第1类整合子中aadA2基因翻译起始位点的确定

目的 确定第1类整合子中aadA2基因能否从上游无核糖体结合位点的密码子ATG起始翻译并合成有功能的蛋白.方法 定点突变含有不同翻译起始密码子的aadA2基因盒,并连同上游的可变区启动子分别克隆入质粒pACYC184中,转化大肠埃希菌JM109,免疫印迹检测含有不同翻译起始密码子的aadA2基因的翻译产物,用微量肉汤稀释法检测链霉素对含有不同翻译起始密码子aadA2基因的大肠埃希菌JM109的最小抑菌浓度.结果 aadA2基因可从上游无核糖体结合位点的密码子ATG及上游具有核糖体结合位点的密码子GTG起始翻译,同时在GTG密码子下游还存在着起始翻译密码子,其翻译产物在免疫印迹中均可与抗氨基糖苷-3″-腺苷酰基转移酶多克隆抗体产生特异性杂交条带,并赋予宿主细菌对链霉素不同水平的耐药.结论 当位于第1类整合子第1位基因盒中,aadA2基因可从上游无核糖体结合位点的密码子ATG起始翻译并合成有功能的蛋白,这一结构特点使得整合入第1类整合子的基因盒,不需带有核糖体结合位点即可起始基因盒中相应读码框的翻译,从而有利于第1类整合子表达从外界环境中捕获的基因.     

银离子对细菌整合子捕获耐药性基因盒频率的抑制作用

目的:讨论银离子对大肠埃希菌BL21(DE3)宿主中第一类整合子整合频率的影响。方法:将pUCINT及pACINAD两种重组质粒依次转化大肠埃希菌BL21(DE3),命名为HS2。实验组分别使用含0.3 μg/ml、0.6 μg/ml和0.8 μg/ml银离子LB液体培养基,对照组使用普通LB液体培养基,银离子通过硝酸...     

产AmpC酶大肠埃希菌和肺炎克雷伯菌中整合子与多重耐药的研究

目的探讨整合子介导的耐药机制在产AmpC酶大肠埃希菌和肺炎克雷伯菌多重耐药中的作用.方法5株产AmpC酶大肠埃希菌和肺炎克雷伯菌分离自2002年1月-2004年5月间我院呼吸科住院的患者,采用E-test试验条进行药敏试验、电转化试验,筛选、分离耐药质粒.PCR扩增Ⅰ型整合子基因盒插入序列,分子克隆和序列分析.结果所有产酶菌株通过电转化试验可将头孢西丁耐药性传递给受体菌,5个产AmpC酶耐药质粒中,有4个检出整合酶序列,其中3个携带2种抗药性基因盒,包括氨基糖苷乙酰转移酶基因aacA4;氨基糖苷腺苷转移酶基因aadA5;二氢叶酸还原酶基因dfrA17;氯霉素外排蛋白编码基因cmL44.结论整合子介导的抗药性基因盒参与了产质粒AmpC酶大肠埃希菌和肺炎克雷伯菌多重耐药的形成,应引起高度重视.     

基于“乳头状试验”检测整合酶介导基因盒剪切频率方法的建立

整合子可通过位点特异性重组捕获外源基因盒并使之表达,同时整合子可位于质粒上,或自身作为转座子的一个组成部分而参与转移,使耐药基因发生播散[1-2].整合子因其在细菌耐药性基因水平传播中的重要作用而受到研究者的关注,然而到目前为止,整合酶催化的基因盒剪切与整合的具体机制仍有待阐明.     

沙门菌中第一类整合子的鉴定及特性分析

目的　基于整合子在细菌耐药机制中的重要作用 ,对来自正常人体内耐药沙门菌的整合子分布及其结构特征进行分析。方法　应用PCR方法 ,设计第一类整合酶基因intI1和耐药基因盒的特异性引物 ,用PCR方法检测整合子阳性菌株并对其整合的耐药基因进行测序和序列分析。结果　发现 1株S .hadar和 3株S .tshiongwe为第一类整合酶阳性菌株。耐药基因盒进行扩增的结果 ,从 2株中分别得到 10 0 9bp的扩增产物。 1株得到 16 6 4bp的扩增产物。 1株菌得到 10 0 9bp和 16 6 4bp的扩增产物。序列分析结果表明 ,10 0 9bp的扩增产物为携带aadA2 ,对氨基糖苷类抗生素药物壮观霉素、链霉素产生耐药的基因盒 ;16 6 4bp为携带aadA5和dfr17,对氨基糖苷类抗生素药物壮观霉素、链霉素和磺胺类药物甲氧氨苄嘧啶产生耐药的基因盒。结论　首次揭示了健康人携带由整合子介导的耐药菌这一现象 ,提示我们要从基因水平上监测细菌的耐药情况     

人DC-SIGN全长编码区基因的克隆及其胞外段的原核表达

目的:克隆人DC-SIGN全长编码区基因, 获得其胞外段的原核表达产物.方法:采用RT-PCR方法, 从健康产妇胎盘中克隆DC-SIGN全长cDNA, 扩增其胞外段基因并构建pET41a-sDC-SIGN重组表达质粒, 在大肠杆菌BL21(DE3)中表达, 以SDS-PAGE和Western blot鉴定表达产物.结果:从健康产妇胎盘总RNA中, 扩增获得约1 300 bp的DNA片段, 克隆至pGM-T载体获得重组质粒pGM-DC-SIGN.从pGM-DC-SIGN扩增DC-SIGN的胞外段基因, 构建重组表达质粒pET- 41a-sDC-SIGN;纯化表达产物sDC-SIGN-GST, 鉴定其相对分子质量( M r)为66 000, Western blot证明其可与抗DC-SIGN抗体特异性结合.结论:成功克隆DC-SIGN全长编码区基因, 并在大肠杆菌中成功表达其胞外段融合蛋白sDC-SIGN-GST, 为进一步研究DC-SIGN的功能奠定了基础.     

抗HIV-1 gp120单链抗体和白喉毒素融合基因的构建及其在大肠杆菌中的表达

目的 构建人源抗HIV-1单链抗体和白喉毒素融合基因的原核表达质粒,并诱导其在大肠杆菌中表达重组蛋白.方法通过PCR扩增,获得目前应用较多的DAB389基因片段,将其克隆入原核表达载体pET-28a,转化大肠杆菌BL21(DE3),IPTG诱导表达,经SDS-PAGE和Western blot方法分析鉴定.结果酶切及DNA序列测定鉴定正确.SDS-PAGE和Western Blot分析证实,重组质粒可表达出相对分子质量为75 200的蛋白,与DT-hs120融合基因子量一致,其表达量约占菌体总蛋白的21%,表达形式主要为包涵体.结论 成功构建了DT-hS120表达载体,并获得了高效表达.     

人胰岛素样生长因子在pET-DsbA载体中的克隆及表达

目的 通过重组技术,对胰岛素样生长因子IGF-Ⅰ基因进行缺失、突变以获得最有利于高水平表达的IGF-Ⅰ cDNA.构建原核表达载体,并进行表达,以获得能够高水平表达高活性产物的基因工程菌珠.方法 提取人肝脏组织总RNA,RT-PCR后琼脂糖电泳初步鉴定产物.将cDNA回收、补平后插入克隆质粒KS,酶切鉴定后,对IGF-Ⅰ基因进行序列分析.采用PCR法从克隆质粒中扩增IGF-Ⅰ片段,亚克隆至pET-DsbA原核表达载体,酶切鉴定,并进行序列测定.转化到大肠杆菌BL21(DE3)PLysS中,IPTG诱导表达,聚丙稀酰胺凝胶电泳、Western Blot分析目的蛋白.结果 成功构建原核表达载体pET-DsbA-IGF-Ⅰ,测序结果与预期序列完全一致,并在大肠杆菌BL21(DE3)plysS表达出IGF-Ⅰ融合蛋白.结论 IGF-Ⅰ融合蛋白在大肠杆菌BL21(DE3) PLysS中的表达,对进一步研究IGF-Ⅰ的功能及开发其生物制品具有重要意义.     

粉尘螨Ⅰ类抗原cDNA的克隆表达和初步鉴定

目的　构建粉尘螨Ⅰ类抗原 (Derf1)cDNA基因的重组表达质粒 ,并于E .coli表达。方法　用BamHⅠ和SacⅠ从重组质粒pMD 18T Derf 1上切下Derf1基因 ,插入表达载体pET32a( )质粒 ,转化大肠杆菌BL2 1,在氨苄青霉素阳性的LB平板上筛选阳性重组子 ,并经双酶切及PCR扩增鉴定。重组质粒pET32a( ) Derf 1转化大肠杆菌 ,IPTG诱导表达后进行SDS PAGE电泳和薄层凝胶扫描定量分析。结果　对重组质粒进行酶切和PCR鉴定 ,与预期结果相符 ,证明已成功构建携带Derf1基因的重组原核表达质粒pET32a( ) Derf 1。核酸序列测定及同源性分析证实所构建的原核表达质粒pET32a( ) Derf1中所含的Derf1基因与GenBank中的Derf1序列同源性达到 99.5 %。Derf 1基因在大肠杆菌诱导表达后获得Mr 约4 5 0 0 0的蛋白 ,蛋白含量占全菌体蛋白含量的 15 %。结论　成功构建了粉尘螨Ⅰ类抗原cDNA基因的重组表达质粒pET32a( ) Derf 1,并在大肠杆菌中获得高效表达 ,为获得重组纯化Derf 1变应原并用于尘螨变应性疾病的诊治奠定基础     

细粒棘球绦虫重组质粒pGEX-Eg95的构建及其在大肠杆菌BL21（DE3）中的表达

目的构建细粒棘球绦虫重组质粒pGEX-Eg95,并研究该质粒在大肠杆菌BL21（DE3）中的表达。方法超声粉碎细粒棘球蚴组织提取总RNA,通过RT—PCR扩增Eg95抗原编码基因;克隆至原核表达载体pGEX—1λT,构建重组质粒pGEX-Eg95;转化大肠杆菌BL21,经异丙基硫代-β—D-半乳糖苷（IPTG）诱导表达后用SDS-PAGE和Western blowing对表达产物进行分析和鉴定。结果RT-PCR扩增出471bp的Eg95抗原编码基因;双酶切证实Eg95抗原编码基因成功插入pGEX-1λT中;SDS-PAGE分析显示表达产物为相对分子质量约42500的重组蛋白,与预期结果一致,表达的蛋白约占菌体总蛋白的21％;Western blot鉴定显示重组蛋白能被细粒棘球蚴感染鼠血清识别。结论成功构建了细粒棘球绦虫重组质粒pGEX-Eg95,该质粒在大肠杆菌BL21中获得了高效融合表达,表达的融合蛋白具有特异的抗原性。     

Detection and typing of integrons in epidemic strains of Acinetobacter baumannii found in the United Kingdom

Integrons were sought in Acinetobacter isolates from hospitals in the United Kingdom by integrase gene PCR. Isolates were compared by pulsed-field gel electrophoresis, and most belonged to a small number of outbreak strains or clones of A. baumannii, which are highly successful in the United Kingdom. Class 1 integrons were found in all of the outbreak isolates but in none of the sporadic isolates. No class 2 integrons were found. Three integrons were identified among the main outbreak strains and clones. While a particular integron was usually associated with a strain or clone, some members carried a different integron. Some integrons were associated with more than one strain. The cassette arrays of two of the integrons were very similar, both containing gene aacC1, which confers resistance to gentamicin, two open reading frames coding for unknown products (orfX, orfX'), and gene aadA1a, which confers resistance to spectinomycin and streptomycin. The larger of these integrons had two copies of the first (orfX) of the gene cassettes coding for unknown products. The third integron, with a cassette array containing gene aacA4, which codes for amikacin, netilmicin, and tobramycin resistance; a chloramphenicol acetyltransferase, catB8; and gene aadA1, conferring resistance to spectinomycin and streptomycin, was associated with an OXA-23 carbapenemase-producing clone, which has spread rapidly in hospitals in the United Kingdom during 2003 and 2004. These integron cassette arrays have been found in other outbreak strains of A. baumannii from other countries. We conclude that integrons are useful markers for epidemic strains of A. baumannii and that integron typing provides valuable information for epidemiological studies.     

Detection and characterization of class 1 integrons in enterohemorrhagic Escherichia coli

Enterohemorrhagic Escherichia coli (EHEC) strains isolated from humans, cattle, and food and belonging to serogroups O26 (7 strains), O111 (19 strains), and O157 (70 strains) were examined for susceptibility to 11 antimicrobial drugs. Fifty-nine strains showing resistance to at least one of the drugs were examined by PCR for the presence of class 1 integrons, which were identified in 17 strains. Integrons were found more frequently in strains belonging to serogroups O111 and O26 than in the O157 isolates. DNA sequence analysis demonstrated that most of the integrons contained the aadA1 gene cassette conferring resistance to streptomycin/ spectinomycin, alone or associated with the drfA1 gene cassette conferring resistance to trimethoprim. One integron, identified in a O157:H7 strain, carried the aadA2 and dfrA12 gene cassettes, conferring resistance to streptomycin/spectinomycin and trimethoprim, and the open reading frame F (OrfF) encoding unknown functions. Most of the integrons were carried by Tn21 derivative transposons and were transferable by conjugation to an E. coli K-12 strain. In conclusion, integrons and antibiotic resistance genes can be frequently found in EHEC strains, particularly E. coli O111 and E. coli O26, and their presence could complicate therapeutic trials.     

纳豆激酶酶原基因的克隆、融合表达及活性测定

目的:利用基因工程技术, 构建表达具溶栓活性的纳豆激酶的大肠杆菌工程菌。方法:利用PCR方法扩增纳豆激酶酶原(pro-NK)基因, 并克隆到表达载体pET3c上, 构建表达pro-NK和载体上22氨基酸短肽的融合蛋白的表达质粒pENK;表达质粒pENK分别转化溶源化宿主菌BL21(DE3)pLysS^-和BL21(DE3)pLysS⁺, 获得表达菌pENK-(DE3)pLysS^-和pENK-(DE3)pLysS⁺。利用SDS-PAGE和纤维蛋白平板法检测目的蛋白的表达及活性。结果:SDS-PAGE显示两株菌株均表达42kD的目的蛋白。纤维蛋白平板法显示表达产物具溶栓的活性。在pENK-(DE3)pLysS^-中融合蛋白不需异丙基硫代-β-D-半乳糖苷(IPTG)诱导就有基础表达, 融合蛋白的表达使表达菌细胞溶解, 菌落中空, 表明表达产物具细胞毒作用。结论:本研究成功实现了在大肠杆菌表达具有溶栓活性的pro-NK融合蛋白, 为开发纳豆激酶成为新一代溶栓药物的研究奠定基础。     

Association of the blaCMY-10 gene with a novel complex class 1 integron carrying an ISCR1 element in clinical isolates from Korea

The bla_CMY-10 gene responsible for β-lactam resistance was located on a new complex class 1 integron within a conjugative plasmid. The sul1-type class 1 integron, containing an aadA2a gene cassette, was identified upstream of bla_CMY-10. A unique gene array (yqgF–yqgE–gshB–orf97—orf105) was identified downstream of bla_CMY-10.     

嗜肺军团菌Ⅳ型菌毛蛋白在大肠杆菌中的表达及纯化

目的扩增编码Ⅳ型菌毛蛋白的嗜肺军团菌pilE基因,构建重组质粒pET32a（＋）-pilE并在原核系统中表达．纯化Ⅳ型菌毛蛋白PILE．为进一步探讨Ⅳ型菌毛蛋白的作用及其作为军团病的诊断抗原提供实验基础。方法采用聚合酶链式反应（PCR）从嗜肺军团菌扩增得到pilE基因,构建重组质粒pET32a（＋）-pilE,转化大肠杆菌BL21（DE3）并用聚合酶链式反应、限制性酶切分析、序列分析鉴定后,IPTG诱导表达PIIJE蛋白,用硫酸十二烷酸钠-聚丙烯酰胺凝胶电泳（SDS—PAGE）、Western印迹鉴定。使用HisTrap^TM HP亲和层析柱纯化Ⅳ型菌毛蛋白PILE。结果扩增出430bp的pilE基因;构建了重组质粒pET32a（＋）-pilE;表达并纯化出35700Mr的目标蛋白。结论成功构建了嗜肺军团菌pilE基因的原核重组质粒,并在原核系统中得到了高效表达。成功纯化Ⅳ型菌毛蛋白PILE。     

Integron-associated antibiotic resistance in enteroaggregative and enteroinvasive Escherichia coli

Ten enteroinvasive (EIEC) and 25 enteroaggregative (EaggEC) E. coli strains isolated from Senegalese patients were analyzed for their integron content. All strains were resistant to at least two antibiotics. Four EIEC and 15 EaggEC were found to carry a class 1 integron. An identical integron carrying a single dfrA5 cassette, conferring resistance to trimethoprim, was identified in all four EIEC strains. Five EaggEC strains harbored an integron with a single cassette, dfrA7, while the remaining 10 strains carried two integrons, one with a single cassette, aadA1a conferring resistance to streptomycin and spectinomycin, and the second one bearing two cassettes, dfrA13 and oxa5, the later being a beta-lactam resistance cassette. The presence of these integrons is worrying, because trimethoprim is largely used for diarrheal disease therapy in Africa. Thus, the presence of integrons in diarrheagenic strains is of public health importance because a limited number of antibiotics are available in developing countries.