2型猪链球菌中国强毒株znuA基因的原核表达及免疫学活性分析

目的克隆2型猪链球菌高亲和力锌吸收蛋白(High-affinity zinc uptake system protein,ZnuA)编码基因并进行原核表达,研究ZnuA蛋白的免疫学活性。方法采用PCR法,从2型猪链球菌中国强毒株05ZYH33基因组扩增基因znuA,构建重组表达质粒pET32a::znuA,转化E.coli BL21(DE3),筛选阳性转化子进行IPTG诱导表达,SDS-PAGE鉴定表达产物;His亲和层析柱纯化重组蛋白;Western blot检测ZnuA蛋白的免疫原性;重组蛋白免疫新西兰兔后收集多抗血清,间接ELISA检测多抗血清效价。结果构建的重组质粒在宿主菌中可高效表达,His亲和层析柱纯化获得较高纯度的重组蛋白;Western blot结果表明ZnuA蛋白具有良好的免疫原性;重组蛋白免疫新西兰兔后获得高效价的多抗血清。结论在原核系统中成功地表达了znuA基因编码的蛋白,且ZnuA重组蛋白具有良好的免疫原性,为进一步研究znuA基因在猪链球菌致病过程中的作用以及猪链球菌疫苗的开发奠定了基础。     

人UNC5CL原核蛋白的表达及多克隆抗体的制备和鉴定

目的:构建UNC5CL基因的原核表达载体,纯化GST-UNC5CL融合蛋白,并以其为抗原免疫家兔,制备抗人UNC5CL多克隆抗体,并鉴定其反应性和特异性。方法:用PCR方法扩增UNC5CL基因(表达其280-518位氨基酸)片段并克隆至pGEX-4T-2原核表达载体中,转化大肠杆菌BL21诱导融合蛋白GST-UNC5CL(aa 280-518)表达;所获得的可溶性蛋白经亲和层析纯化,免疫家兔制备抗血清,再采用ELISA、Western blot和免疫组化的方法检测抗体的效价及特异性。结果:测序证实原核重组质粒构建成功;经IPTG诱导,可表达Mr为52 000的GST-UNC5CL(aa280-518)的融合蛋白;谷胱甘肽亲和层析柱纯化的融合蛋白免疫家兔制备的抗血清经Western blot和免疫组化检测证实可识别目的蛋白。结论:制备了兔抗人UNC5CL的多克隆抗体,且抗体对内源性UNC5CL具有较好的反应性和特异性,为进一步研究UNC5CL的功能奠定了基础。     

梅毒螺旋体0751基因的克隆、表达及鉴定

目的构建梅毒螺旋体0751基因的原核表达质粒,诱导重组蛋白表达及进行免疫学鉴定。方法以梅毒螺旋体基因组DNA为模板,PCR扩增梅毒螺旋体0751基因,构建其重组原核表达载体,并采用Western-blot方法初步鉴定该蛋白的特异性。结果成功扩增出梅毒螺旋体0751基因,该基因在大肠杆菌表达系统中得到表达,并且特异性识别梅毒患者血清。结论在大肠杆菌中成功表达梅毒螺旋体0751基因,为梅毒螺旋体的诊断提供了新的特异性抗原。     

肠道病毒71 VP1基因的原核表达及其抗血清的制备

目的克隆并表达重组肠道病毒71(EV71)VP1基因,进行抗血清的制备并检测抗血清效价。方法利用原核表达系统将PCR获得的VP1片段构建成原核表达载体pET32a(+)-VP1,诱导其表达VP1融合蛋白并利用包涵体纯化方法进行重组蛋白的纯化,将纯化的重组蛋白免疫新西兰兔制备VP1抗血清,ELISA检测抗体效价。同时构建真核表达载体,利用其在真核细胞中的瞬时表达检测抗血清的特异性。结果通过克隆获得VP1原核表达载体,IPTG诱导VP1蛋白表达并纯化,SDS-PAGE结果显示重组蛋白表达且纯化浓度较高,将重组蛋白乳化后免疫新西兰兔3次,取血检测抗血清的效价,ELISA结果显示抗体效价为1∶64 000,利用所构建真核表达载体pcDNA3.1(+)-VP1表达VP1对抗血清进行检测,Western blot结果表明抗血清可较好的与VP1特异性结合。结论制备具有免疫原性的VP1蛋白及其效价较高的抗血清,为进一步研究抗EV71诊断方法、血清学诊断试剂盒及抗病毒疫苗的研制奠定基础。     

RPA32蛋白原核表达、多克隆抗体制备及初步鉴定

目的原核表达人RPA32蛋白并制备其多克隆抗体,为后续RPA32的功能研究奠定基础。方法以人cDNA文库为模板,构建原核表达质粒PET41a-RPA32,转化受体菌E.coli BL21,经IPTG诱导表达,采用GST亲和纯化方法获得GST-RPA32融合蛋白,将该融合蛋白免疫家兔制备RPA32多抗血清,最后采用Western blot检测其在多种肝细胞株中的特异性。结果在受体菌E.coli BL21中成功诱导表达GST-RPA32融合蛋白,免疫家兔后获得高效价RPA32抗血清,Western blot检测证实该血清能够识别多种肝细胞株中的RPA32及CPT作用后出现的RPA32磷酸化条带。结论成功制备兔抗人RPA32多克隆抗体,为RPA32在DNA损伤中的功能研究奠定了基础。     

恶性疟原虫复制蛋白PfRPA2的原核表达及多抗制备

目的克隆表达恶性疟原虫复制蛋白PfRPA2亚基,制备多抗,为该蛋白的功能研究奠定基础。方法PCR扩增目的基因片段,克隆到表达载体pGEX．KG中,构建PjRPA2／pGEX-KG原核表达载体,IPTG诱导表达,SDS—PAGE电泳分析表达产物．谷胱甘肽柱纯化蛋白,Western blot检测其抗原性,以纯化的GST-PfRPA2免疫小鼠,制备抗tyRPA2的多抗．间接ELISA法检测鼠血清效价,Western blot鉴定多抗特异性。结果成功构建了重组踯PA2／pGEX．KG原核表达质粒,并在大肠杆菌中以可济眭形式高效表达,纯化表达产物,制备抗邸PA2的鼠多抗,效价为10-7,Western blot证实此抗体可识别恶性疟原虫内与PJRPA2蛋白位置对应的特异性条带。结论恶性疟原虫复制蛋白PfRPA2亚基在大肠杆菌中获得可溶性高效表达,纯化表达产物能诱导小鼠生产能识别天然蛋白的特异性抗体。     

釉成熟蛋白Amelotin抗体的制备和初步鉴定

目的:表达并纯化小鼠釉成熟蛋白(Amelotin), 制备多克隆抗体, 并进行初步鉴定.方法:RT-PCR获得Amelotin成熟肽片段, 构建pET32a-Amelotin, IPTG诱导表达Amelotin, 纯化后免疫新西兰大白兔制备多克隆抗体, ELISA检测抗体滴度.Western blot和免疫组织化学鉴定抗体特异性.结果:成功构建了Amelotin重组表达载体pET32a-Amelotin,表达的重组蛋白纯化后免疫新西兰大白兔, 得到的多克隆抗体ELISA显示抗体效价可以达到1:12 800.Western blot分析表明该抗体能特异结合Amelotin, 免疫组化分析表明自制的抗体能特异的与Amelotin相互作用.结论:以纯化的Amelotin为免疫原, 成功地制备了效价及特异性较高的兔抗Amelotin抗体,为进一步建立简便的Amelotin检测方法及研究Amelotin生物学功能奠定了良好的基础.     

人survivin蛋白的原核表达、纯化及抗原活性鉴定

目的 构建人肿瘤抗原survivin的原核表达载体,优化在大肠杆菌中的表达条件,并对survivin/His融合蛋白进行纯化和抗原活性鉴定.方法 设计针对survivin基因序列的特异引物,通过聚合酶链式反应(PCR)扩增人survivin全长基因序列(538 bp)克隆至原核表达载体pET28a(+),构建重组表达载体pET28 a-survivin,并将该载体转化大肠杆菌BL21(DE3),经IPTG诱导表达survivin/His融合蛋白,并采用Ni亲和层析凝胶纯化重组蛋白.纯化后的重组蛋白经Western blot法、ELISA鉴定其抗原活性.结果 重组表达载体经BamH Ⅰ和HindⅢ鉴定正确;IPTG诱导后经SDS-PAGE分析表明获得了相对分子质量(Mr)24000大小的重组蛋白;纯化后的蛋白纯度达到90％.Western blot法和ELISA检测证实纯化的survivin蛋白能够与特异性抗体发生反应,表明其具有良好的抗原活性.结论 成功构建了原核表达载体pET28 a-survivin,利用大肠杆菌表达系统实现了融合蛋白的可溶性表达并进行纯化,纯化后survivin蛋白经鉴定具备较高的抗原活性.     

小鼠IL-1α多克隆抗体的制备及应用

目的:构建小鼠白细胞介素-1α原核表达载体,表达并纯化IL-1α蛋白,制备兔抗鼠IL-1α多克隆抗体,并对抗体特性进行初步的鉴定.方法:利用RT-PCR技术,从BALB/c小鼠脾脏cDNA中扩增出IL-1α的全长基因,酶切后连接至pET32a(+)原核表达载体,重组载体测序正确后转化至BL21( DE3)大肠杆菌.利用蛋白质原核表达自动诱导方案成功表达重组蛋白.重组蛋白经电洗脱纯化后用以免疫新西兰大白兔,获得了抗小鼠IL-1α的多克隆抗体,ELISA检测抗体效价.Western blot和流式细胞术(FCM)检测抗血清的特异性.结果:成功构建了重组表达载体pET32a(+)-IL-1α,表达的重组蛋白纯化后免疫新西兰大白兔,得到的多克隆抗体ELISA显示抗体效价可达1∶25 600.Western blot和FCM分析该抗体能特异性结合IL-1α.结论:利用重组的IL-1α蛋白成功制备了高效价、高特异性的兔抗IL-1α抗体,为进一步研究IL-1α的生物学功能奠定了基础.     

小鼠FcγRⅢ原核表达、纯化及鉴定

目的: 制备纯化重组蛋白mFcγRⅢ,并鉴定其生物学活性.方法: 从克隆载体mRⅢ-T中扩增mFcγRⅢ胞外区,构建原核表达载体pETmRⅢ,转化大肠杆菌BL21(DE3),IPTG诱导重组蛋白表达,SDS-PAGE和Western blot对重组表达蛋白进行鉴定;表达产物通过包涵体纯化后用稀释方法复性;ELISA检测复性蛋白活性.结果: 成功构建了重组表达质粒pETmRⅢ,纯化和复性了重组蛋白mFcγRⅢ;复性的重组蛋白mFcγRⅢ具有较强的体外活性.结论: 原核重组蛋白mFcγRⅢ复性后具有较强的体外结合配体特性,为探索重组蛋白治疗自身免疫病奠定了基础.     

梅毒螺旋体黏附素Tp0751 核酸菌影的构建及免疫原性研究

目的:构建梅毒螺旋体(Tp)黏附素Tp0751 的重组真核大肠埃希菌菌影(EBG)并检测其在免疫鼠中的免疫原性,为探讨新型梅毒疫苗奠定基础。方法:构建pcDNA3.1(+) / Tp0751 真核表达载体,将其装载入已构建的空EBG 中,形成重组核酸菌影pcD/ Tp0751-BG,计算装载率;将核酸菌影转染鼠源性巨噬细胞RAW264.7,Western blot(WB)鉴定目的蛋白表达。将雌性BALB/ c 鼠随机分为A(PBS)、B(空EBG)、C(空pcDNA3.1)三个对照组和D(pcD/ Tp0751)、E(pcD/ Tp0751-BG)、F(pcD/ Tp0751-BG+rTp0751)三个实验组,各组间隔两周肌注免疫共三次,检测特异性血清IgG 及生殖道黏膜SIgA、小鼠脾细胞增殖水平和分泌IFN-γ水平。结果:重组真核质粒对菌影的装载率为76.1%;WB 显示此转染细胞能有效表达重组目的蛋白。D、E、F 实验组小鼠特异性血清IgG 与生殖道SIgA 效价均随免疫次数增加而增加,各时间点均显著高于三个对照组(P<0.01),于末次加免后第8 周达到峰值,此时F 组IgG 与SIgA 效价分别为1 :102 400 与1 :12 800;首次加免2 周后,E、F 组均显著高于D 组(P<0.01);末次加免2 周后,F 组显著高于E 组(P<0.01)。末次加免后第8 周,D、E、F 组的刺激指数(SI)值与IFN-γ水平均分别显著高于三个对照组(P<0.01);E、F 组均分别显著高于D 组(P<0.01);F 组分别均高于E 组(P<0.05)。结论:Tp0751 真核质粒菌影具有良好的免疫原性,在小鼠体内诱生了有效的系统和黏膜的体液应答以及系统细胞免疫应答;异源加免较同源加免免疫效果更好。     

Expression and purification of the recombinant outermembrane protein Tp0453 of Treponema pallidum and its characterization of immuno-competence

ELISA test has been shown to have some advan tages in relation to the tests used for the diagnosisof syphilis because of its easy and quick perfor mance and result readings. With recent develop ment of the gene engineering technology and eluci dation of the whole genome of Nichols strain ofTreponema pallidum, new protein coding openreading frames (ORFs) are available for testing,and the study on the serological tests based on therecombinant protein have been become the focus ofinter…     

Identification of a Treponema pallidum laminin-binding protein

Host extracellular matrix (ECM) components represent ideal microbial adhesion targets that many pathogens use for colonization of tissues and initiation of infection. This study investigated the interaction of the spirochete Treponema pallidum with the ECM component laminin. To identify candidate laminin-binding adhesins, the T. pallidum genome was analyzed to predict open reading frames that encode putative outer membrane proteins, as these proteins interact directly with host ECM components. Subsequent recombinant expression of these proteins and analysis of their laminin-binding potential identified one protein, Tp0751, that demonstrated specific attachment to laminin. Tp0751 attached to laminin in a dose-dependent, saturable manner but did not attach to the ECM component collagen type I or IV or to the negative control proteins fetuin or bovine serum albumin. Sodium metaperiodate treatment of laminin reduced the Tp0751-laminin interaction in a concentration-dependent manner, suggesting that oligosaccharides play a role in this interaction. In addition, Tp0751-specific antibodies were detected in serum samples collected from both experimental and natural syphilis infections, indicating that Tp0751 is expressed in vivo during the course of infection. Collectively, these experiments identified Tp0751 as a laminin-binding protein that is expressed during infection and may be involved in attachment of T. pallidum to host tissues.     

梅毒螺旋体膜蛋白Tp0971 经TLR2 / NF-κB 诱导巨噬细胞分泌细胞因子

目的:探讨梅毒螺旋体(Treponema pallidum,Tp)膜蛋白Tp0971 诱导巨噬细胞分泌细胞因子的分子机制。方法:以Tp Nichols 株基因组为模板,PCR 扩增Tp0971 基因并构建重组质粒pET28a(+) / Tp0971,随后将其转化至表达宿主菌E.coli Rosseta 中,IPTG 诱导表达,Ni-NTA 亲和层析柱纯化重组蛋白。经去除Tp0971 重组蛋白中的内毒素后,将其刺激巨噬细胞,ELISA 检测细胞因子IL-8、IL-1β和IL-6 的分泌水平。并采用Toll 样受体2(TLR2)中和抗体或TLR2 siRNA,以及核转录因子资B(NF-κB)抑制剂PDTC 处理细胞,观察TLR2 和NF-κB 在介导细胞因子分泌中的作用。结果:成功构建pET28a(+) /Tp0791 原核表达载体,并表达出一相对分子量为29 kD 的重组蛋白。ELISA 结果显示,0.5 ~ 10 g/ ml Tp0791 能以剂量依赖性方式诱导巨噬细胞分泌IL-8、IL-1β和IL-6。采用siRNA 沉默TLR2 表达,或用TLR2 中和抗体封闭后,IL-8、IL-1β和IL-6 分泌明显减少。此外,Tp0791 也能增加细胞核中NF-κB p65 的表达水平,采用NF-κB 抑制剂PDTC 处理后,细胞因子分泌水平也显著降低。结论:Tp0971 经TLR2/ NF-κB 可诱导巨噬细胞分泌细胞因子。     

Serodiagnosis of syphilis: antibodies to recombinant Tp0453, Tp92, and Gpd proteins are sensitive and specific indicators of infection by Treponema pallidum

Syphilis serodiagnosis relies on a combination of nonspecific screening tests (antilipoidal antibodies) and Treponema pallidum-specific tests (anti-T. pallidum antibodies). We studied a group of six recombinant T. pallidum antigens for their sensitivities and specificities with sera from individuals with syphilis (n = 43), relapsing fever (n = 8), Lyme disease (n = 8), and leptospirosis (n = 9) and from uninfected individuals (n = 15). Three recombinant proteins, Tp0155, Tp0483, and Tp0751, demonstrated sensitivity values that ranged from 28 to 42%. In contrast, three other recombinant proteins exhibited the following sensitivity and specificity values: Tp0453, 100% sensitivity and 100% specificity; Tp92 (Tp0326), 98% sensitivity and 97% specificity; and Gpd (Tp0257), 91% sensitivity and 93% specificity. Tp0453, Tp92, and Gpd also were recognized by sera from individuals with early primary syphilis that were nonreactive with the antilipoidal Venereal Disease Research Laboratory test. The reactivities of syphilis patient sera with Tp0453, Tp92, and Gpd were proportional to the titers of these sera with the treponemal test MHA-TP (microhemagglutination assay for T. pallidum). Thus, the recombinant T. pallidum antigens Tp0453, Tp92, and Gpd show promise as diagnostic antigens in the enzyme-linked immunosorbent assay-based assay.     

Defining the interaction of the Treponema pallidum adhesin Tp0751 with laminin

Various invasive pathogens attach to host tissues via the extracellular matrix component laminin, the major glycoprotein found within basement membranes. Previous investigations identified the laminin-binding adhesin Tp0751 within the spirochete bacterium Treponema pallidum. In the current study, Tp0751 was shown to attach to a variety of laminin isoforms that are widely distributed throughout the host, including laminins 1, 2, 4, 8, and 10. Such universal attachment is conducive for an adhesin present within a highly invasive pathogen that encounters a variety of tissue sites during the course of infection. Additional studies systematically identified the amino acid residues within Tp0751 that contribute to laminin binding using synthetic peptides designed from the mature protein sequence. The minimum laminin-binding region of the adhesin was localized to 10 amino acids; peptides containing these residues inhibited attachment of Tp0751 and T. pallidum to laminin. Further, Tp0751-specific antibodies inhibited attachment of T. pallidum to laminin. This study furthers our knowledge of the interaction of T. pallidum with laminin, an association that is proposed to facilitate bacterial traversal of basement membranes and subsequent entry into the circulation and tissue invasion. As such, these investigations will reveal new targets for possible prevention of bacterial dissemination and establishment of chronic infection.     

中国株HIV-1核心蛋白Gag核酸疫苗的构建与表达

目的 构建含HIV－1中国流行株B亚型核心蛋白Gag基因的真核表达质粒，并在体外进行表达和鉴定。方法 将Gag基因插入到核酸疫苗载体质粒pVAX1中，构建真核表达质粒pVAXGAG。用脂质体法。将重组质粒转染人Hela细胞后进行表达产物的检测。结果 间接免疫荧光检测显示，转染重组质粒的细胞表面有绿色荧光。Western blot和Dot ELISA分析显示，重组质粒转染细胞的裂解物中存在表达的Gag蛋白。结论 构建的核酸苗可在体外进行表达，且表达的蛋白具有特异性。     

梅毒螺旋体Tp0136活性肽段的可溶性表达、纯化及鉴定

目的 筛选梅毒螺旋体特异性抗原Tp0136的活性肽段,可溶性表达和纯化该肽段,并鉴定其免疫活性,探索Tp0136活性肽段在早期梅毒诊断中的价值.方法 通过生物信息学方法对Tp0136亲水性、B细胞表位和二级结构等进行分析,筛选出Tp0136活性肽段(Tp0136B)替代全蛋白.将Tp0136B基因插入到pET22b(+)上,在E.coli BL21中表达.镍离子亲和色谱纯化表达产物,Western blot检测其免疫反应性,免疫日本大耳白兔评价其免疫原性,免疫双扩检测其效价,以重组Tp0136B蛋白为包被抗原的间接ELISA检测早期梅毒血清抗体.结果 重组工程菌可溶性表达相对分子质量约为28×103的rTp0136B,表达率为21%,制备得到纯度大于98%的rTp0136B.纯化的rTp0136B能诱导大耳白兔产生特异性免疫应答,免疫双扩测得其效价为1:16.Western blot检测重组蛋白能与兔抗Tp0136多克隆抗体发生特异性反应.间接ELISA检测正常人血清均为阴性,而早期梅毒血清抗体的阳性率为85.5%.结论 重组表达的Tp0136活性肽段具有良好的免疫活性,预示其在早期梅毒血清学诊断中具有良好的前景.

Abstract：

Objective To express and purify recombinant Tp0136 epitope fragment, and study the immunity activity. Methods The Tp0136 selective fragment(Tp0136B) gene was devised by the surface property analysis, solvent-accessible suface calculateions, secondary structure function region analysis, and was inserted between the sites of Nde Ⅰ and Not Ⅰ in pET22b ( + ) . The recombinant plasmid was expressed in E. coli BI21. After nickel ion metal affinity chromatography, the antigenic and immune reactivity of rTp0136B was confirmed. Then indirect ELISA with the rTp0136B as coating antigen was performed to detect the anti-Tp0136 antibody in sera from 100 normal human controls and 131 primary syphilis patients. Results The rTp0136B was soluble expressed with a molecular weight of about 28 000 and was obtained with a purity of ＞98% by chromatography. Western blot proved that the rTp0136B could specifically react with anti-Tp0136 polyclonal antibody. Specific humoral response was elicited by the recombinant protein in Japan negative. The positive detection rate in sera from primary syphilis patients was 85.5%. Conclusion This result suggested that the recombinant Tp0136 epitope fragments have a satisfactory immunocompetence,which may have applications in the serodiagnosis of primary syphilis.