sCR1在原核中表达、纯化、复性及活性鉴定

目的采用原核表达载体pET28a在E.coli BL21(DE3)中获得高表达、高活性的重组人sCR1融合蛋白。方法从人外周血中提取总RNA,应用RT-PCR获得人sCR1全长cDNA,将其克隆入原核表达载体pET28a中,构建含人sCR1的重组质粒(pET28a-sCR1),经测序鉴定正确,转化入E.coli BL21(DE3)中,经IPTG诱导,表达产物经SDS-PAGE分析和Western blot鉴定,通过Ni2+-NTA agarose亲和层析纯化后进行复性及生物学活性鉴定。结果获得原核表达载体pET28a-sCR1,经PCR鉴定及测序鉴定,得到重组E.coli BL21(DE3)克隆菌株,经IPTG诱导含有pET28a-sCR1的E.coli BL21(DE3)克隆菌,表达出重组人sCR1融合蛋白。此蛋白在SDS-PAGE上表现为Mr大于29000的蛋白区带,在Western blot分析中可被sCR1的CD35单克隆单抗体(mAb)识别。经Ni2+-NTA agarose亲和层析纯化、复性后得到高纯度的sCR1融合蛋白表达及较高的生物学活性。结论人sCR1融合蛋白在E.coli BL21(DE3)表达系统中的高水平表达,并且有与人体天然蛋白相同的抗原性及其生物学活性。     

AcAP5蛋白N端片段的表达、纯化与活性评价(英文)

犬钩虫抗凝肽5(Ancylostoma anticoagulant peptide 5,AcAP5)N端40个氨基酸片段(N40)含有与FXa结合的结构域。为研究N40的FXa抑制作用,我们克隆、表达和纯化了N40,并检测其生物活性。用PCR扩增N40基因;将PCR产物克隆至原核表达载体pET-30a中,构建质粒pET30a-N40;将其转化入大肠杆菌E.coli.BL21(DE3)中,IPTG诱导目的蛋白表达,SDS-PAGE电泳鉴定,经过镍柱亲和层析纯化,用BCA法进行蛋白定量,测定该蛋白抑制FXa的活性。限制性酶切鉴定和基因测序结果显示重组质粒构建成功;SDS-PAGE结果显示目的蛋白在E.coli.BL21(DE3)中为可溶性表达,亲和层析纯化后获得了纯度约90%的蛋白,经活性鉴定该蛋白确有抑制FXa的活性。     

儿茶酚胺氧位甲基转移酶的克隆表达及纯化

目的将儿茶酚胺氧位甲基转移酶基因(COMT)克隆到原核表达载体进行可溶性表达,制备纯化COMT蛋白,为深入研究COMT的功能提供材料。方法运用分子生物学方法构建融合表达质粒Pet22b+-COMT。将Pet22b+-COMT转入E.coli BL21(DE3)表达菌株,利用IPTG诱导表达,并用亲和色谱纯化COMT。结果经测序分析成功构建了融合表达质粒Pet22b+-COMT。COMT基因在E.coli BL21(DE3)中表达相对分子质量约为28000的蛋白质,纯化后,COMT蛋白的纯度大于90%。结论COMT基因在原核中得到良好的表达,得到了纯度较高的蛋白质,为酶学活性研究奠定了基础。     

脂多糖应激新分子融合蛋白的高表达及纯化

目的在原核表达系统中表达脂多糖应激新分子编码的蛋白质。方法PCR扩增HlrgcDNA编码区,克隆入表达载体pcTAT中,构建成融合6His的表达载体pcTAT-lrg,转化E.coli BL21(DE3),以IPTG诱导。表达产物用SDS-PAGE及凝胶光密度扫描分析,用Ni-NTA亲和层析柱纯化。结果经过IPTG诱导4 h,表达出相对分子质量(Mr)约25 000的蛋白,占菌体总蛋白的51%。表达产物为可溶性的,用Ni-NTA亲和层析柱纯化的表达蛋白达到电泳纯。加入培养液中的纯化蛋白,可在30 min内进入HEK293细胞。结论在E.coli中成功地高表达人Lrg融合蛋白,并对其进行初步纯化,为人Lrg功能的研究打下基础。     

胞苷磷酸激酶基因的克隆与原核表达

目的:克隆胞苷磷酸激酶(CMPK)基因,构建携带CMPK基因的原核表达载体,诱导表达具有活性的重组CMPK融合蛋白,获得转基因工程菌.方法:利用PCR法扩增大肠杆菌基因组DNA,纯化的PCR产物链接至pMD18-T载体上,得到重组质粒pMD18-T-CMPK,转化E-coli BL21(DE3)感受态细胞,酶切鉴定;分别用BamH I和XhoI限制性内切酶双酶切重组质粒pMD18-TCMPK和pET28a(+)表达载体.连接后,转入E.coli BL21(DE3)感受态细胞,酶切鉴定.用终浓度1 mmol/L的IPTG诱导一定时间(3、6h)后,取E.coli上清液做电泳分析.结果:所获CMPK基因全长为786 bp,编码含有262个氨基酸的蛋白,当A值为0.6～0.8时重组质粒经IPTG诱导3h后,相对分子质量约30000处出现目的蛋白条带,随着时间的延长,蛋白表达量增加不明显.结论:成功地克隆CMPK基因,表达了大肠杆菌BL21(DE3)重组CMPK融合蛋白,为胞苷磷酸激酶进一步开发和应用奠定基础.     

黑色素瘤抗原-3原核重组质粒的构建及表达

目的 构建MAGE-3目的 基因原核重组表达质粒pGEX-4T-1-MAGE-3并检测其在大肠杆茵(E.coli)B121(DE3)中的表达.方法 RT-PCR法制备MAGE-3目的 基因,连接入原核表达载体pGEX-4T-1.构建MAGE-3目的 基因的原核重组表达质粒pGEX-4T-1-MAGE-3并测序,将该质粒转化E.coli BL21(DE3)菌株,经IPTG诱导,12%SDS-PAGE和Western Blot表达鉴定.结果 扩增出349bp的MAGE-3 目的 基因并构建了原核重组表达栽体pGEX-4T-1-MAGE-3;测序结果与GenBank收录序列相一致;在E.coli BL21(DE3)中检测到含该重组表达载体的转化菌表达出分子量约35kD的融合蛋白并证实其为目的 蛋白.结论 成功构建的原核重组表达栽体pGEX-4T-1-MAGE-3及其所表达的融合蛋白,为以MAGE-3为基础的肽疫苗及特异诊断试剂的研制提供抗原打下了基础,为后续实验提供了依据.     

重组质粒Eg.EF-1/pGEX-6P-1的构建及原核诱导表达

将细粒棘球蚴（Echinococcus granulosus,Eg.）延伸因子（Elongation factor 1,EF-1）与pGEX-6P-1构建成重组质粒并进行表达、纯化及对其特性进行鉴定.将构建好的EF-1/pGEM-T经双酶切后获取目的基因片段,定向重组于表达载体pGEX-6P-1并转化大肠杆菌（Escherichia coli）E.coli BL21,IPTG诱导表达,用蛋白亲和层析柱GSTrap FF对表达产物进行纯化,特异性蛋白解离酶PreScission^TM Protease酶切融合蛋白从中获取重组Eg.EF-1;SDS-PAGE和Western blot方法鉴定表达产物.获得的Eg.EF-1/pGEX-6P-1/E.coli BL21菌株,诱导表达融合蛋白和纯化分离得到的31 ku Eg.EF-1均能被细粒棘球蚴天然抗原免疫的兔多克隆抗血清识别.证明成功构建出具有有效表达的Eg.EF-1/pGEX-6P-1菌株,初步证实重组蛋白具有较好的抗原性.     

内源性血管生成抑制因子Arresten的序列测定及在E.coli BL21中的表达

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,并对其进行序列测定和在E.coli BL21中进行表达。方法从健康产妇的胎盘组织中提取总RNA,经反转录-聚合酶链式反应(RT-PCR)扩增出Arresten基因,对其序列测定后构建重组质粒pBV220-Arr转化E.coli BL21进行原核表达。结果成功筛选出Arresten基因,并将基因序列和蛋白序列提交基因库,成功构建了重组质粒pBV220-Arr,重组质粒在菌株中获得表达。结论构建的原核表达重组体pBV220-Arr能高效表达重组Arresten蛋白。     

抗菌抗癌肽CM4N在大肠杆菌中的融合表达与活性鉴定

为探讨内含肽intein在抗菌肽表达与纯化中的应用,采用递归PCR法合成CM4末端添加Asn的基因CM4N,克隆至E.coli表达载体pTYB11中,构建了与intein的C端融合的表达载体pTYYB11-CM4N.重组质粒转化至E.coli BL21(DE3)中进行IPTG诱导表达,融合蛋白intein-CM4N主要以可溶形式存在于胞内.利用载体中intein中的chitin结合域,将融合蛋白通过chitin亲和层析一步纯化,经DTT诱导intein的自我切割活性,实现CM4N在亲和柱上的切割与分离,透析冻干后得到了纯度95%以上的重组CM4N.活性检测结果显示,重组CM4N具有抗大肠杆菌、沙门氏菌和K562肿瘤细胞的活性.研究认为,内含肽在抗菌肽的基因工程中可能具有重要的应用前景.     

犬尿氨酸-3-单加氧酶SibC的体外蛋白表达纯化与生化反应

目的通过体外基因合成西比罗霉素(sibiromycin)生物合成途径中推测但未验证功能的犬尿氨酸-3-单加氧酶编码基因sibC(GenBank:FJ768674.1:1380-2810),研究该犬尿氨酸-3-单加氧酶SibC的体外生化功能,为解析放线菌素D中相关结构单元的生物合成途径提供参考。方法通过生物信息学分析体外全合成基因sibC的序列并克隆至表达载体,将重组质粒pET28a-sibC转化至E.coli BL21(DE3)中进行表达,利用Ni-NTA亲和层析法纯化SibC,以犬尿氨酸(kynurenine,kyn,1)为底物进行SibC的体外酶活性测试,利用高效液相色谱(HPLC)对酶反应产物进行分析。结果PCR验证证明表达菌株E.coli BL21(DE3)/pET28a/sibC构建成功,SibC蛋白在E.coli BL21(DE3)中获得可溶性表达,经Ni-NTA亲和层析法纯化获得SibC蛋白,纯化的SibC能够催化犬尿氨酸生成3-羟基犬尿氨酸(3-hydroxykynurenine,3-HK,2)。结论体外验证了sibiromycin中SibC的功能为犬尿氨酸-3-羟化酶,为进一步研究犬尿氨酸途径及放线菌素的生物合成奠定了基础。     

Escherichia coli Subtilase Cytotoxin

Subtilase cytotoxin (SubAB) is the prototype of a new AB₅ toxin family produced by a subset of Shiga toxigenic Escherichia coli (STEC) strains. Its A subunit is a subtilase-like serine protease and cytotoxicity for eukaryotic cells is due to a highly specific, single-site cleavage of BiP/GRP78, an essential Hsp70 family chaperone located in the endoplasmic reticulum (ER). This cleavage triggers a severe and unresolved ER stress response, ultimately triggering apoptosis. The B subunit has specificity for glycans terminating in the sialic acid N-glycolylneuraminic acid. Although its actual role in human disease pathogenesis is yet to be established, SubAB is lethal for mice and induces pathological features overlapping those seen in the haemolytic uraemic syndrome, a life-threatening complication of STEC infection. The toxin is also proving to be a useful tool for probing the role of BiP and ER stress in a variety of cellular functions.     

Escherichia coli Shiga toxin

The Stx family contains two types called Stx1 (verotoxin 1: VT1 or Shiga-like toxin: SLT1) and Stx2 (VT2, SLT2); both toxins are encoded by bacteriophages. Stx1 is identical to Shiga toxin produced by Shigella dysenteriae type I. Stx2 is heterogeneous and immunologically different from Stx1. Although many variations are found in Stx family, all Stx has an A-B structure: the A subunit has N-glycosidase activity and the B subunit binds to a membrane glycolipid, globotriaosylceramide (Gb3). The A subunit cleaves a single adenine residue from the 28S rRNA component of eukaryotic ribosomes, resulting in inhibition of protein synthesis. Stx-producing Escherichia coli (STEC) is known to cause hemorrhagic enterocolitis and hemolytic-uremic syndrome (HUS). Stx plays a role in the occurrence of blood in the feces and in the HUS by their action on the endothelial cells of blood vessels in the intestinal submucosa and in the renal glomeruli. Epidemiologically, Stx2 seems to be more important than Stx1 in development of HUS. The action of Stx is not limited to inhibition of protein synthesis. Stx induces macrophages to express tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and interleukin-6 (IL-6) in vitro. These cytokines and lipopolysaccharide (LPS) are reported to increase the susceptibility of cells to Stx. A variety of cells such as tubular epithelial cells, may be targets for Stx-mediated apoptosis. Apoptosis is considered to contribute to the pathogenesis of HUS caused by STEC. In this review, recent progress in Stx-related research is summarized.     

药品在被少量大肠杆菌污染而造成漏检的有关问题的探讨

目的:探讨药品在被少量大肠杆菌污染后,按中国药典现行方法检验是否存在漏检可能,建立了在该种情况下可行的大肠杆菌检查的方法.方法:对8个品种人为污染少量大肠杆菌后,按中国药典检查.结果:药品在被少量大肠杆菌污染后,按药典法大肠杆菌阳性检出率为0～66%,改进法为100%.结论:药品在被少量大肠杆菌污染后,按中国药典现行方法检验存在漏检可能.     

Virulence factors of uropathogenic Escherichia coli

Virulence factors of Escherichia coli are of two main types; those produced on the surface of the cell and those produced within the cell and then exported to the site of action. Those on the surface include different sorts of fimbriae that have a role in adhesion to the surface of host cells but may also have additional roles such as tissue invasion, biofilm formation or cytokine induction. The activities of cell wall components are discussed and several exported virulence factors are described that have anti host cell activities. Others virulence factors enable the bacteria to grow in an environment of iron restriction.