热诱导FLP重组酶删除转基因烟草外源基因

利用热激启动子Hsp18.2、FLP重组酶基因及其专一识别位点构建了重组酶删除系统的植物表达载体转化烟草.热激处理后转基因植株中FLP重组酶表达并识别两个同向loxP-frt重组酶识别位点,使两位点间的DNA插入片段(包含kn1、gus、nptⅡ基因)从转基因烟草基因组中删除,由kn1基因引起的转基因烟草特殊表型及gus基因产生的蓝色表型消失.     

抗牛精子sp18蛋白单链抗体基因的克隆及表达

应用重组噬菌体抗体技术,从小鼠抗牛精子sp18蛋白的A5杂交瘤细胞系中分离总RNA,构建重组噬菌体抗体库。以牛精子作为包被抗原,经过三轮“亲和吸附-洗脱-扩增”淘选后,用phage-ELISA筛选出特异性阳性克隆。取其中特异结合牛精子细胞的重组噬菌体抗体pCSA1克隆进行序列测定。将其ScFv(single chain fragment variable)因克隆到pET-30a( )上,用IPTG进行诱导表达,成功得到了抗sp18单链抗体基因蛋白。     

来源于芽孢杆菌(Bacillus circulans)的乳糖酶在毕赤酵母中的高效表达

根据毕赤酵母(Pichia pastoris)密码子选择偏好对来源于芽孢杆菌(Bacillus circulans)的乳糖酶基因lacO进行了分子改造.改造后的基因lacM按正确的阅读框架插入到毕赤酵母表达载体pPIC9上,构建得到重组毕赤酵母表达质粒.PCR检测、SDS-PAGE电泳分析和酶活力测定的结果表明,lacM基因已重组到酵母染色体上,并能正常分泌表达.与未改造的基因lacO的毕赤酵母重组子相比,酶蛋白表达量明显增加,约为改造前的3倍以上.     

华根霉(Rhizopus chinensis)前导肽脂肪酶基因的克隆及其在Pichia pastoris中的表达

通过3次PCR程序克隆得到了华根霉(Rhizopus chinensis CCTCCM201021)脂肪酶全基因序列,该基因的开放阅读框长1170bp,不含内含子,编码一个389个氨基酸残基的蛋白质,包括26个氨基酸的信号肽,94个氨基酸的前导序列和269个氨基酸的成熟肽,其推断的氨基酸序列与一些已报道的根霉脂肪酶序列同源性为86%.在巴斯德毕赤酵母(Pichia pastoris)GS115中分泌表达前导肽序列.SDS-PAGE分析表明表达蛋白的分子量约37kD.N-端氨基酸序列分析表明该重组蛋白分泌过程中前导序列N-端的67个氨基酸被切割掉,表达的重组脂肪酶由前导序列C-端的27个氨基酸和成熟肽的269个氨基酸组成.发酵132h后上清中重组脂肪酶的表达量最高,蛋白含量约5.4mg/mL,橄榄油乳化法测水解酶活为161U/mL,其比活比野生型华根霉脂肪酶高约43倍.     

一个新的重组酶系统在转基因植物外源基因删除中的应用

为了解决转基因植物可能带来的生物安全性问题,设计了一个新的位点特异性重组酶系统pLFHFGN,该系统中重组酶FLP基因由热激蛋白Hsp18．2基因的启动子控制,所有外源基因(包括重组酶基因nP、GUS报告基因和NPTI筛选标记基因)都置于两个融合识别位点loxFRT之间。将上述重组酶系统通过根癌农杆菌(Agrobacterium tumefaciens)介导转化烟草,获得的再生植株分别通过GUS组织染色和PCR分析等方法进行鉴定。然后在37℃下热激处理转基因植株,利用GUS组织染色检测重组酶系统在转基因植株T1代幼苗中删除外源基因的效率,结果显示,该系统删除效率达到了48．2％-84．9％,说明通过重组酶系统pLFHFGN能够有效地删除转基因植物中的外源基因,这为解决转基因植物潜在的安全性问题提供了一条新的途径。     

人TNF—α分泌型,膜型和膜稳定型重组体的构建,表达及杀瘤效应研究

通过用ＩＬ－２信号肽编码序列置换ＴＮＦ前导肽编码序列，构建出一种在真核细胞中仅产生分泌型ＴＮＦ的重组体；通过缺失二型ＴＮＦ转换的酶银部位而构建出跨膜表达的膜稳定型ＴＮＦ重组体，并建立分别只表达分泌型或膜型及可同时表达两型ＴＮＦ的真核细胞株，比较二型ＴＮＦ的杀瘤效应。结果显示，膜型ＴＮＦ主要经效－靶细胞间直接接触发挥胞毒效应，杀瘤谱比分泌型ＴＮＦ广，ＴＮＦ的分泌型和膜型具有协同效应，通过基因直接注射     

人凝血因子IX在脆壁克鲁维酵母中的表达与分泌

通过改造人凝血因子IX基因(h-FIX)使其与乳酸克鲁维酵母(Kluyveromyces lactis)的分泌信号序列(Kss)融合，并置于脆壁克鲁维酵母(K．fragilis)LAC4基因调控型启动子的控制之下，构建成h-FIX基因表达盒。将此表达盒插入克鲁维酵母的整合质粒，得到了一个高稳定的h-FIX基因整合表达载体pHKB202-FP。经摇瓶培养并以半乳糖诱导此表达载体转化的酵母菌株，ELISA检测显示酵母上清液中的人凝血因子IX表达量可达到约200ng/ml。此结果表明人凝血因子IX蛋白首次成功地在脆壁克鲁维酵母中得到了表达和分泌。这对于促进人凝血因子IX的基因工程产业化具有实际意义。     

甜蛋白Monellin在毕赤酵母中的分泌表达

在西非热带植物Dioscoreophyllum cumminsii中存在着一种由两条多肽构成的甜度极高的蛋白Monellin。本研究根据已知的Monellin晶体衍射分析结果和毕赤酵母(Pichia pastoris)基因偏爱密码子设计并合成了连接两条多肽的重组基因，插入到毕赤酵母分泌表达质粒pGAPZαA中。扩增后，电击穿孔转化毕赤酵母GS115，筛选高产菌株放大培养。以5升罐发酵培养，表达量为150mg／L。经纯化，可以获得大于130mg／L的具有高甜度的活性蛋白。     

利用Cre/loxP重组系统构建甜菜碱合成酶多基因表达载体

通过使用一套基于Cre/loxP重组系统的植物多基因表达载体构建系统,将辽宁碱蓬(Suaeda liaotungesis kitag)的胆碱单加氧酶(CMO)基因、甜菜碱醛脱氢酶(BADH)基因以及烟草的核基质结合区(MAR)序列构建到同一表达载体上,得到可直接用于农杆菌转化的植物表达载体pYLTAC747H-MAR-BADH-CMO-MAR.该甜菜碱合成酶多基因表达载体的成功构建为进一步进行植物的遗传转化,以有效提高转基因植株的耐盐性提供了实验基础.实验中,用热激法替代了电击法进行质粒的大肠杆菌转化,并去掉了透析等步骤,简化了构建过程.     

用农杆菌真空渗透法在烟草中瞬时表达人酸性成纤维细胞生长因子

利用农杆菌真空渗透法在烟草中瞬时表达了人酸性成纤维细胞生长因子(haFGF)基因,并对影响表达量的几个因素进行了研究.结果表明,在真空渗透后4天,外源基因获得最高水平的表达,烟草Nicotiana benthamiana(N.b)的表达量明显低于烟草N.H(转基因沉默抑制子Hc-pro的N.b植株).pM390-haFGF、pM390-haFGF/KDEL、pM390-SP/haFGF/KDEL三种表达框架被用来表达人酸性成纤维细胞生长因子,结果表明框架pM390-haFGF/KDEL的表达效果最好,受伤叶片和Valentine的培养方法要比完整叶片和Kapila的培养方法的表达量高.     

Preparation of a single-chain variable fragment and a recombinant antigen-binding fragment against the anti-malarial drugs, artemisinin and artesunate, and their application in an ELISA

Two different recombinant antibodies, a single-chain variable fragment (scFv) and an antigen-binding fragment (Fab), were prepared against artemisinin (AM) and artesunate (AS) and were developed for use in an enzyme-linked immunosorbent assay (ELISA). The recombinant antibodies, which were derived from a single monoclonal antibody against AM and AS (mAb 1C1) prepared by us, were expressed by Escherichia coli cells and their reactivity and specificity were characterized. As a result, to obtain sufficient signal in indirect ELISA, a much greater amount of a first antibody was needed in the use of scFv due to the differences of the secondary antibody and conformational stability. Therefore, we focused on the development of the recombinant Fab antibodies and applied it to indirect competitive ELISA. The specificity of the Fab was similar to that of mAb 1C1 in that it showed specific reactivity toward AM and AS only. The sensitivity of the icELISA (0.16 μg/mL to 40 μg/mL for AM and 8.0 ng/mL to 60 ng/mL for AS) was sufficient for analysis of antimalarial drugs, and its utility for quality control of analysis of Artemisia spp. was validated. The Fab expression and refolding systems provided a good yield of high-quality antibodies. The recombinant antibody against AM and AS provides an essential component of an economically attractive immunoassay and will be useful in other immunochemical applications for the analysis and purification of antimalarial drugs.     

一种新型双向启动子--棉花曲叶病毒启动子的分离、序列分析与功能初探

以棉花曲叶病毒(CLCuV)侵染的烟草叶片组织总DNA为模板,通过聚合酶链反应扩增CLCuV双向启动子片段并插入克隆载体.序列分析和同源性比较表明,克隆的启动子长436bp,与目前发现的9种CLCuV株系的启动子序列均不相同,同源性最高达99.32%.将启动子片段分别以不同方向与GUS报告基因和nos终止子融合,构建了瞬时表达载体.通过基因枪法将质粒载体导入烟草和棉花叶片细胞中进行瞬时表达,结果表明,互补链基因方向启动子属强启动子,在叶肉及维管组织均有较高的活性;病毒链基因方向启动子表达活性较低.本文初步证实分离的互补链基因启动子可作为新型强启动子应用于双子叶植物尤其棉花的遗传转化.     

抗CD20嵌合抗体Fab'片段在大肠杆菌中高效表达

利用ＰＣＲ方法从抗ＣＤ２０ＳｃＦｖ表达载体上扩增重链可变区、轻链可变区基因,然后将ＶＨ、ＶＬ基因重组到Ｆａｂ’表达载体中,构建成抗ＣＤ２０嵌合抗体Ｆａｂ’片段表达载体ｐＹＺＦｃｄ２０,用ｐＹＺＦｃｄ２０转化大肠杆菌１６Ｃ９,在１６Ｃ９菌中分泌表达可溶性抗ＣＤ２０Ｆａｂ’片段,经分离纯化获得具有ＣＤ２０抗原特异结合活性的Ｆａｂ’片段,竞争性竞争荧光抑制实验表明,抗ＣＤ２０Ｆａｂ’片段竞争性抑亲本属源     

Isolation and characterization of a thermally stable recombinant anti-caffeine heavy-chain antibody fragment

We have isolated and characterized a caffeine-specific, heavy-chain-only antibody fragment (V(HH)) from llama that is capable of being utilized to analyze caffeine in hot and cold beverages. Camelid species (llama and camel) were selected for immunization because of their potential to make heat-stable, heavy-chain-only antibodies. Llamas and camels were immunized with caffeine covalently linked to keyhole limpet hemocyanin, and recombinant antibody techniques were used to create phage displayed libraries of variable region fragments of the heavy-chain antibodies. Caffeine-specific V(HH) fragments were selected by their ability to bind to caffeine/bovine serum albumin (BSA) and confirmed by a positive reaction in a caffeine enzyme-linked immunosorbent assay (caffeine ELISA). One of these V(HH) fragments (VSA2) was expressed as a soluble protein and shown to recover its reactivity after exposure to temperatures up to 90 degrees C. In addition, VSA2 was able to bind caffeine at 70 degrees C. A competition caffeine ELISA was developed for the measurement of caffeine in beverages, and concentrations of caffeine obtained for coffee, Coca-Cola Classic, and Diet Coke agreed well with high performance liquid chromatography (HPLC) determination and literature values. VSA2 showed minimal cross reactivity with structurally related methylxanthines.     

Multiplexed detection of antibodies to nonstructural proteins of foot-and-mouth disease virus

Liquid array technology was used to develop a multiplexed assay for the detection of antibodies to viral nonstructural proteins (NSPs), raised in cattle in response to infection with foot-and-mouth disease (FMD) virus. Two assays, one based on recombinant NSPs and the other on synthetically produced peptides, were developed and compared side-by-side. Serum samples from serial bleeds of cattle, each experimentally infected with one of the seven serotypes (C, A, O, Asia, SAT1, SAT2, SAT3) of FMD virus were analyzed. A distinct pattern in the detection of NSP antibodies and a close correlation of the recombinant protein and peptide-based assays were observed. The detection of antibodies to NSPs is a method to differentiate FMD-infected and FMD-vaccinated animals, and a high-throughput assay would be an invaluable tool in the case of an outbreak of FMD in North America, when emergency vaccination may be utilized to spare vaccinated, noninfected animals from slaughter and subsequent disposal.     

Natural radioactivity contents in tobacco and radiation dose induced from smoking

One of the causative factors for cancer-inducing mechanisms in humans is radioactive elements present in tobacco leaves used in the manufacture of cigarettes. Smoking of tobacco and its products increases the internal intake and radiation dose due to naturally occurring radionuclides that are considered to be one of the most significant causes of lung cancer. In this work, different commercial types of cigarettes, cigar and moassel were collected from market. Naturally occurring radionuclides (226)Ra and (214)Bi ((238)U series), (228)Ac and (228)Ra ((232)Th series), (40)K and man-made (137)Cs were measured in tobacco using gamma-ray spectrometer. Results show that the average concentrations of (238)U, (232)Th and (40)K were 4.564, 3.940 and 1289.53 Bq kg(-1), respectively. This reflects their origin from the soil by root uptake and fertilisers used in the cultivation of tobacco plants. Concentration of (137)Cs was 0.348 Bq kg(-1) due to root uptake or deposition onto the leaf foliage. For smokers, the annual effective dose due to inhalation of (238)U varied from 49.35 to 139.40 μSv(-1) (average 104.27 μSv y(-1)), while of (232)Th from 23.86 to 111.06 μSv y(-1) (average 65.52 μSv y(-1)). The annual effective dose resulting from (137)Cs was varied from 10.96 to 24.01 nSv y(-1) (average 19.41 nSv y(-1)).     

Single-chain variable fragment (scFv) antibodies optimized for environmental analysis of uranium

Recombinant single-chain variable fragment antibodies (scFv) were specifically generated and selected for the measurement of environmental uranium with an antibody-based sensor. These sFvs, which recognized UO(2)(2+) complexed to 2,9-dicarboxyl-1,10-phenanthroline-acid (DCP), were produced using genetic material obtained from the spleen cells of rabbits immunized with UO(2)(2+)-DCP conjugated to keyhole limpet hemocyanin. Immunoglobulin light chain and heavy chain genes were amplified and cloned into the phagemid pSD3 for generation of a recombinant antibody library and phage-displayed antibodies. The screening process was designed to isolate antibodies that bound to a "loaded" noncovalent complex with high affinity, while selecting against binding to an "unloaded" complex. After five rounds of panning, individual positive scFv clones were used to infect E. coli TG1 and soluble scFv antibodies were purified and characterized. Binding studies showed that the best scFv bound tightly to the UO(2)(2+)-DCP complex (K(d), 19.6 nM). However, because of the depletion experiments performed on this library during the panning process, this scFv bound 1200-fold less tightly (K(d), 23.5 μM) to metal-free DCP. This scFv (clone 3A) was subsequently used to accurately determine the UO(2)(2+) concentrations in environmental water samples using a sensor based on kinetic exclusion analysis. The present studies demonstrate that recombinant scFvs with properties engineered for specific applications (i.e., biosensor-based measurement of metals in groundwater) can be prepared if the correct genetic material and techniques are employed. The phage display system permitted the generation of proteins with very specific binding properties (in this case, high affinity for a metal-chelate complex and low affinity for metal-free chelator). The recombinant scFvs isolated in these studies will be the basis for rapid and affordable assays for the detection of residual uranium in environmental water samples.     

互补的烟草花叶病毒介导表达丙型肝炎的核心抗原

ＴＭＶ两种突变体ＴＳＨｃ和ＴＢＤ，共同接种于烟草叶片。Ｎｏｒｔｈｅｒｎ杂交的结果表明这两种突变体由于功能上互补而产生了系统侵染。ＥＬＩＳＡ和Ｗｅｓｔｅｒｎ的检测结果表明烟草的上部叶片表达了丙型肝炎的核心抗原和ＴＭＶ外壳蛋白质。     

Detection of the recombinant proteins in single transgenic microbial cell using laser tweezers and Raman spectroscopy

Laser tweezers Raman spectroscopy (LTRS) has been used for the rapid detection of recombinant somatolactin protein produced in single Escherichia coli bacteria and Pichia pastoris yeast cell in the current study. A cDNA sequence encoding mature peptide of zebrafish somatolactin beta was inserted into two different expression vectors and transfected into E. coli or P. pastoris yeast cells. We measured Raman spectra of single E. coli cells at different culture times following the induction with isopropyl beta-d-1-thiogalactopyranoside, from which the amount of the generated somatolactin proteins was obtained by the projection of the entire cell's spectrum onto the spectrum of the pure somatolactin proteins or the dot product between these two spectral vectors. We found that the intensity of the somatolactin beta protein-associated spectra from single E. coli cells increased as the function of the culture time, which correlates with the accumulation of recombinant proteins inside the cells. This spectral observation was supported by evidence obtained by conventional methods of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting analyses. The increased intensities of recombinant protein-associated Raman bands were also observed in another expression system, P. pastoris yeast cells. These findings demonstrate that the LTRS is a useful method for rapid sensing of recombination production in single host microorganism in vivo.