大肠杆菌表达抗原结合片段的策略

背景:抗原结合片段在大肠杆菌中表达量低是制约其大规模生产和应用的主要因素,通过对宿主细胞、表达载体、表达条件和纯化条件等进行改造与优化,将有可能获得基因工程抗体的高效表达.目的:总结并介绍抗原结合片断抗体在大肠杆菌中的表达和纯化的最新策略.方法:由第一作者用计算机检索中国期刊全文数据库(CNKI:1990/2010)和Medline数据库(1990/2010),检索词分别为"大肠杆菌,抗原结合片段,表达,纯化,抗体"和"Escherichia coli,Fab,Expression,antibody,purification",语言分别设定为中文和英文.从Fab抗体在大肠杆菌中表达方式及纯化2方面进行总结,对抗原结合片断抗体在大肠杆菌中最新的表达和纯化等方面进行介绍.结果与结论:共检索到125篇文章,按纳入和排除标准对文献进行筛选,共纳入30篇文章.结果表明抗原结合片段抗体在大肠杆菌中表达方式主要为细胞质表达和分泌表达,其纯化主要有金属离子螯和纯化和特异蛋白亲和纯化,抗原结合片断在大肠杆菌中表达的影响因素是多方面且相互关联的.目前研究对大幅提高抗原结合片断抗体在大肠杆菌中最新的表达量和最适纯化策略尚不一致.     

人心肌型脂肪酸结合蛋白(H-FABP)在大肠杆菌中的表达和纯化

目的获得重组人心肌型脂肪酸结合蛋白,为进一步开发应用奠定基础。方法根据已报道的心肌型脂肪酸结合蛋白(H-FABP)基因序列,采用RT-PCR的方法获得心肌型脂肪酸结合蛋白基因。将所得的PCR产物插入原核表达载体pQE30中,得重组质粒(pQE-H-FABP)并转化大肠杆菌(E.coli)M15,通过IPTG诱导表达出带有六个组氨酸的融合蛋白。经镍固定金属亲和层析纯化。结果序列分析表明:H-FABP基因成熟肽编码区含有396bp,编码132个氨基酸;与GenBank(NM004012)中已报道的H-FABP分离物的核苷酸序列有99·9%同源性。经IPTG诱导表达,SDS-PAGE电泳和免疫印迹分析显示,表达的融合蛋白占菌体蛋白总量的27%,分子质量约为15kDa并与商品化的H-FABP单抗(Clone6B6)呈特异性反应。经Ni 2-NTAagarose纯化获得SDS-PAGE电泳下单一条带。结论在大肠杆菌中获得了人心肌型脂肪酸结合蛋白的高效表达,为研究其生物学功能和制备单克隆抗体奠定了基础。     

解脲支原体1型MB抗原保守区基因片段的克隆、表达及纯化

目的 以解脲支原体1型培养液为模板，PER获得其MB抗原保守区部分基因，进行体外表达和纯化。方法用ExPasy网上软件Protscale分析Uu14个血清型的MB蛋白结构特点及抗原特性，选定14个血清型均在该蛋白同一保守区域有较好的抗原性，针对该序列设计特异性引物，并以UU血清1型DNA为模板进行PER，获得其MB抗原基因相应的片断，克隆入表达载体经测序鉴定后。在大肠杆菌中表达并纯化，以Western—blot鉴定表达产物。结果 准确扩增了解脲支原体1型MB抗原保守区基因片段，并在大肠杆菌中获得表达。结论 成功构建了pQE30／MB抗原部分保守序列的原核表达载体，在体外成功表达并纯化。表达和纯化的产物具有免疫反应性。     

胰腺癌相关自身抗原47原核表达、纯化及其抗体检测胰腺癌的价值初探

目的 验证胰腺癌相关性自身抗原47(PAA47)的质谱鉴定结果，初探抗PAA47检测在诊断胰腺癌中的意义。方法 构建pQE—30—PAA47重组表达载体；在大肠杆菌M15中诱导表达KIAA0111编码蛋白质；经纯化后用点印迹、免疫印证法验证表达产物与相应胰腺癌相关性自身抗体阳性血清的抗原反应性；建立间接酶联免疫吸附实验(ELISA)，检测60名健康体检者、60例胰腺癌、20例慢性胰腺炎及120例其他肿瘤(大肠癌、肝癌、胃癌及肺癌各30例)患者血清中的抗PAA47，并与回顾性糖链抗原19—9(CAl9—9)检测结果相比较。结果 表达产物的序列与KIAA0111编码序列一致；点印迹法和免疫印迹法显示纯化表达产物与抗PAA47血清具有抗原反应性。ELISA结果显示，胰腺癌患者中，该抗体阳性率为31．67％(19／60)，大肠癌、肝癌、胃癌分别为10．00％、3．33％、6．67％，正常人、慢性胰腺炎及肺癌患者中未见阳性。抗PAA47对胰腺癌的灵敏度为31．67％，特异性为95．89％。与慢性胰腺炎患者比较，PAA47的灵敏度为31．67％，特异性为100％，CAl9—9的灵敏度为75．00％，特异性为86．96％。平行法联合检测2项指标，既保持了相同的特异性，又提高了检测的灵敏度(90％，P＜0．05)。采用顺序法联合检测，灵敏度和特异性分别为70％和100％。结论 本研究成功克隆了人KIAA0111编码基因，并将其在大肠杆菌中成功表达；新鉴定的PAA47，其抗体血清学检测与CAl9—9等肿瘤标志物联合，对提高胰腺癌诊断的灵敏度和特异性具有一定的意义。     

乙型肝炎病毒核心抗原在大肠杆菌中的构建及表达

目的用基因工程的方法获得乙型肝炎病毒核心抗原(HBcAg)编码区的基因片段,构建重组质粒并在大肠杆菌中表达抗原蛋白。方法用PCR法扩增HBcAg基因片段,构建含有HBcAg基因的克隆质粒及表达质粒,在大肠杆菌BL21(DE3)plys中以IPTG诱导重组蛋白的表达,并用Western blot对重组蛋白进行检测。结果重组HBcAg在大肠杆菌中得到表达,Western blot检测显示在预期位置出现特异性条带。结论成功构建HBcAg原核表达系统并获得重组HBcAg,为该重组蛋白的相关功能研究奠定了基础。     

HCV单片段抗原EIA与常规EIA检测丙型肝炎病毒抗体的比较

目的比较国产重组HCV单片段抗原酶免疫(EIA)检测试剂与国产第三代HCV常规抗体(EIA)检测试剂的敏感性和特异性。方法利用基因工程技术重组表达的HCV单片段(HCV-C、NS、NS4、NS)EIA检测试剂和常规EIA试剂检测国家第三代抗-HCV血清考核盘(40份阳性和40份阴性血清样品)及20份不符血样，对检测结果进行统计分析。结果HCV单片段EIA检测试剂总符合率100％，常规试剂为96．2％。结论HCV抗体单片段试剂可作为对HCV常规EIA试剂检测不符血样的确认试剂。     

肿瘤相关抗原p16的表达和纯化及其在癌症检测中的应用

肿瘤抑制蛋白p16是一个细胞cyclin依赖的细胞激酶抑制因子，具有调节细胞分裂周期的重要功能，p16基因的突变与肿瘤发生密切相关。以往的研究表明与细胞恶性转变有关联的一些蛋白如p53，c—mye，p62和survivin等可以诱发机体产生自身抗体，而这些自身抗体可以作为监测肿瘤发展的标志。是否血清中p16抗体也可作为一个肿瘤诊断的标志仍需要进行评价。本研究用GST融合蛋白纯化系统对体外表达的p16重组蛋白进行纯化，并进一步将其用做肿瘤抗原对癌症病人血清中存在的p16抗体进行检测。初步结果表明在多种肿瘤病人的血清中都可检测到p16抗体。由于本研究使用的样本数有限，是否p16抗体可以作为肿瘤的一个诊断标志仍需要做进一步的研究证实。     

结核分枝杆菌抗原Rv0173的克隆、表达与纯化

目的 在大肠杆菌中表达、纯化结核分枝杆菌Rv0173抗原,为研制新型结核病疫苗打下基础.方法 将结核杆菌抗原Rv0173的全长cDNA插入到原核表达载体pGES-4T-1中,构建成Rv0173重组质粒.将重组质粒转入大肠杆菌BL21后用IPTG进行诱导表达,通过SDS-PAGE和Western-blot鉴定重组表达蛋白.结果 获得了pGEX4T-Rv0173 重组子,Rv0173蛋白在BL21菌中获得表达,表达的蛋白条带大小约45KD,与预期结果相符.结论 成功地对结核杆菌免疫保护性抗原Rv0173进行了基因克隆与表达,为进一步研究其在结核病新型疫苗研制中的应用奠定了基础.     

重组人干扰素α8在大肠杆菌中高可溶性表达及纯化

目的:在大肠杆菌表达体系中获得产量较高且有活性的重组人干扰素α8蛋白.方法:重组人干扰素α8的cDNA通过基因合成和PCR获得,构建重组人干扰素α8表达菌株.结果:表达的目的蛋白多以可溶性形式存在,占总上清液的52%,表达蛋白带有组氨酸标签经SDS-PAGE及Western Blot分析证实为重组人干扰素α8,通过Ni2+螯合柱及Q Sepharose FF柱层析纯化,纯度可达95.6%.结论:通过对重组人干扰素α8的cDNA序列进行适优化组合及选择合适的载体,在大肠杆菌表达体系中可获得产量较高且有活性的重组人干扰素α8蛋白.     

重组人Flt3配体在大肠杆菌中的表达、纯化与功能鉴定

目的：建立重组人Flt3配体（rhFL)的原核高效表达系统及目标蛋白的纯化途径,为大规模获得rhFL产品,促进干细胞体外扩增,移植等新技术在临床的应用创造条件,方法：将FL细胞段cDNA与pProEXFT载体连接,重组体引入大肠菌菌并在异丙基－β－D－硫化半乳糖苷诱导下表达,提取包涵体,经变性,复性等处理,用金属离子螯合层析纯化表达产物,观察纯化所得rhFL刺激CD^ 34细胞的增殖情况,结果：rhFL的表达约占菌体蛋白总量的15％,经亲和纯化纯度达90％以上,rhFL CG-CSF＋Fpo刺激CD34^ 细胞增殖约400倍,结论：获得了rhFL在大肠肝菌中的高效表达,并初步建立了产物纯化途径,纯化后的rhFL具有产强的刺激造血干／祖细胞增殖的能力。     

Escherichia coli O antigen typing using DNA microarrays

DNA microarrays were developed for rapid identification of different serogroups of Escherichia coli in a single platform. Oligonucleotides, as well as PCR products from genes in the O antigen gene clusters of E. coli serogroups O7, O104, O111, and O157 were spotted onto glass slides. This was followed by hybridization with labeled long PCR products of the entire O antigen gene clusters of these serogroups. Results demonstrated that microarrays consisting of either oligonucleotides or PCR products generated specific signals for each serogroup. This is the first report describing the development of model DNA microarrays for determining the serogroup of E. coli strains.     

曲霉菌内切型壳聚糖酶基因克隆及在大肠杆菌中的表达

背景：壳聚糖酶是高效、特异降解壳聚糖的酶,因此高效稳定地表达具有较高活性的壳聚糖酶可有效提高壳聚糖介导的基因治疗效果。目的：构建一种可高效降解壳聚糖的壳聚糖酶基因,探讨其在大肠杆菌中的表达及影响其活性的主要因素。方法：根据GenBank公布的曲霉菌CJ22-326内切型壳聚糖酶基因序列信息（EU302818）,设计并合成23条重叠引物,PCR 法扩增壳聚糖酶基因片段,构建原核表达质粒 pET28a-His6-CSN,将其转化大肠杆菌,收集融合蛋白His6-CSN,检测融合蛋白的表达及酶活性,同时检测不同pH值及温度对壳聚糖酶活性的影响。结果与结论：Western-blot证实融合蛋白His6-CSN成功表达,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳检测显示表达的融合蛋白相对分子质量约为29000,二硝基水杨酸比色法测定壳聚糖酶对壳聚糖的降解活性显著高于溶菌酶（P 〈0.05）,但低于灰色链霉菌壳聚糖酶（P 〈0.05）。壳聚糖酶的最适pH值和最适温度分别为6.0和50℃,当pH值在4.0-7.0、温度在30-50℃范围内具有较高的酶活性。     

Correlation between human lactoferrin binding and colicin susceptibility in Escherichia coli.

Escherichia coli H10407 demonstrated low 125I-human lactoferrin (HLf) binding (7%) and was insusceptible to group A (A, E1, E2, E3, E6, and K) and group B (B, D, Ia, Ib, and V) colicins. Conversely, a spontaneous HLf high-binding (44%) variant, H10407(Lf), demonstrated an increase susceptibility to both colicin groups. Colicin-insusceptible E. coli wild-type strains 75ColT, 84ColT, and 981ColT showed a low degree of HLf binding, i.e., 4, 8, and 10%, respectively. The HLf binding capacity was high in the corresponding colicin-susceptible mutants 75ColS (43%), 84ColS (32%), and 981ColS (43%). Furthermore, HLf low- (less than 5%) and high- (greater than 35%) binding E. coli clinical isolates (10 in each category) were tested for susceptibility against 11 colicins. Colicin V susceptibility did not correlate with HLf binding in either categories. However, with the remaining colicins, three distinct HLf-binding, colicin susceptibility patterns were observed; (i) 10 of 10 HLf low-binding strains were colicin insusceptible, (ii) 6 of 10 HLf high-binding strains were also colicin insusceptible, and (iii) the remaining HLf high binders were highly colicin susceptible. Certain proteins in the cell envelope and outer membrane of wild-type H10407 (HLf low binder, colicin insusceptible) showed a lower mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis compared to the corresponding proteins of mutant H10407(Lf) (HLf high binder, colicin susceptible). These mobility differences were also associated with HLf-binding proteins in Western blot (ligand blot) analysis. The wild type showed a smooth form of lipopolysaccharide (LPS) with a distinct ladder of O-chains, compared to the rough LPS of the mutant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fimbriae of enterotoxigenic Escherichia coli function as a mucosal carrier for a coupled heterologous antigen.

Receptor-mediated uptake of orally administered antigen can lead to an antigen-specific immune response, whereas oral administration of most other non-replicating soluble antigens results in the induction of oral tolerance. In the present study, it is shown that fimbriae purified from an F4(K88)(+) enterotoxigenic Escherichia coli strain can function as a mucosal carrier molecule for the model antigen human serum albumin (HSA). Glutaraldehyde-coupled F4/HSA conjugates were able to bind F4 receptor positive (F4R(+)) enterocytes, but not to F4R(-) enterocytes. Moreover, oral immunization of F4R(+) pigs with F4/HSA conjugates induced a HSA-specific immune response, whereas oral immunization with HSA/HSA conjugates did not. This mucosal carrier function of F4 fimbriae was improved following oral co-administration of the F4/HSA conjugates with the mucosal adjuvant cholera toxin (CT) to F4R(+) pigs, since both humoral and cellular HSA-specific responses were significantly increased. In comparison with F4R(+) pigs, the HSA-specific response was reduced following oral F4/HSA+CT immunization of F4R(-) pigs. This indicates that F4 fimbriae as mucosal carrier and CT as adjuvant synergistically improve the induction of a HSA-specific immune response following oral immunization of pigs. These results could open new perspectives in the development of vaccines against enteropathogens.     

Improved sensitivity in assays for binding of novel beta-lactam antibiotics to penicillin-binding proteins of Escherichia coli.

Tigemonam and temocillin, but not aztreonam, bound to penicillin-binding proteins (PBPs) 1a and 3 of Escherichia coli with apparent improved affinity when challenged with benzylpenicillin at lowered temperatures. Half times for deacylation of the tigemonam-PBP complexes were shorter than were those of the corresponding aztreonam-PBP complexes. The implications of the routine testing of PBP affinities are discussed.     

Adhesion-promoting receptors on human macrophages recognize Escherichia coli by binding to lipopolysaccharide

We report here that human macrophages bind Escherichia coli by recognizing bacterial lipopolysaccharide (LPS). Purified LPS was inserted into erythrocyte membranes, and the resulting LPS-coated red cells were bound by macrophages with the same temperature and cation dependence as observed for E. coli. When receptors for LPS were withdrawn from the plasma membrane by spreading the macrophages on LPS-coated surfaces, the binding of E. coli was blocked. We have also identified the receptors on macrophages that recognize LPS. Macrophages express three structurally homologous cell surface proteins, CR3, lymphocyte function-associated antigen (LFA-1), and p150,95. We used surface-bound monoclonal antireceptor antibodies to selectively remove these proteins from the apical surface of macrophages. We found that each of these proteins mediated the binding of E. coli to macrophages.