猪流行性腹泻病毒N-E.coli OmpA融合基因构建、表达及其免疫原性评价

为获得免疫原性更强的猪流行性腹泻病毒(PEDV)结构蛋白,试验将PEDV N蛋白基因和大肠杆菌(E.coli)OmpA蛋白基因进行融合,采用柔性linker联接,得到OmpA-linker-PEDV N融合基因,并克隆至pCold I质粒,构建重组质粒pCold I-OmpA-linker-PEDV N。重组质粒转入BL21感受态细胞,SDS-PAGE分析重组质粒的表达,并对其编码的融合蛋白进行二级和三级结构预测。Western-blot和ELISA分析融合蛋白的免疫原性。结果表明:构建了融合基因表达质粒pCold I-OmpA-linker-PEDV N,表达了约88 ku的融合蛋白;融合蛋白的二级结构未发生明显变化,三级结构预测可折叠为正确的空间构像。该融合蛋白同时具有PEDV N蛋白和OmpA蛋白的免疫原性,并且其免疫原性显著高于单一PEDV N蛋白。说明试验获得了免疫原性较好的融合基因表达蛋白,可以为PEDV N蛋白功能研究、特异性抗体制备以及PEDV亚单位疫苗的研制提供材料和研究基础。

E.coli的Prlc基因产物对OmpA前体蛋白翻译后转位的功能

使用OmpA为指示蛋白,我们证实了prlc基因产物主要定位在胞液中,在分泌蛋白的转位过程中,它不是一个必需因子,但它的确涉及OmpA前体的转位定域。使用印迹试验证明它可能是一个信号肽水解酶。

Comparison of extended spectrum β-lactamasesproducing Escherichia coli with non-ESBLsproducing E.coli:drug-resistance and virulence

The virulent factors of Escherichia coil （E.cofi） play an important role in the process of pathopoiesis. The study aimed to compare drug-resistant genes and virulence genes between extended spectrum β-1actamases （ESBLs）-producing E.coli and non-ESBLs-producing E.cofi to provide a reference for physicians in management of hospital infection. From October 2010 to August 2011,96 drug-resistant strains of E. coli isolated were collected from the specimens in Qingdao Municipal Hospital, Qingdao, China. These bacteria strains were divided into a ESBLs-producing group and a non-ESBLs-producing group. Drug sensitivity tests were performed using the Kirby-Bauer （K-B） method. Disinfectant gene, qacEAl-sull and 8 virulence genes （CNF2, hlyA, eaeA, VT1, est, bfpA, elt, and CNF1） were tested by polymerase chain reaction （PCR）. Among the 96 E.coli isolates, the ESBLs-producing E.coli comprised 46 （47.9%） strains and the non-ESBLs-producing E.cofi consisted of 50 （52.1%） strains. The detection rates of multiple drug-resistant strain, qacEAl-sull, CNF2, hlyA, eaeA,VT1, est, bfpA, elt, and CNF1 in 46 ESBLs-producing E.coli isolates were 89.1%, 76.1%, 6.5%, 69.6%, 69.6%, 89.1%, 10.9%, 26.1%, 8.7%, and 19.6%, respectively. In the non-ESBLs-producing E.cofi strains, the positive rates of multiple drug-resistant strain, qacEAl-sull, CNF2, hlyA, eaeA, VT1, est, bfpA, elt, and CNF1 were 62.0%, 80.0%, 16.0%, 28.0%, 64.0%, 38.0%, 6.0%, 34.0%, 10.0%, and 24.0%, respectively. The difference in the detection rates of multiple drug-resistant strain, hlyA and VT1 between the ESBLs-producing E.cofi strains and the non-ESBLs-producing E.cofi strains was statistically significant （P〈0.05）. The positive rate of multiple drug-resistant strains is higher in the ESBLs-producing strains than in the non-ESBLs-producing strains. The expression of some virulence genes hlyA and VT1 varies between the ESBLs-producing strains and the non-ESBLs-producing strains. Increased awareness of clinicians and enhanced testing by laboratories are required to reduce treatment failures and prevent the spread of multiple drug-resistant strains.

Performance Parameters:Demobilization Antibiotic Resistant Bacteria(ARB)and Carrying Genes(ARG)in Wastewater Disinfection

The UV irradiation is used for removing Antibiotic Resistant Bacteria(ARB)and Antibiotic Resistance Genes(ARG)from wastewater treatment.Bacteriophages are viruses that infect within bacteria,are recognized for bacterial control.The influence of some parameters in quantification and performance influencing of pathogen demobilization could be considered in disinfection of wastewater.The comparison of Polyvalent phage(NE1)versus Coliphage(NE4)in suppressing a bacterium Escherichia coli(NDM-1:b-lactam-resistant)with UV irradiation was observed the efficacy in reduction of cells in the disinfection and parameter process.The results with the effect of UV-C irradiation on NDM-1 infected with 1%of NE4 showed a decrease of cells from 8×10^(6)to 2×10^(5)in 60 min with UV-C dose.The NDM1(E.coli)was infected with 1%of NE4(Polyvalent Phage)under magnetic stirring for 1 h,the cells count was 8×10^(6).After 1 h in UV-C e×posure,the cells number reached 3×10^(5).The NDM1 that was e×posed in 1 h of UV-C irradiation and then was infected with 1%of NE4.Cells counting were done 24 h after this procedure.These cells were e×posed in UV-C and showed a reduction in the number of cells from 1×10^(8)to 4×10^(5)after 60 min.The results indicate that bacteriophages can mitigate bacteria species,and combined the conventional water disinfection technologies that can support the microbial safety control strategies.

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.

猪卵透明带-3β融合蛋白在E.coli中的表达和鉴定

对全长猪卵透明带－３β（ｐＺＰ３β）ｃＤＮＡ的５端重新测序分析，发现文献报道的该克隆基因５端非编码序列中漏读了两个碱基，继而选择符合ｐＺＰ３βｃＤＮＡ阅读框的ｐＷＲ４５０－２载体质粒，通过双酶切构建了β－半乳糖苷酶／ｐＺＰ３β融合蛋白基因的细菌表达质粒。转化宿主菌后用ＩＰＴＧ诱导，ＳＤＳ－ＰＡＧＥ分析表明ｐＺＰ３β融合蛋白在Ｅ．ｃｏｌｉ中获得表达，并在蛋白印迹鉴定中能同兔抗猪ＺＰＩｇＧ呈特异性免疫反应。

秦皇岛地区狐源致病性E.coli对四环素类药物耐药性和耐药基因的检测

为了确定秦皇岛地区狐源大肠杆菌(E.coli)对四环素类药物的耐药性和耐药基因分布,采用常规的鉴定方法,从不同养狐场送检的腹泻的狐狸体内分离鉴定出20株E.coli。致病性试验表明,该菌为致病性E.coli。药敏试验结果表明:分离菌株对四环素和强力霉素药率分别达到95%和90%。通过PCR方法检测分离菌株四环素类药物的耐药基因,结果显示,tet A和tet B基因的检出率分别为100%和95%。本研究为秦皇岛地区防治狐源致病性大肠杆菌病提供实验基础。

抗生素耐药性大肠杆菌外膜蛋白mOmpA N端序列分析

从抗生素耐药大肠杆菌中克隆了mOmpA(突变OmpA)并构建表达载体pET32a-mOmpA。序列比对分析表明,mOmpA N端与OmpA N端DNA序列同源性为79.93%,氨基酸同源性为81.17%。Swiss-Model蛋白结构预测表明,mOmpA loop环的方向发生了显著变化。研究结果有助于进一步了解大肠杆菌OmpA的结构和功能以及大肠杆菌耐药机制。

中国白兔白介素-15基因的克隆、序列分析及其在E.coli中的表达

以RT-PCR技术对用ConA刺激的中国白兔外周血淋巴细胞（PMBCr）进行扩增,将纯化后的PCR产物克隆入pMD18-T中进行核苷酸序列测定,并与不同物种的IL-15基因进行序列比较。结果IL-15基因全长489 bp,编码162个氨基酸,其中前29个氨基酸残基构成信号肽序列。与不同物种IL-15基因相比,核苷酸和推导的氨基酸序列有一定的差异。在推导的中国白兔IL-15氨基酸序列中,在108～110、119～121、127～129和143～146位存在4个潜在的N-联糖基化位点,同时存在6个Cys残基。将pTIL-15双酶切,回收目的基因片段克隆到大肠杆菌表达载体pET28a中构建了重组质粒pETIL-15,转化大肠杆菌BL21（DE3）,并用IPTG进行了诱导。结果重组菌菌体裂解物经SDS-PAGE电泳可检测到相对分子质量为20 500的重组目的蛋白。经凝胶薄层扫描,目的蛋白表达量可占菌体蛋白的13.6%。

内源性血管生成抑制因子A rresten在E.coli JM109中的表达及抗新生血管生成的药理学研究

目的构建内源性血管生成抑制因子Arresten基因的原核表达载体，并进行表达，抑制新生血管的药理学实验中发现，该表达产物具有抑制鸡胚绒毛尿囊膜血管生长的功能。方法从健康产妇的胎盘组织中提取总RNA，经逆转录-聚合酶链式反应（RT-PCR）扩增出Arresten基因，构建重组质粒pBV220-Art转化E．coli JM109进行原核表达，大量表达提取Arresten蛋白，用鸡胚绒毛尿囊膜实验进行活性测定。结果成功构建的重组质粒pBV220-Arr在E．coli JM109菌株中2～8h均可获得表达，其中诱导4h表达效率最高，Arresten蛋白可明显抑制鸡胚绒毛尿囊膜血管生长，活性功能明显强于血管抑素。结论成功构建Arresten基因重组质粒pBV220-Arr，并可在E．coli JM109菌株中获得表达，Arresten蛋白具有明显的抑制血管生成的作用。

指状青霉提取物诱发E.coli ND-160及K12 infA基因突变

背景与目的:研究指状青霉(Penicillium digitatum)提取物对大肠杆菌菌株的致突变性。材料与方法:采用E.coli ND-160菌株回复突变试验、K12infA基因突变试验及其突变序列分析。结果:指状青霉提取物:①可明显地诱导ND-160菌株回复突变;②对K12茼株可诱发其infA基因DNA序列中5个碱基位点突变,且其中1个位点的突变还可导致编码相应氨基酸的改变(Lys→val)。结论:指状青霉对大肠杆菌基因有明显的致突变性。

用E.coli表达Canstatin-N及其表达条件优化

以重组质粒pET-CN为模板设计引物CASN1和CASN2,PCR方法扩增约267bp的人血管能抑素N端1～89氨基酸基因片段,用EcoR I和Sal I双酶切将其克隆进pET-22b(+)载体获得重组表达质粒pET-22b(+)-CN,转化E.coli BL21(DE3),用IPTG诱导表达Canstatin-N,产物以包涵体形式存在。本文在摇瓶发酵条件下研究了诱导剂浓度、诱导培养时间对目标蛋白表达的影响,结果表明IPTG的最佳诱导浓度为0.1mmol/L;37℃下诱导培养2h时产物表达量最高。纯化获得的融合his6的重组Canstatin-N具有免疫和抑制鸡胚绒毛尿囊膜(CAM)新生血管生成活性。

人胰岛素样生长因子Ⅰ型在E.coli和家蚕中的表达

将hIGF-I基因克隆进原核表达载体pET-28a(+),在E.coli中进行了融合表达,West-ern blotting显示在26 kD附近有一条特异条带。将hIGF-I基因克隆进pBacPAK-8,获得了杆状病毒转移载体pBacPAK-8-IGF-I,在脂质体的介导下,与线性化的家蚕杆状病毒共转染家蚕培养细胞Bm-N,经空斑筛选,PCR检测,获得了重组病毒Bm-Bac-hIGF-Ⅰ。SDS-PAGE检测表明,在感染重组病毒后,家蚕幼虫血淋巴中可以检测到一条分子量约为7.5 kD的特异性条带,ELISA检测表达量达4.51μg/mL蚕血淋巴。

衍生自大肠杆菌OmpA信号肽的多肽P-KKK杀菌作用研究

目的研究和比较大肠杆菌OmpA信号肽衍生多肽P-KKK在体外对不同细菌的杀菌效应。方法采用琼脂铺板计数法测定衍生多肽P-KKK对革兰阳性(G+)菌和革兰阴性(G-)菌的杀菌活性,将不同浓度的衍生多肽P-KKK分别与3种G+菌(金黄色葡萄球菌、屎肠球菌、枯草芽孢杆菌)和3种G-菌(大肠杆菌、肺炎克雷伯杆菌、绿脓杆菌)在37℃孵育2 h,铺板后置于37℃中培养18~24 h,记录每一琼脂板上菌落形成数量(CFU),计算多肽P-KKK的杀菌率。结果衍生肽P-KKK对G+菌具有较强的杀菌活性,浓度在200 ug/m L时,能杀灭98.5%以上的G+菌,浓度为40 ug/m L时杀菌率也达80.2%以上;对G-菌也有较强的杀菌活性,浓度在200 ug/m L时,能杀灭93.8%以上的G-菌,浓度为40 ug/m L时杀菌率也达15.2%~99.7%。结论大肠杆菌OmpA信号肽衍生肽P-KKK对G+菌及G-菌均具有明显的杀菌活性,进一步对其结构与功能进行研究将有助于开发出新型抗菌药物。

An Improved Strategy for Efficient Expression and Purification of Soluble HIV-1 Tat Protein in E.coli

Although the endogenous function of Tat has been elucidated in the past twenty years, the study of its exogenous activity has been hampered due to the difficulty of large scale preparation of the active Tat protein. To express the full-length Tat protein in E.coli, the tat gene was cloned from an HIV infected patient by overlapping PCR. Rare codon usage analysis showed that rare E.coli codons, especially consecutive rare codons for Arg, account for 14% (14 of 101) rare E.coli codons in the tat gene. The expression of the HIV-1 tat gene was verified to be very poor in strain BL21 (DE3) due to the abundance of rare codons; however, tat gene expression was found to be very efficient in the host strain of Rosetta-gami B (DE3), which was supplemented with six rare tRNAs for Arg, Leu, Ile and Pro. Subsequent purification revealed that the proteins are soluble and unusually, the tagged Tat can form dimers independent of cystine disulfide bonds. The purity, integrity and molecular weight of the Tat protein were demonstrated by MALDI-TOF mass spectrometry. Reporter gene activating assay was further confirmed by investigating the transactivation activity of the recombinant Tat protein. Our improved strategy for efficient high level expression and purification of soluble Tat protein has paved the way to fully investigate its exogenous function.

Amino terminus mutant OmpA from an isolated antibiotic resistant <i>Escherichia</i><i>coli</i>still possess resistance to environmental stresses

Antibiotic resistant Escherichia coli strains are becoming more common recently. OmpA is a very important antigen protein of E. coli, which consists of two separate domains, N-terminal and C-terminal domain. The N-terminal domain contains eight β- barrel regions that plays important roles in the multifaceted functions of OmpA. In the present study, we cloned a mutant OmpA gene from a multi-antibiotic resistant E. coli strain. Sequence analysis indicated that the N-terminal DNA sequence of the mutant OmpA shared 81.05% homology with the modeled OmpA from E. coli K12 and the N-terminal amino acid sequence of the mutant OmpA was 81.22% identical to that of the E. coli K12 OmpA. Moreover, several amino acids located in the β-barrel region were mutated. The mutant OmpA was expressed in BL21 suggested by SDS-PAGE. Resistance to environmental stress assay indicated that the N-terminus mutant OmpA still possessed excellent activities in pH, temperature and osmotic pressure resistance. Our pre- sent study may supply insights into better and deeper understand the relationships between OmpA N-terminal regions and its functions in environmental stress conditions and the mechanisms on antibiotic resistance of E. coli.

Expression of Core Domain of Porcine Zona Pellucida 3β in E.coli

To obtain the recombinant core domain of porcine zone pellucida 3β （cZP3β） for the further research on its functions Methods The nucleotide sequence region from 44 to 306 codons of pZP3β entire eDNA was obtained by PCR and then was cloned into pET-3c vector. After being identified, recon was transformed into E.coli BL21 （DE3） pLysS and then induced by IPTG. Results The recombinant cZP3β was expressed in E. coli up to 15% of total cellular proteins, and was made sure by Western blot analysis. Conclusion The research on expression of core domain of pZP3β could benefit to further investigation of its immunogenicity and the development of antigen preparation.

Detection of new antibiotic resistance gene profile in Escherichia coliassociated with avian leukosis virus infection from broiler chickens

The Escherichia coli(E.coli)is prevailing worldwide,but the epidemiology of E.coli infections feature regional distribution characteristics to some extent.E.coli,as a zoonotic pathogen,can be transferred from animals to humans through food chain or via contact with wounds,causing a public health risk.We reported the swelling of proventriculus and tracheal bleeding following the death in two broiler chickens(Gallus gallus domesticus)from Beijing,China.To investigate whether a virus was involved in the infection,Madin Darby Bovine Kidney(MDCK)cells were co-cultured with supernatants of proventriculus,trachea and spleen homogenates.The avian leucosis virus was detected in the samples of proventriculus and trachea,but the avian influenza virus,the Newcastle disease virus and the avian infectious laryngotracheitis virus were not detected.E.coli isolates were resistant to almost all the antimicrobial as tested except for the combinations of amoxicillin/clavulanic acid and sulfamethoxazole/trimethoprim.PCR tests demonstrated the presence of antibiotic resistance genes in these E.coli isolates and further research revealed a novel gene profile with the presence of CTX-M-1,gyrA,gyrB,oqxA,oqxB,parC and Sul2 antibiotic resistance genes in a strain isolated from a proventriculus sample.These results demonstrated that the presence of antibiotic resistant E.coli would not necessarily cause outbreak of large-scale disease.However,when the bacteria carrying new antibiotic resistance genes enter the environment,it may result in the development of more virulent strains which will potentially impact human and animal health.

<i>Escherichia coli</i>Harbouring Resistance Genes, Virulence Genes and Integron 1 Isolated from Athi River in Kenya

Rivers can act as reservoirs of highly resistant strains and facilitate the dissemination of resistance, virulence and integron 1 genes. A cross-sectional study was carried out where 318 water samples were collected (53 from each site) and from the samples, 318 E. coli isolates were analysed for resistance genes, virulence genes and integron 1 using Polymerase Chain Reaction. 22% of the isolates had blaTEM, 33% had blaCTX-M and 28% had blaCMY. Prevalence of typical Enteropathogenic E. coli strains (carrying both eae and bfp genes) was 5% while the prevalence of atypical Enteropathogenic E. coli (carying only eae) was 1.8%. The prevalence of Enteroaggregative E. coli carrying the aggr genes was 11%. The prevalence of Enterotoxigenic E. coli encoding only lt toxin was 16 (5%) and while those carrying only st toxin was 6.9%. The prevalence of Enteroinvasive E. coli strains encoding as IpaH was 5% while that of strains, adherent invasive E. coli, carrying adherent invasive gene inv was 8.7%. 36% isolates were positive for class 1 integrons which were mostly isolated near the sewage effluent from waste treatment plant. Anthropogenic activities and close proximity to sewage treatment plant were found to play a key role in pollution of water body and accumulation of resistance and virulence genes. These results suggest that waste treatment plant may act as reservoir of resistance, virulence and integron 1 genes and is a potential risk to human and animal health in the region.

Presence of Virulence-Associated Genes and Ability to Form Biofilm among Clinical Isolates of <i>Escherichia coli</i>Causing Urinary Infection in Domestic Animals

Background: Urinary tract infection caused by Escherichia coli is a frequently observed condition both in humans and animals. Uropathogenic E. coli (UPEC) has been shown to have a pathogenicity island that enables them to infect the urinary tract. Because there is little information about the presence of UPEC-associated virulent genes in animal isolates this work was carried out with the intent to enhance the understanding about the strains of E.coli that cause infections in animals. Results: We screened 21 E. coli strains isolated causing urinary tract infection in domestic animals. Primers were designed to amplify urinary infection-associated genes. Nine genes, papA, tcpC, fyuA, tpbA, Lma, hylA, picU, tonB, and flicC were then amplified and sequenced. Different from the human isolate CFT073, all the animals E. coli lack some of the pathogenesis-associated genes. Genes encoding for proteins used to scavenge iron appear not to be so necessary during animal infections as they are in human infection. In further investigation of phenotypic properties, it was observed that animal UPECs have significantly more impaired ability to form biofilms than human UPEC strain. Conclusions: This study identified significant differences between human and animal UPECs. This may have its roots in the fact that it is difficult to determine if an animal has symptoms. Future studies will focus on some of the observations.