融合蛋白Trx-T4 DL可溶性表达、纯化与其生物活性应用

构建含SUMO、IF2、GST、NusA、MsyB、Trx和MBP融合标签的重组表达载体,转化到大肠杆菌E.coli Transetta (DE3)中进行自诱导(auto-induction,AI)表达,以提高T4 DNA连接酶(T4 DL)的表达产量。通过磁珠法检测融合蛋白的可溶性表达情况,10% SDS-PAGE电泳结果显示,重组菌Transetta (DE3)(pNBEVⅡ-T4 DL)诱导表达的可溶性融合蛋白Trx-T4 DL的产量最多,经诱导培养条件优化后,Trx-T4 DL的可溶性大幅度提高,确定了最佳诱导条件为30℃、装瓶量50 mL/250 mL、接种量2‰、pH7。分别用镍柱和MagNi磁珠纯化重组菌破碎后上清中的融合蛋白Trx-T4 DL,结果显示后者纯化效率更高,最终获得的融合蛋白浓度为1700.462 mg/L。与其他公司T4 DL活性进行比较,检测其酶活性约为500 U/μL,并使其成功应用于低背景重组克隆载体构建中,为融合蛋白Trx-T4 DL的生产及应用提供理论基础。     

一种新型凋亡素融合蛋白的克隆表达及活性测定

目的 克隆表达EC-SOD3-凋亡素融合蛋白,并检测其生物活性.方法 PCR扩增出apopfin序列,与表达载体EC-SOD3-Pet-28a连接后在大肠杆菌BL21(DE3)中经IPTG诱导,表达产物进行Ni2+-NTA纯化和MIT活性检测.结果 表达载体Pet-28a-EC-SOD3-apoptin经酶切鉴定和序列分析,证明质粒构建正确,转化E. Coli BL21(DE3)后,重组蛋白获得表达,Ni2+-NTA纯化后的凋亡素融合蛋白纯度达到85%以上,经噻唑蓝(MTT)法测定具有活性.结论 成功构建了表达载体Pet-28a-EC-SOD3-apopfin,并在大肠杆菌中表达了EC-SOD3-凋亡素融合蛋白,纯化的蛋白质具有诱导HeLa细胞凋亡的能力.     

一种新型凋亡素融合蛋白的克隆表达及活性测定

目的克隆表达EC-SOD3-凋亡素融合蛋白,并检测其生物活性。方法PCR扩增出apoptin序列,与表达载体EC-SOD3-pET-28a连接后在大肠杆菌BL21(DE3)中经IPTG诱导,表达产物进行Ni2+-NTA纯化和MTT活性检测。结果表达载体pET-28a-EC-SOD3-apoptin经酶切鉴定和序列分析,证明质粒构建正确,转化E.coliBL21(DE3)后,重组蛋白获得表达,Ni2+-NTA纯化后的凋亡素融合蛋白纯度达到85%以上,经噻唑蓝(MTT)法测定具有活性。结论成功构建了表达载体pET-28a-EC-SOD3-apoptin,并在大肠杆菌中表达了EC-SOD3-凋亡素融合蛋白,纯化的蛋白质具有诱导HeLa细胞凋亡的能力。     

重组融合蛋白MBP-PAI的表达、纯化及酶活测定

为了解决亚油酸异构酶(Linoleic Acid Isomerase,PAI)在大肠杆菌中形成包涵体的问题,选择麦芽糖结合蛋白(Maltose-Binding Protein,MBP)标签和低温诱导表达系统pColdV,构建了大肠杆菌重组表达菌株E.coli BL21(pCold-Mpai),并对其诱导表达条件进行了优化。SDS-PAGE电泳结果显示,融合蛋白MBP-PAI成功表达,重组菌的最佳诱导表达条件为:诱导温度15℃、IPTG添加量0.1 mmol/L、诱导时间12 h,在该诱导条件下,与未融合MBP的PAI相比,MBP-PAI的可溶性表达量为后者的18倍、酶活为后者的1.5倍。经过MBPTrap HP亲和层析柱纯化后,MBP-PAI纯蛋白质的比酶活为1.58 U/mg,能够转化亚油酸形成反10,顺12-共轭亚油酸。     

硫氧还蛋白促进人胰岛素样生长因子-1在大肠杆菌中高效可溶表达

为实现人胰岛素样生长因子-1(Insulin-like growth factor-1,IGF-1)在大肠杆菌中的高效可溶表达,获得大量具生物活性的IGF-1。应用融合PCR技术构建trx-igf1融合基因,克隆至p ET30a载体。重组载体pET30a-Trx-IGF-1转化大肠杆菌C43(DE3),并进行条件优化大量表达可溶性蛋白Trx-IGF-1。利用His标签纯化表达产物,对纯化蛋白进行Western blot与生物活性分析。构建的pET30a-Trx-IGF-1重组载体转化大肠杆菌C43(DE3)后,在30℃培养条件下1 mmol/L IPTG诱导表达5 h,获得大量以可溶形式表达的融合蛋白Trx-IGF-1;采用Ni离子亲和层析,获得纯度达90%以上的融合蛋白,经Western blot检测具有IGF-1抗原活性。生物学活性检测显示,融合蛋白Trx-IGF-1能明显促进NIH3T3细胞增殖及细胞周期进展。本研究应用的载体蛋白Trx,能实现在大肠杆菌中高效可溶表达具生物活性的IGF-1,为包涵体蛋白在大肠杆菌中的可溶性表达提供借鉴。     

重组融合蛋白MBP-BSH 在大肠杆菌中的表达及其纯化、功能鉴定

选择IF2融合标签,另选择含SUMO、GST、NusA和MBP融合标签的质粒构建表达载体。SDS-PAGE电泳结果显示,重组菌Escherichia Rosetta(DE3) (pLS932-BSH)的诱导表达细胞提取液上清出现了明显的融合蛋白MBP-BSH条带,经诱导表达条件优化后,MBP-BSH可溶性大幅度提高。用Ni-NTA Resin和Amylose Resin分别进行目标蛋白纯化,结果显示前者效果更好,并且C端融合His-Tag更有利于其与Ni离子结合,MBP-BSH蛋白纯化量更大,杂带更少。茚三酮显色反应测定酶活力,结果显示纯化后的MBP-BSH的酶比活力为2.4282U/mg。     

水稻密码子优化的cry2A^＊基因在大肠杆菌中的表达及其表达产物的纯化

通过PCR从克隆载体puC18-3Z/Cry2A*上扩增水稻偏爱型密码子优化的抗虫基因cry2A*,经限制性内切酶Nde I和BamH I双酶切定向插入到原核表达载体pET-28a( ),成功构建了在表达蛋白的N端只带有6个组氨酸标签的融合蛋白表达载体pET-28a( )/Cry2A*,并转入人肠杆菌BL21(DE3)中.通过对其表达条件进行优化,发现在IPTG浓度为0.05mmol/L、诱导时问为3h、诱导温度为20℃的表达条件下目的蛋白大部分以可溶形式进行表达.采用Ni-NTA亲和柱纯化得到高纯度目的蛋白,薄层扫描分析蛋白纯度达到95%.     

鲎素Tachyplesin Ⅰ在大肠杆菌中的重组表达及其抑菌活性

为了高效制备抗菌肽,本文将鲎素抗菌肽目的基因Tachyplesin-1(TP1)在大肠杆菌BL21中进行表达。首先构建表达质粒pET32a-TP1并转化大肠杆菌BL21(DE3),在IPTG诱导下进行目的融合蛋白表达,并利用His标签与镍柱亲和层析对融合蛋白进行纯化。纯化后的融合蛋白经羟胺裂解液切割和质谱分析后,获得单一的TP1重组蛋白,最后对其抑菌活性进行表征。结果表明,重组菌在37℃经IPTG诱导后,融合蛋白表达成功,TrxA-TP1融合蛋白的分子量在20 kDa左右。经羟胺裂解得到的重组蛋白TP1对于金黄色葡萄球菌(Staphylococcus aureus)与枯草芽孢杆菌(Bacillus subtilis)均具有良好抑菌活性,最小抑菌浓度分别为6和10 mg/L。本研究为鲎素抗菌肽TP1的开发应用和大量生产奠定了基础。     

小鼠核糖核酸酶抑制剂异源高效可溶性表达、纯化及活性研究

为获得高效可溶性表达的小鼠核糖核酸酶抑制剂(MRNI),本文通过构建含SUMO、IF2、GST、NusA、MsyB、Trx和 MBP融合标签的重组表达载体,以大肠杆菌BL21(DE3)作为宿主菌进行自诱导(auto-induction,AI)表达,利用MagNi磁珠检测及电泳分析MRNI的表达状况,并对其培养温度及时间进行优化,同时,与其他公司核糖核酸酶抑制剂(RI)活性进行对比。结果表明,重组菌BL21(DE3)(pNBEⅡ-MRNI)诱导表达的可溶性融合蛋白IF2-MRNI的产量最高,最适诱导条件为37 ℃,诱导培养6 h,20 ℃培养24 h。磁珠纯化后获得的融合蛋白IF2-MRNI浓度为3621.3 mg/L,检测其酶活性约为40 U/μL。该酶具有抑制RNase A活性,防止RNA被降解的作用,为RI的生产及应用提供理论基础。     

抗克伦特罗单链抗体-碱性磷酸酶融合蛋白表达条件的初步研究

初步研究抗克伦特罗单链抗体-碱性磷酸酶融合蛋白(CBLscFv-AP)的表达条件,提高CBLscFv-AP融合蛋白的表达量。采用液体发酵法培养抗克伦特罗单链抗体-碱性磷酸酶融合蛋白(CBLscFv-AP)基因工程菌,研究不同宿主菌、初始pH、诱导时期、诱导时间和培养基对CBLscFv-AP融合蛋白表达量的影响以及重组质粒的稳定性。选用E·coliBL21(DE3)pLysS为宿主菌,在初始pH为7·4的LB培养基中培养至A600nm为0·8～0·9时,诱导表达8h,为CBLscFv-AP融合蛋白最佳表达条件。而且重组质粒pBV220-scFv-phoA具有较好的结构稳定性。在该表达条件下,提高了CBLscFv-AP融合蛋白的表达量,为进行大规模发酵生产CBLscFv-AP融合蛋白奠定基础。     

Reliable identification of grapevine rootstock varieties using RAPD PCR on woody samples

Since grapevine ( Vitis spp .) rootstock material is being traded increasingly as disbudded woody material a lack of distinctive morphological features on such material necessitates an alternative and reliable means of identification. Methods described here were developed for rapid and efficient extraction of DNA from woody samples rich in phenolic compounds and polysaccharides, and for subsequent identification of varieties by RAPD PCR. Using these methods, and with the application of only one selected RAPD primer, we were able to differentiate sixteen rootstock varieties, including the seven varieties most commonly used in Germany. Problems commonly encountered with reproducibility of RAPD patterns were avoided by choosing primers with a dinucleotide sequence and a high G/C content that allowed a rather high annealing temperature of 45°C. Methods described here should also be useful for other horticultural crops, especially those with woody tissues rich in phenolic compounds and polysaccharides.     

An internet compendium of analytical methods and spectroscopic information for monomers and additives used in food packaging plastics

An internet website (http://cpf.jrc.it/smt/) has been produced as a means of dissemination of methods of analysis and supporting spectroscopic information on monomers and additives used for food contact materials (principally packaging). The site which is aimed primarily at assisting food control laboratories in the European Union contains analytical information on monomers, starting substances and additives used in the manufacture of plastics materials. A searchable index is provided giving PM and CAS numbers for each of 255 substances. For each substance a data sheet gives regulatory information, chemical structures, physico-chemical information and background information on the use of the substance in particular plastics, and the food packaging applications. For monomers and starting substances (155 compounds) the infra-red and mass spectra are provided, and for additives (100 compounds); additionally proton NMR are available for about 50% of the entries. Where analytical methods have been developed for determining these substances as residual amounts in plastics or as trace amounts in food simulants these methods are also on the website. All information is provided in portable document file (PDF) format which means that high quality copies can be readily printed, using freely available Adobe Acrobat Reader software. The website will in future be maintained and up-dated by the European Commission's Joint Research Centre (JRC) as new substances are authorized for use by the European Commission (DG-ENTR formerly DGIII). Where analytical laboratories (food control or other) require reference substances these can be obtained free-ofcharge from a reference collection housed at the JRC and maintained in conjunction with this website compendium.     

Chlorohydrins of bisphenol A diglycidyl ether (BADGE) and of bisphenol F diglycidyl ether (BFDGE) in canned foods and ready-to-drink coffees from the Japanese market

BADGE.2HCl and BFDGE.2HCl were determined in 28 samples of ready-to-drink canned coffee and 18 samples of canned vegetables (10 corn, 5 tomatoes and 3 others), all from the Japanese market. HPLC was used as the principal analytical method and GCMS for confirmation of relevant LC fractions. BADGE.2HCl was found to be present in one canned coffee and five samples of corn, BFDGE.2HCl in four samples of canned tomatoes and in one canned corn. No sample was found which exceeded the 1mg/kg limit of the EU for the BADGE chlorohydrins. However the highest concentration was found for the sum of BFDGE.2HCl and BFDGE.HCl.H₂O at a level of 1.5mg/kg. A Beilstein test confirmed that all cans containing foods contaminated with BADGE.2HCl or BFDGE.2HCl had at lest one part coated with a PVC organosol.     

Aroma characterization of various apricot varieties using headspace–solid phase microextraction combined with gas chromatography–mass spectrometry and gas chromatography–olfactometry

The characterization of the aromatic profile of several apricot cultivars with molecular tracers in order to obtain objective data concerning the aromatic quality of this fruit was undertaken using headspace–solid phase microextraction (HS–SPME). Six apricot cultivars were selected according to their organoleptic characteristics: Iranien, Orangered, Goldrich, Hargrand, Rouge du Roussillon and A4025. The aromatic intensity of these varieties measured by HS–SPME–Olfactometry were defined and classified according to the presence and the intensity of grassy, fruity and apricot like notes. In the six varieties, 23 common volatile compounds were identified by HS–SPME–GC–MS. Finally, 10 compounds, ethyl acetate, hexyl acetate, limonene, β-cyclocitral, γ-decalactone, 6-methyl-5-hepten-2-one, linalool, β-ionone, menthone and (E)-hexen-2-al were recognized by HS–SPME–GC–O as responsible of the aromatic notes involved in apricot aroma and considered as molecular tracers of apricot aromatic quality which could be utilized to discriminate apricot varieties.     

European priorities for research to support legislation in the area of food contact materials and articles

A strong science base is required to underpin the planning and decision-making process involved in determining future European community legislation on materials and articles in contact with food. Significant progress has been made in the past 5 years in European funded work in this area, with many developments contributing to a much better understanding of the migration process, and better and simpler approaches to food control. In this paper this progress is reviewed against previously identified work-areas (identified in 1994) and conclusions are reached about future requirements for R&D to support legislation on food contact materials and articles over the next 5 or so years.     

Textural Properties of Cold-set Gels Induced from Heat-denatured Whey Protein Isolates

A 9% whey protein (WP) isolate solution at pH 7.0 was heat-denatured at 80°C for 30 min. Size-exclusion HPLC showed that native WP formed soluble aggregates after heat-treatment. Additions of CaCl2 (10–40 mM), NaCl (50–400 mM) or glucono-delta-lactone (GDL, 0.4–2.0%, w/v) or hydrolysis by a protease from Bacillus licheniformis caused gelation of the denatured solution at 45°C. Textural parameters, hardness, adhesiveness, and cohesiveness of the gels so formed changed markedly with concentration of added salts or pH by added GDL. Maximum gel hardness occurred at 200 mM NaCl or pH 4.7. Increasing CaCl2 concentration continuously increased gel hardness. Generally, GDL-induced gels were harder than salt-induced gels, and much harder than the protease-induced gel.     

Tests of potential functional barriers for laminated multilayer food packages. Part I: Low molecular weight permeants

The advent of the functional barrier concept in food packaging has brought with it a requirement for fast tests of permeation through potential barrier materials. In such tests it would be convenient for both foodstuffs and materials below the functional barrier (sub-barrier materials) to be represented by standard simulants. By means of inverse gas chromatography, liquid paraffin spiked with appropriate permeants was considered as a potential simulant of sub-barrier materials based on polypropylene (PP) or similar polyolefins. Experiments were performed to characterize the kinetics of the permeation of low molecular weight model permeants (octene, toluene and isopropanol) from liquid paraffin, through a surrogate potential functional barrier (25 μm-thick oriented PP) into the food simulants olive oil and 3% (w/v) acetic acid. These permeation results were interpreted in terms of three permeation kinetic models regarding the solubility of a particular model permeant in the post-barrier medium (i.e. the food simulant). The results obtained justify the development and evaluation of liquid sub-barrier simulants that would allow flexible yet rigorous testing of new laminated multilayer packaging materials.     

Occurrence of bisphenol-F-diglycidyl ether (BFDGE) in fish canned in oil

The levels of bisphenol-F-diglycidyl ether (BFDGE) were quantified as part of a European survey on the migration of residues of epoxy resins into oil from canned fish. The contents of BFDGE in cans, lids and fish collected from all 15 Member States of the European Union and Switzerland were analysed in 382 samples. Cans and lids were separately extracted with acetonitrile. The extraction from fish was carried out with hexane followed by re-extraction with acetonitrile. The analysis was performed by reverse phase HPL C with fluorescence detection. BFDGE could be detected in 12% of the fish, 24% of the cans and 18% of the lids. Only 3% of the fish contained BFDGE in concentrations considerably above 1mg/kg. In addition to the presented data, a comparison was made with the levels of BADGE (bisphenol-A-diglycidyl ether)analysed in the same products in the context of a previous study.     

Contribution of European research to risk analysis

The European Commission's, Quality of Life Research Programme, Key Action 1—Health, Food & Nutrition is mission-oriented and aims, amongst other things, at providing a healthy, safe and high-quality food supply leading to reinforced consumer confidence in the safety of European food. Its objectives also include the enhancing of the competitiveness of the European food supply. Key Action 1 is currently supporting a number of different types of European collaborative projects in the area of risk analysis. The objectives of these projects range from the development and validation of prevention strategies including the reduction of consumers risks; development and validation of new modelling approaches; harmonization of risk assessment principles, methodologies, and terminology; standardization of methods and systems used for the safety evaluation of transgenic food; providing of tools for the evaluation of human viral contamination of shellfish and quality control; new methodologies for assessing the potential of unintended effects of genetically modified (genetically modified) foods; development of a risk assessment model for Cryptosporidium parvum related to the food and water industries; to the development of a communication platform for genetically modified organism, producers, retailers, regulatory authorities and consumer groups to improve safety assessment procedures, risk management strategies and risk communication; development and validation of new methods for safety testing of transgenic food; evaluation of the safety and efficacy of iron supplementation in pregnant women; evaluation of the potential cancer-preventing activity of pro- and pre-biotic ('synbiotic') combinations in human volunteers. An overview of these projects is presented here.