O157:H7

Background:

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an infectious zoonotic pathogen causing human infections. These infections, in some cases, can lead to hemolytic uremic syndrome and its life-threatening complications and even death worldwide. The first intimate bacterial adhesion, intimin (I), with its own receptor translocated intimin receptor (Tir) and E. coli secreted protein A, acting as Tir conduit, are highly immunogenic proteins for vaccine development against E. coli O157:H7.

Methods:

A chimeric trivalent recombinant protein was previously found to be a suitable strategy for developing vaccines against E. coli O157:H7. In this study, the recombinant EIT (rEIT) was used to design a protective EHEC nasal nanovaccine. Chitosan and its water-soluble derivative, trimethylated chitosan (TMC), as muco-adhesive biopolymers, are good candidates for preparation of nanovaccines.  Using the electrospraying technique, as a novel method, we could obtain particles of rEIT loaded with chitosan and TMC on a nanometer scale. Mice were immunized with intranasal administration or intrapretoneal injection of rEIT.

Results:

The rEIT-specific immune responses (IgG and IgA) were measured by indirect ELISA. Only nasal administration of chitosan electrospray and TMC formulation produced significant secretion IgA. Intranasal administration of nanovaccine reduced the duration of bacterial fecal shedding on mice challenged with E. coli O157:H7.

Conclusion:

Since development of mucosal vaccines for the prevention of infectious diseases requires efficient antigen delivery; therefore, this research could be a new strategy for developing vaccine against E. coli O157:H7.Key Words: EnterohemorrhagicEscherichia coli, Nanoparticles, Intranasal vaccination     

Durable antimicrobial cotton fabrics treated with a novel N-halamine compound

5,5-Dimethyl-3-((3’-triethoxysilylpropylamido)propyl)hydantoin (Si-Hy), a novel N-halamine precursor, has been synthesized in this work. The traditional pad-dry-cure process was used to coat the produced Si-Hy onto cotton fabrics. The coated fabric was characterized by SEM, FTIR and XPS. After exposure to chlorine bleach, the treated fabric presented good antimicrobial ability. The chlorinated sample demonstrated potent antibacterial ability against S. aureus (ATCC 6538) and E. coli O157:H7 (ATCC 43895) in brief contact time. Sixty seven percent of oxidative chlorine was retained and over 85 % of chlorine could be recharged after storage for 15 days and rechlorination. The antibacterial materials with good biocidal efficacies have potential applications in the healthcare industry.     

茶氨酸生物合成工程菌构建

通过PCR扩增E.coli DH5α的γ-ggt基因,产物经纯化后用Kpn I和Xho I双酶切,回收γ-谷氨酰转肽酶基因目的片断,并与经相同双酶切的表达载体pET-32a连接,得到重组质粒pET-GGT。将重组质粒转化到E.coli BL21中,获得工程菌。工程菌株经0.05βmol/L IPTG,32℃诱导表达,湿菌体的酶活达到2.0βU/g,大约是出发菌株E.coli DH5α的15倍。工程菌催化L-谷氨酰胺和盐酸乙胺反应生成茶氨酸的产量达到29.40βg/L,L-Gln的转化率为48.22%,其催化L-谷氨酰胺和盐酸乙胺反应生成茶氨酸的能力比出发菌株E.coli DH5α提高了100多倍。     

N-halamine-bonded cotton fabric with antimicrobial and easy-care properties

N-halamine precursor 2,2,6,6-tetramethyl piperidinol (TMP), a hindered amine light stabilizer, was bonded onto cotton fabric by using 1,2,3,4-butanetetracarboxylic acid (BTCA) as a crosslinking agent. A variety of treating conditions including TMP concentration, curing temperature and time, and catalyst were studied. The treated fabrics were characterized using FTIR spectra and scanning electron microscope (SEM). The cotton fabric treated with TMP precursor could be rendered biocidal upon exposure to dilute household bleach. The chlorinated cotton swatches showed great efficacy and inactivated 100 % of Staphylococcus aureus with 7.1 log reduction with 5 min of contact and 83.25 % of E. coli O157:H7 at 10 min of contact. In addition, the wrinkle recovery angle of the treated cotton fabrics increased from 229 ° of untreated cotton fabrics to 253 °. This study provided a practical finishing process to produce cotton fabrics with easy care and antibacterial functionalities at the same time.     

Controlled expression of cholera toxin B subunit from Vibrio cholerae in Escherichia coli

The ctxB gene, the causative agent of cholera epidemic was successfully cloned from V. cholerae in E. coli. The insertion of the gene was confirmed by PCR as well as restriction digestion analyses. The sequencing results for the gene confirmed that the insert was in the correct orientation and in-frame with the P(BAD) promoter and it showed that the gene was 99% homologous to the published ctxB sequence. The CTB protein was successfully expressed in E. coli using the pBAD/His vector system. The expected protein of approximately 14 kDa was detected by SDS-PAGE and Western blot. The use of pBAD/His vector to express the cholera toxin gene in E. coli would facilitate future study of toxin gene products.     

Antimicrobial and antioxidant activities of lignin from residue of corn stover to ethanol production

To improve the economic viability of the biofuel production from biomass resource, a value-added lignin byproduct from this process is increasingly important. Antioxidant and antimicrobial activities of lignin extracted from residue of corn stover to ethanol production were investigated. The lignin extracts exhibited strong antioxidant activities in hydrophilic oxygen radical absorbance capacity (ORAC) assay and Folin-Ciocalteu test. The extracts also exhibited antimicrobial activities against Gram-positive bacteria (Listeria monocytogenes and Staphylococcus aureus) and yeast (Candida lipolytica), but not Gram-negative bacteria (Escherichia coli O157:H7 and Salmonella Enteritidis) or bacteriophage MS2. Different extraction conditions (temperature and residue/solvent ratio) affected the antioxidant and antimicrobial activities of lignin extracts. Generally, the bioactivities of lignin extracts were consistent with FTIR analysis results. Lignin byproducts showed the potential for their antioxidant and antimicrobial application.     

Antibacterial Coating of Cellulose by Iso-bifunctional Reactive N-halamine with the Dyeing Process of Reactive Dye

In this study, we synthesized a novel N-halamine precursor, sulfuric acid mono-[2-(4-[4-chloro-6-(2-[4,4- dimethyl-2,5-dioxo-imidazolidin-1-yl]-ethylamino)-[1,3,5]triazin-2-ylamino]-benzenesulfonyl)-ethyl] ester sodium (TB), which contains two reactive groups of monochloro triazine reactive groups and bis-sulphatoethylsolphone reactive groups. The structure of TB is similar to iso-bifunctional group reactive dyes and could be coated on cotton fabrics by covalent bonds through a reactive dyeing process. The cotton coated with TB was characterized by FTIR and SEM. After chlorination, the treated cotton fabrics showed excellent antibacterial efficacy and inactivated all inoculated S. aureus (ATCC 6538) and E. coli O157: H7 (ATCC 43895) within 1 min of contact. Over 85 % of tensile strength retained both in warp and weft directions after treatment and chlorination. Almost 80 % of active chlorine can be regained by treating with household bleach after extensive washing and long time storage.     

一种E.coli-Free的酵母同源重组系统稳定快速构建PLDMV侵染性克隆的新方法

侵染性克隆是研究病毒的重要工具,而偏大的病毒基因组及其在大肠杆菌中的不稳定给构建侵染性克隆造成很大困难。通常采用插入内含子和酵母同源重组等方式可以获得稳定克隆,但本实验最初利用酵母同源重组系统并未成功构建番木瓜畸形花叶病毒(PLDMV)的侵染性克隆。经研究证实,该不稳定现象确实存在于大肠杆菌中,而非酵母和农杆菌细胞。通过改良酵母同源重组方法,成功构建了有/无内含子intron 2的PLDMV侵染性克隆p35S-FL和p35S-FL-In2,农杆菌注射接种番木瓜均发病,侵染效率达64.7%~69.7%。本研究通过酵母同源重组质粒直接转化农杆菌,建立了一种10 d内即可稳定快速构建PLDMV侵染性克隆的E.coli-Free酵母同源重组新方法。该方法对其他在大肠杆菌中不稳定的植物病毒侵染性克隆的构建具有重要意义。     

几种野生稻抽提物对斜纹夜蛾幼虫的拒食作用

用甲醇对6种野生稻E6-1, E6-3/6-4(大颖野生稻的不同生态型),E7-4(颗粒野生稻), E9-10、E9-20(阔叶野生稻的不同生态型),E13-13（小粒野生稻）,E15-13（药用野生稻）和E16-3（斑点野生稻）进行了索氏提取,提取率分别为19.62%,15.60%,18.90%,8.50%,16.27%, 24.86%,10.04%和13.32%。通过测定这6种野生稻抽提物对3龄斜纹夜蛾幼虫的选择性拒食活性和非选择性拒食活性,筛选出大颖野生稻抽提物E6-1对斜纹夜蛾有较强的拒食作用。用E6-1对叶碟处理后,在1%的浓度下,24 h和48 h测得其对3龄斜纹夜蛾选择性拒食率均为100%,而非选择性拒食率分别为40.95%和47.27%;毒力测定表明2龄斜纹夜蛾幼虫24 h和48 h后的拒食中浓度([i]AFC[/i][sub]50[/sub]）分别为120 mg/mL和50 mg/mL。     

几种野生稻甲醇提取物对害虫的行为干扰及对酯酶同工酶的影响

采用索氏回流法,经甲醇提取,获得大颖野生稻（E6 3/6 4、E6 1）、小粒野生稻（E13 9、E13 13）、药用野生稻（E15 8、E15 13）、斑点野生稻（E16 1、E16 3、E16 13）、阔叶野生稻（101392、E9 1、E9 10）和颗粒野生稻（E7 4）甲醇提取物。室内生物活性测定表明这些野生稻甲醇提取物对柑橘全爪螨和绣线菊蚜具有一定的忌避活性,其中药用野生稻E15 8的甲醇抽提物效果更为显著,5×104 μg/mL的浓度处理,测得24 h和48 h对柑橘全爪螨的忌避率分别为83.26%和87.95%;用同样的浓度处理,对绣线菊蚜的忌避率分别为87.86%和82.43%。经聚丙烯酰胺凝胶电泳检测,药用野生稻E15 8的甲醇抽提物对两种害虫的酯酶同工酶具有明显的抑制效果。     

齿兰环斑病毒外壳蛋白基因的表达及其表达产物抗血清的制备

用pET－21d构建齿兰环斑病毒（ORSV）外壳蛋白基因大肠杆菌表达载体pEO。经SDS－PAGE和Westernblotting分析，含pEO的大肠杆菌正确表达了ORSV外壳蛋白基因。此表达产物为融合蛋白。用表达产物制备抗血清，并应用于间接酶联法检测ORSV，具有较高的灵敏度（10ng／mLORSV）和特异性。     

Potential anti-HIV agents from marine resources: an overview

Human immunodeficiency virus (HIV) infection causes acquired immune deficiency syndrome (AIDS) and is a global public health issue. Anti-HIV therapy involving chemical drugs has improved the life quality of HIV/AIDS patients. However, emergence of HIV drug resistance, side effects and the necessity for long-term anti-HIV treatment are the main reasons for failure of anti-HIV therapy. Therefore, it is essential to isolate novel anti-HIV therapeutics from natural resources. Recently, a great deal of interest has been expressed regarding marine-derived anti-HIV agents such as phlorotannins, sulfated chitooligosaccharides, sulfated polysaccharides, lectins and bioactive peptides. This contribution presents an overview of anti-HIV therapeutics derived from marine resources and their potential application in HIV therapy.     

短短芽胞杆菌FJAT-0809-GLX超氧化物歧化酶基因的克隆及原核表达

短短芽胞杆菌FJAT-0809-GLX具有抑菌、抗褐变和保鲜功能。以短短芽胞杆菌菌株FJAT-0809-GLX总DNA为模板,采用PCR扩增超氧化物歧化酶(Supseroxide dismutase,SOD)基因,将该片段纯化回收后与p MD18-T连接并转入大肠杆菌(Escherichia coli)DH5α,进行序列测定,结果显示SOD基因序列长度为609 bp(Gen Bank登录号:KM255665),编码202个氨基酸残基。将SOD基因片段与同样酶切的表达载体p ET-28a连接,构建重组表达载体p ET-28a-SOD,转入大肠杆菌BL-21,采用异丙基-β-D-硫代吡喃半乳糖苷(Isopropyl-β-D-thiogalactopyranoside,IPTG)进行诱导表达。SDS-PAGE结果表明,在菌体中存在约25 ku的蛋白表达产物,与该基因ORF预期大小接近。以上结果为进一步研究该酶的生理生化特性奠定了基础。     

Repellent Activity of Extracts of Wild Rice Species against Panonychus citri and Aphis citricola in Associated with Esterase Isoenzyme in Insests

China is one of the eight centers of origin for crop plants in the world and also the origin center of Asian cultivated rice. There are three species of wild rice i.e. Oryza rufipogon, O. officinalis and O. meyeriama in the South of China. Wild rice enjoys the second-class national protection in China due to its high research values and possession of abundant stress resistance genes. Wild rice has developed certain level of resistance against stress due to better genetic characteristics sin…     

水稻粳粳交花药培养继代愈伤组织分化培养基的优化

以继代的粳粳交（空育131／稻花香2号）F。花药培养的愈伤组织为试验材料,研究培养基中的影响因素,对已有的培养基进行优化。共用20种培养基进行分化,涉及7种影响因素：谷氨酰胺,糖类,激素,山梨醇,铜、银离子组合,硅素和活性炭。结果表明：（1）长时间继代的愈伤组织仍具有分化能力。（2）可以显著提高绿苗分化率的因素为60mg／L硅素,0.25mg／LAgNOs和0.75mg／L CuSO4·5H2O。（3）加160目活性炭的培养基中愈伤生长状况得到很大改善。（4）最佳分化培养基为G1W3。     

叶面喷施Mg、Fe、Zn对菠萝生长和产量的影响

采用大田试验方法,研究了玄武岩发育而成酸性土壤上叶面喷施七水硫酸镁、一水硫酸亚铁、七水硫酸锌对菠萝生长和产量影响,为在此类土壤上种植的菠萝施用中微量元素提供依据。试验结果表明：与对照(喷清水)相比,喷施硫酸亚铁显著提高了菠萝叶片长度、宽度、叶片数及叶片中叶绿素含量,提高幅度分别为9.1%、14.9%、15.9%、62.6%,且显著提高菠萝产量、单果重和商品果率,提高幅度分别为11.8%、11.5%、7.7%;叶面喷施硫酸镁显著提高叶片叶绿素含量,但对菠萝叶片长度、宽度、叶片数和产量没有显著影响;叶面喷施硫酸锌对菠萝生长和产量都没有显著影响。     

斑茅SAMDC基因的克隆及其原核表达分析

以斑茅(Erianthus arundinaceus)samdc基因的cDNA为基础,采用基因重组技术,将该基因按正确的阅读框架定向克隆于原核表达载体pET-29a(+)中,转化大肠杆菌BL21(DE3),用IPTG诱导表达,并对表达产物进行SDS-PAGE分析.结果表明:重组斑茅samdc基因在大肠杆菌中获得高效表达,其分子量约为43.281kDa.斑茅samdc基因原核表达载体的成功构建和重组斑茅SAMDC蛋白在大肠杆菌中的高效表达,为进一步研究其生物学功能奠定了基础.     

Immunogenicity of a Fusion Protein Comprising Coli Surface Antigen 3 and Labile B Subunit of Enterotoxigenic Escherichia coli

Background: Enterotoxigenic Escherichia coli (ETEC) strains are the major causes of diarrheal disease in humans and animals. Colonization factors and enterotoxins are the major virulence factors in ETEC pathogenesis. For the broad-spectrum protection against ETEC, one could focus on colonization factors and non-toxic heat labile as a vaccine candidate. Methods: A fusion protein is composed of a major fimbrial subunit of coli surface antigen 3, and the heat-labile B subunit (LTB) was constructed as a chimeric immunogen. For optimum level expression of protein, the gene was synthesized with codon bias of E. coli. Also, recombinant protein was expressed in E. coli BL21DE3. ELISA and Western tests were carried out for determination of antigen and specificity of antibody raised against recombinant protein in animals. The anti-toxicity and anti-adherence properties of the immune sera against ETEC were also evaluated. Results: Immunological analyses showed the production of high titer of specific antibody in immunized mice. The built-in LTB retains native toxin properties which were approved by GM₁ binding assay. Pre-treatment of the ETEC cells with anti-sera significantly decreased their adhesion to Caco-2 cells. Conclusion: The results indicated the efficacy of the recombinant chimeric protein as an effective immunogen inducing strong humoral response. The designated chimer would be an interesting prototype for a vaccine and worthy of further investigation. Key Words: Recombinant vaccine, Enterotoxigenic Escherichia coli (ETEC), cstH, eltB     

Recombinant Production of a Novel Fusion Protein: Listeriolysin O Fragment Fused to S1 Subunit of Pertussis Toxin

Background:Some resources have suggested that genetically inactivated PTs bear a more protective effect than chemically inactivated products. This study aimed to produce new version of PT, by cloning an inactive PTS1 in a fusion form with N-terminal half of the LLO pore-forming toxin. Methods:Deposited pdb structure file of the PT was used to model an extra disulfide bond. Codon-optimized ORF of the PTS1 was used to make recombinant constructs of PTS1 and LLO-PTS1 in the pPSG-IBA35 vector. The recombinant PTS1 and LLO-PTS1 proteins were expressed in BL21 DE3 and SHuffle T7 strains of E. coli and purified by affinity chromatography. Cytotoxic effects of the recombinant proteins were examined in the MCF-7 cell line. Results:The purity of the products proved to be more than 85%, and the efficiency of the disulfide bond formation in SHuffle T7 strain was higher than BL21 DE3 strain. No cytotoxicity of the recombinant proteins was observed in MCF-7 cells. Soluble recombinant PTS1 and LLO-PTS1 proteins were produced in SHuffle T7 strain of E. coli with high efficiency of disulfide bonds formation. Conclusion:The LLO-PTS1 with corrected disulfide bonds was successfully expressed in E. coli SHuffle T7 strain. Due to the safety for human cells, this chimeric molecule can be an option to prevent pertussis disease if its immunostimulatory effects would be confirmed in the future. Key Words: Adjuvant, Cloning, Fusion protein, Pertussis toxin