放线菌噬菌体重组酶Sre在大肠杆菌内高效介导位点特异性重组

目的证明放线菌噬菌体重组酶Sre在大肠杆菌细胞内介导高效的位点特异性重组。方法在大肠杆菌内构建分子内重组分析系统。质粒pBZP含有方向相同的attB和attP位点，分别位于LacZ基因的两侧。将pBZP电击导入含有sre基因的大肠杆菌细胞。结果重组的发生导致LacZ基因的切除，使含有X—Gal成分的培养基上细菌菌落从蓝色变成白色。整合后DNA经酶切、PCR和DNA测序证实。发生100％重组率的attB和attP最小片段分别是50bp和47bp。结论在大肠杆菌宿主环境中放线菌噬菌体重组酶sre高效催化位点特异性重组。本分子内重组分析系统是一个简便而有效的方法。     

噬菌体展示系统的研究进展

噬菌体展示是一种用于筛选和改造功能性多肽/蛋白质强有力的生物技术,广泛应用在蛋白组学,未知基因的克隆和测序等多个分子生物学领域。展示技术使用的噬菌体系统有丝状噬菌体展示系统、λ噬菌体展示系统、T4噬菌体展示系统和T7噬菌体展示系统。丝状噬菌体pⅢ系统单价展示,可以筛选高亲和力配体,pⅧ系统拷贝数高,利于疫苗的研制,但丝状噬菌体分泌释放,不易展示大分子蛋白;λ噬菌体和T4噬菌体系统容量大,拷贝数高,但不易筛选高亲和力配体;T7噬菌体系统可以高、中、低拷贝展示不同分子量的蛋白,是目前最为理想的展示系统。     

Daudi细胞系特异性Fab噬菌体抗体库的构建、筛选及鉴定

本研究以人B细胞淋巴瘤Daudi细胞免疫的小鼠脾淋巴细胞RNA出发，用基因克隆技术将B细胞全套k轻链和重链Fd片段基因克隆出来，组装到表达载体pComb3H—SS内并表达到噬菌体表面，构建出Daudi细胞系特异性Fab噬菌体抗体库。以Daudi细胞为抗原对抗体库筛选后，获得针对B细胞淋巴瘤Daudi细胞系膜抗原的抗体。     

HAV噬菌体抗体特异性的鉴定

ＨＡＶ噬菌体抗体特异性的鉴定万泽生王海涛姜绍谆近年出现的噬菌体抗体库技术，使人们能够不经过细胞融合，采用基因工程的方法，从人外周血淋巴细胞ＩｇｍＲＮＡ直接制备抗体。我们利用这一技术，已成功地构建了噬菌体抗体库〔１〕。本文报道从库中筛选具有抗甲肝病毒（...     

人LIGHT胞外段基因的克隆及在大肠杆菌中的表达

目的:构建人LIGHT胞外段基因的原核表达载体,并在大肠杆菌中诱导表达.方法:从人外周血单核细胞来源的未成熟树突状细胞中提取总RNA,通过RT-PCR得到LIGHT胞外段基因,并将其克隆至pET32a(+)原核表达载体中,经双酶切鉴定及序列测定后的重组质粒转化入E.coli BL21,IPTG诱导表达目的蛋白,并用SDS-PAGE和Western blot进行检测.结果:RT-PCR扩增出了大小为543 bp 的LIGHT胞外段基因,经测序证明序列正确.SDS-PAGE和Western blot分析证实重组质粒可表达出Mr约为41 000的蛋白.结论:成功地克隆了人LIGHT胞外段基因并在大肠杆菌中进行了表达为进一步研究LIGHT的功能打下了基础.     

大容量噬菌体抗体库的构建及鉴定

目的　构建大容量噬菌体单链抗体库 ,从中筛选人源单链抗体 (ScFv)。方法　从正常成人外周血和新生儿脐血分离淋巴细胞 ,用RT PCR扩增轻链可变区基因 (VL)和重链可变区基因(VH) ,通过重叠PCR法将VH 和VL 拼接形成ScFv基因 ,并克隆入噬菌体表达载体PDF ,得到ScFv初级噬菌体抗体库。以高MOI超感染cre 菌株BS136 5 ,通过loxp cre定位重组系统 ,介导轻重链的组合配对 ,得到大容量抗体库 ,用多种抗原对抗体库进行生物淘筛 ,鉴定抗体库的性能。结果　获得了 6×10 10 的大容量单链噬菌体抗体库。分别用卵清蛋白、胃蛋白酶、铁蛋白、人角蛋白、人TNF α、地高辛等6种抗原进行筛选 ,均得到多样性的特异性噬菌体抗体。结论　经loxp cre定位重组系统在单细胞内重组成功地构建了大容量单链噬菌体抗体库 ,初步尝试对 6种抗原进行筛选均获成功 ,提示该抗体库可用于制备具有应用前景的人源抗体     

人CD137胞外区噬菌体展示系统的构建及鉴定

目的：以噬菌体展示具有天然结构的人CD137分子胞外区，检测其抗原性和生物活性，为以CD137为靶点体外筛选CD137拮抗性类肽药物奠定基础。方法：PCR方法从CIS质粒扩增人CD137胞外区，将其克隆人噬菌粒载体pComb3HSS，构建的重组载体电转入受体菌XLl-Blue，并加辅助噬菌体VCSM13共感染，使CD137胞外区展示在噬菌体表面。采用ELISA法检测噬菌体展示CD137的抗原性，MTT法检测CD137噬菌体对抗CD137抗体刺激的人外周血淋巴细胞增殖作用的影响检测其生物活性。结果：成功构建表达人CD137分子胞外区的重组噬菌粒载体pComb3H-CD137，并以噬菌体展示系统展示CD137分子胞外区。ELISA结果显示噬菌体展示CD137可与抗人CD137抗体特异性结合，证实CD137分子成功展示于噬菌体表面，且具有抗原性。体外生物活性实验显示，展示在噬菌体表面的CD137可抑制抗CD137抗体刺激的人外周血淋巴细胞增殖反应，证实噬菌体展示的CD137有与其配体结合的生物活性结构域。结论：利用噬菌体展示系统成功展示CD137胞外区，噬菌体展示CD137具有抗原性及生物活性。     

铜绿假单胞菌噬菌体的分离鉴定及其控制生物被膜的应用研究

目的 分离鉴定铜绿假单胞菌烈性噬菌体,研究噬菌体控制宿主菌形成牛物被膜的效率.方法 以铜绿假单胞菌临床菌株为指示菌,从不同环境样品中分离噬菌体;采用限制性内切酶图谱分析和宿主范围测定方法,对分离的噬菌体进行分类;利用透射电子显微镜对分离噬菌体进行形态学研究;以TJC729为指示菌,开展噬菌体控制牛物被膜形成的应用研究.结果 分别以14株铜绿假单胞菌临床分离菌株为指示菌,共分离得到13株烈性噬菌体,命名为C1～C13.利用限制性内切酶EcoR Ⅰ分析结果表明,13株噬菌体基因组均为双链DNA,并可被分成8组;宿主谱测定结果显示,c1和C13、C6和C7、C9和C11分别具有相同的宿主范围,其余7株噬菌体的宿主范围各不相同.随机挑选噬菌体C1进行形态学研究,发现噬菌体C1头部具有二十面体结构,尾部较长且无收缩性尾鞘,属于长尾噬菌体科.生物被膜控制实验结果显示,混合噬菌体能够较好地抑制TJC729生物被膜的形成.进一步实验结果显示,噬菌体C1、C10和C12分别与牛物被膜混合培养24 h后,牛物被膜的量分别下降到初始量的32.7%、57.6%、32.8%.结论 分离了13株铜绿假单胞菌烈性噬菌体,它们能够显著抑制宿主菌生物被膜的形成,并对生物被膜造成一定程度的破坏,为控制铜绿假单胞菌引起的感染提供了一个新方法.

Abstract：

Objective To isolate and classigy the bacteriophages specific to Pseudomonas aetuginosa and to investigate biofilm control efficaey of the isolated virulent phages.Methods With P. aeruginosa clinical strains as indicators.bacteriophages were isolated by screening difierent environmental samples.Classification of the isolated phages was done with the methods of restriction fragment analysis of phage genome and host range analysis.Transmission electron microscopy(TEM)was used in phage morphology study.In biogilm control tests,TJC729 was used as the jndicator strain to study the biofilm control efficacy of the isolated phages.Results Total 13 lytic phages specific to P.aeruginosa strains were isolated and named as C1-C13.According to the result of restriction fragment analysis.all 13 phages were double-stranded DNA viruses and could be divided into eight groups.Host range experiments were conducted with 5 laboratory strains and 12 clinical strains of P. aeruginosa.The same infection profiles were observed among phage C1 and C13,C6 and C7,and C9 and C11,respectively.While the remaining 7 phages each had different unique infection profile.Phage C1 was selected randomly to study its morphology.The obtained images showed that phage C1 had an icosahedral head with a non-contractile tail,belonging to the Siphoviridae family.Compared with the single phage,phage cocktail had the best effect on biofilm control.Further experiment results showed that phage C1.C10 and C12 can destroy biofilm after treatment of the biofilm for 24 h.The biofilm amounts were deceased to 32.7%,57.6%and 32.8%of the initial values,respectively.Conclusion Thirteen virulent phages specific to P. aeruginosa had been isolated.The phages could significantly inhibit the biofilm formation and had a certain degree of damage on the biofilm.The results suggested an alternative method for the treatments of P.aeruginosa infections.     

人亲环素A基因的克隆及在大肠杆菌中的表达

人亲环素Ａ基因的克隆及在大肠杆菌中的表达李芳秋赵权武建国单祥年环孢素Ａ（ＣｓＡ）是防治器官移植免疫排斥及自身免疫病的重要药物，该药发挥作用必须由体内受体蛋白，即亲环素（ＣｙＰ）来介导。ＣｙＰ是一个功能相关的蛋白家族，具有肽基脯氨酸顺／反异构酶（ＰＰＩ...     

人源性天然噬菌体展示文库的构建及鉴定

目的:构建人源性天然噬菌体展示文库并对文库进行鉴定。方法:从未经主动免疫健康志愿者的外周血淋巴细胞中提取总RNA,反转录合成cDNA,用直接PCR及半巢式PCR扩增人抗体重链(VH)及轻链(Vκ和Vλ)可变区基因。采用改进的重叠延伸PCR法将VH基因和VL(Vκ和Vλ)基因连接成人单链抗体(scFv)基因,并将scFv文库基因克隆到噬菌体载体pCANTAB-5E中,电转化E.coliTG1,构建单链抗体文库。结果:采用直接PCR和半巢式PCR扩增得到42条VH基因片段,16条Vκ基因片段,18条Vλ基因片段。混合的VH和VL等摩尔比连接后,产生的单链抗体文库基因大小为750bp左右;转化后构建了文库容量为1.35×108的单链抗体文库。BstNⅠ酶切分析文库中的单链抗体基因结果显示,酶切得到的scFv指纹图谱均不相同。结论:构建的抗体文库多样性好,可用于多种人源性单链抗体的筛选。     

人PD-L2基因克隆及其在大肠杆菌中的表达

目的 克隆人PD-L2基因并构建PD-L2胞外区的原核表达载体，在大肠杆菌中进行表达。方法 以RT-PCR方法从活化的人外周血单个核细胞总RNA中克隆PD-L2基因的cDNA，构建PD-L2胞外区的原核表达载体，在大肠杆菌BL21(ED3)中进行表达并鉴定。结果 克隆到PD-L2基因cDNA编码区全长序列，经DNA测序证明其与已报道的序列一致。进而构建了PD-L2胞外区的原核表达载体，并在大肠杆菌表达，免疫印迹分析表明在IPTG诱导后表达PD-L2胞外区蛋白，相对分子质量Mr为22000，与理论值大小相符。结论 成功克隆PD-L2基因，其胞外区蛋白在大肠杆菌中获得表达，为进一步研究PD-L2功能提供了条件。     

Subtyping of pathogenic Escherichia coli strains using flagellar (H)-antigens: serotyping versus fliC polymorphisms

Serotyping of O- and H-antigens is regarded as the gold standard in classification of E. coli for taxonomic and epidemiological purposes similar to the Kaufmann-White scheme for Salmonella enterica. Molecular methods to replace or to support the serotyping have been applied recently. Using the molecular polymorphism of the flagella (H-antigen) gene fliC, more than 220 E. coli strains derived from the E. coli reference collection for O- and H-antigens (The International Escherichia and Klebsiella Centre (WHO)) and from clinical origins have been characterised and a reproducible and clear cut classification with very good correlation to serotyping was found. Only some of the H-antigens have revealed multiple fliC classes and vice versa only rarely some of the fliC classes belong to various H-antigen groups. Since also H-antigen-negative and H-antigen non-typeable strains subjected to fliC classification could be typed properly, it is recommended here to use this rapid approach to classify E. coli under routine conditions rather than using classical serotyping. However, scrotyping--in particular using hyperimmune rabbit sera--will remain the gold standard and the task of Reference Centres only, e.g. for defining novel H-antigen types.     

抗-D抗体Fab段基因的克隆及其在大肠杆菌中的表达

目的：于大肠杆菌表达抗-D抗体Fab段基因，为真核载体表达完整的人抗-D抗体奠定基础。 方法： 将已克隆测序的Fab段基因亚克隆到pComb3噬粒载体，用PCR和限制性内切酶酶切鉴定后于大肠杆菌进行表达，表达产物用SDS-PAGE和ELISA等方法分析。 结果： SDS-PAGE电泳表明重组克隆表达了1条约48 kD的蛋白条带, ELISA结果显示，所表达的抗体和Rh+的人红细胞呈阳性反应，和Rh-红细胞反应为阴性，阴性对照和Rh+及Rh-红细胞反应均为阴性。 结论： 于大肠杆菌表达了具有抗原结合特性的人抗-D抗体Fab段抗体分子。     

Flooding adds pathogenic Escherichia coli strains to the water sources in southern Khyber Pakhtunkhwa,Pakistan

Purpose: Seasonal rains in Pakistan result in heavy floods across the country, whereby faecal contaminants will be added to the water bodies and cause numerous food-borne outbreaks. The present study was aimed to determine the prevalence of diarrheagenic Escherichia coli (DEC) strains in the water sources. Materials and Methods: Two hundred water samples collected during (2011–2012) were processed for the isolation of E. coli (EC) strains. EC strains were further analysed for antibiotic susceptibility patterns, and pathogroups-specific virulence factors stx1, stx2, stx2c, eae, tir, hlyA, bfpA, estA and eltA were detected using multiplex polymerase chain reaction. Results: Thirty-three percent of the water samples were contaminated with EC pathotypes. Fifty percent (33/66) of the DEC pathotypes were identified as enterotoxigenic EC (ETEC). Seventy-two percent (13/18) of the enteropathogenic EC (EPEC) strains were identified as typical EPEC and 28% (5/18) as atypical EPEC. Eleven percent (7/66) of the Shiga toxin EC (STEC) isolates carried a combination of stx1 and stx2 genes. Summer was found as a peak season with 47% (31/66) for EC pathogroups’ activities. Eighty-nine percent of the strains showed resistance against tetracycline. Conclusion: ETEC and EPEC are the primary causes of water contamination in southern regions of Khyber Pakhtunkhwa province, Pakistan. Firm adherence to the prescribed drugs can decrease trends in antibiotic resistance.     

大肠杆菌中的小RNA调节子

小RNA(smallRNAs或sRNAs)是一类长度在40至400个核苷酸、广泛存在于从细菌到哺乳动物多种不同的生物体内、执行多种生物学功能的非编码的RNA分子。对于sRNA分子的研究过去主要集中在哺乳动物。近年来,随着生物信息学和分子生物学技术的不断发展,在原核生物体内也陆续发现了一些sRNA分子,这些sRNA分子具有多样的生物学功能。大肠杆菌作为原核生物的代表在其体内已发现了近70种的sRNA分子。同真核细胞中的sRNA一样,细菌中的sRNA绝大多数也是通过与靶mRNA配对来影响mRNA的稳定性或翻译,从而对基因的表达进行调节。Hfq蛋白作为RNA的分子伴侣对于这些RNA分子的正确折叠和行使功能具有重要的作用。     

尿路致病性大肠埃希菌FYUA基因敲除株的构建及其生物学特性

目的构建尿路致病性大肠埃希菌FYUA基因敲除株,比较野生株和敲除株之间生物学特性的改变。方法利用λRed同源重组系统构建FYUA基因敲除株△FYUA;测定A600绘制CFT073和△FYUA在LB液体培养基和无菌尿液中的增殖曲线;平板菌落计数法比较CFT073和△FYUA菌落形成能力;利用96孔板结晶紫染色法比较CFT073和△FYUA生物膜形成能力。结果成功构建CFT073 FYUA基因敲除株△FYUA;CFT073和△FYUA在LB液体培养基中具有相似的增殖曲线,△FYUA在无菌尿液中的增殖速率明显低于CFT073(P0.05);CFT073和△FYUA菌落形成能力无明显区别;△FYUA的生物膜形成能力低于CFT073(P0.05)。结论 FYUA基因对于菌株在无菌尿液中的生长繁殖以及生物膜形成可能发挥重要作用。     

Incidence of extended spectrum beta lactamase producing Escherichia coli among patients,healthy individuals and in the environment

We investigated the faecal carriage of extended spectrum β-lactamases (ESBL) producing Escherichia coli in different groups of human subjects and in the environment. A total of 363 E. coli strains were isolated from stool samples of patients (n = 77), healthy subjects (n = 170) and from different environmental samples (n = 116). A total of 124 ESBL producing E. coli strains were isolated in this study. The frequency of ESBL producing E. coli was found to be highest (60.3%) among the strains isolated from patients, followed by healthy individuals (38%) and the environment (10.5%). The environment was observed to have a very low number of ESBL producing E. coli.     

Prevalence of the new,SPI1-like,pathogenicity island ETT2 among Escherichia coli

The new pathogenicity island ETT2 has been identified by PCR and gene probes among various intestinal serovars and pathovars of E. coli, in particular among EHEC/STEC. However, ETT2 was not detected among extra-intestinal and non-pathogenic E. coli strains or other enteric bacteria including various S. enterica serovars. A considerable molecular diversity of ETT2 among various E. coli serovars was found. The occurrence of ETT2 among E. coli is independent of the presence of other virulence properties, e.g. the pathogenicity islands LEE, LPA, or HPI.     

Diarrhoeagenic Escherichia coli isolated from children with acute diarrhoea at Rakai hospital,Southern Uganda

BackgroundDiarrhoeagenic Escherichia coli (DEC) is a leading cause of childhood diarrhoea. This study estimated the prevalence of DEC and DEC pathotypes among children with acute diarrhoea in Southern Uganda.MethodsA cross-sectional study was conducted on 267 children less than 5 years with acute diarrhoea, admitted to Rakai General Hospital in Southern Uganda. Faecal samples were collected from the children and processed for isolation of E. coli. The presence of DEC and the distribution of DEC pathotypes were determined by polymerase chain reaction.ResultsA total of 102 (38.2%, 102/267) children had DEC of various pathotypes – enteroaggregative E. coli (EAEC) (14.2%); enteropathogenic E. coli (EPEC) (6.7%); enterotoxigenic E. coli (ETEC) (6%); enteroinvasive E. coli (EIEC) (7.5%); enterohemorrhagic E. coli (EHEC) (3%); and cell-detaching E. coli (CDEC) (0.75%). The difference in the overall prevalence of DEC was not significant regarding HIV but individually, EAEC and CDEC were associated with HIV-positive status while ETEC was associated with HIV-negative status.ConclusionsDEC is prevalent in children with acute diarrhoea in Southern Uganda and its identification in children should be considered among strategies for combatting childhood diarrhoea in Africa.     

致泻性大肠杆菌疫苗研究进展

大肠杆菌引起的腹泻多发生在儿童及发展中国家,发展致泻性大肠杆菌疫苗具有特殊的意义。除了灭活疫苗、减毒疫苗、结合多糖疫苗和亚单位疫苗外,新近出现的DNA疫苗、转基因植物疫苗和ghost疫苗技术为疫苗的研制提供了新的思路。本文综述了目前致泻性大肠杆菌疫苗研究的新进展。