乳源性免疫活性肽在E.coli BL21中分泌表达的诱导条件优化

设计4个乳源性免疫活性肽的目的基因后,利用基因重组技术成功构建原核表达载体pTYB11,并将重组质粒转化到E.coli BL21中,通过改变异丙基-β-D-硫代吡喃半乳糖苷 (IPTG)的浓度、培养时间和培养温度,设计正交试验来优化乳源活性小肽的IPTG诱导表达条件,使目的蛋白可在E.coli BL21中高效表达。依据15% SDS-PAGE电泳分析得到融合蛋白的表达量,选出最适诱导条件为IPTG终浓度0.1～0.2mmol/L,在12～15℃条件下培养20h,得到大小为59.2kD左右的融合目的蛋白,其表达量可占总蛋白表达量的40%,经Western Blotting鉴定正确。     

Nisin-rbLF-N融合基因的构建及其在大肠杆菌中的表达

针对乳链菌肽(nisin)抑菌谱窄的缺点,选择对抗菌谱较广且具有较强抗性的牛乳铁蛋白氨基末端多肽(rbLF-N)为材料,构建融合基因Nisin-rbLF-N。将融合基因克隆到原核表达载体pGEX-4T1 中,转化E. coli BL21(DE3)进行诱导表达,将诱导表达的产物进行Tricine-SDS-PAGE 蛋白电泳及抑菌活性检测。结果表明,克隆菌经诱导后可表达出可观的融合蛋白,融合蛋白以包涵体形式存在,包涵体经洗涤、尿素溶解、复性后具有抗菌生物活性。     

Mincle受体蛋白克隆、原核表达与纯化

本研究通过应用基因工程技术,构建融合基因pET14b-Mincle的原核表达载体,并将其转化到大肠杆菌BL21(DE3)中诱导表达目的蛋白Mincle,并对其表达产物进行纯化、鉴定、透析复性。采用RT-PCR技术扩增小鼠Mincle的基因片段,将其克隆于载体pMD18-T中,并克隆到带有His-tag的原核表达载体pET14b中;重组质粒经酶切鉴定、序列比对验证正确后转化大肠杆菌BL21(DE3)中经IPTG诱导表达目的蛋白。经1mmol/LIPTG在37℃下诱导5h获得了分子量约为22kDa的重组融合蛋白的优化表达,SDS-PAGE、Western Blot和ELISA证实了重组蛋白的特异性。Mincle以包涵体形式在宿主中表达,利用Ni2+亲和柱进行纯化和生物膜透析复性,纯化和透析后的蛋白经WesternBlot、ELISA法定性鉴别以及用白色念珠菌(SC5314)整个灭活细胞对复性后蛋白的活性测定,透析后的Mincle重组融合蛋白能与白色念珠菌的特异性结合体现其生物活性,表明获得了具有活性的蛋白,为后续研究打下基础。     

TRPV4原核表达纯化系统的构建及生物信息学分析

目的:构建非选择性阳离子通道蛋白TRPV4的原核表达纯化系统,并对其进行生物信息学分析,为与该目标蛋白相关疾病的研究和药物活性成分筛选提供技术支持.方法:将人源TRPV4基因分别克隆至pET-28a(+)、pET-32a、pET-15b、pGEX-5X-1、pEX-4T和pGEX-6p-1等6种表达载体,并利用BL21...     

重组牛乳铁素融合表达质粒的构建及其抑菌活性分析

为建立生物合成牛乳铁素的方法,根据牛乳铁素氨基酸序列确定其编码序列,利用PCR 技术获得基因扩增产物,接着将该产物与编码鱼腥蓝细菌Anabaena sp. PCC 7120 的DNA 聚合酶基因dnaENI 中断裂的内含肽基因Asp dnaEIn 的PCR 产物进行重组PCR,将获得的重组融合基因再克隆到表达载体pET-28a 构建重组融合表达质粒。结果显示,融合表达质粒转化大肠杆菌后经诱导能在大肠杆菌表达宿主Escherichia.coli BL21(DE3) 中大量表达,且细胞裂解液与纯化的Anabaena sp. PCC 7120中含断裂内含肽Asp DnaEIc的DNA聚合酶蛋白DnaECI混合反应后对金黄色葡萄球菌具有抑制作用。     

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.     

桑树泛素延伸蛋白基因的原核表达

对前期克隆的桑树泛素延伸蛋白基因进行了生物信息学及原核表达分析,以pGEX-4T-1为载体,构建了桑树泛素延伸蛋白基因重组表达质粒,转入受体菌E.coli BL21。经IPTG诱导后,基因在大肠杆菌中得到有效表达。为提高表达效果,从不同诱导时间对目的蛋白表达条件进行优化,用SDS-PAGE电泳检测,结果表明在经37℃条件下,经1.0 mmol/L的IPTG诱导6 h后,原核表达效果明显提高。研究表明,桑树泛素延伸蛋白基因与其他植物具有较高的同源性,因此利用模式植物研究其功能及分子进化分析更加高效,为进一步探讨桑树的逆境生理机制提供了理论依据。     

碱性蛋白酶抑制剂lupI-MSSS原核表达载体的构建及其蛋白表达、纯化

低温碱性金属蛋白酶MP是从菌株YS-80-122中提取纯化的,属于沙雷氏蛋白酶家族,与该家族报道的其他酶的结构类似,在MP基因下游有一抑制剂基因lup I,该抑制剂可以完全抑制蛋白酶MP的活性。在野生型抑制剂基因的基础上,通过定点突变将Lup I的N端增加Met-Ser-Ser-Ser,命名为Lup I-MSSS,通过构建p ET28alup I-MSSS原核表达载体,转化大肠杆菌BL21(DE3)并诱导表达,SDS-PAGE凝胶电泳结果表明该蛋白的大小约11 kDa,与预测的蛋白分子量一致。对影响蛋白诱导表达的诱导剂添加时间、诱导温度、异丙基硫代半乳糖苷(IPTG)浓度、诱导时间4个因素进行优化,并经初步优化得到了其诱导表达的条件为:接种量2%,诱导剂添加时间3 h,诱导剂IPTG终浓度0.5 mmol/L,诱导温度为37℃,诱导时间为8 h。表达的蛋白依次通过超滤,Superdex 200凝胶过滤层析,Q-Sepharose离子交换层析进行纯化,结果显示目的蛋白纯化倍数为20,比活力为15 720U/mg,活性回收率达60.7%,纯化后的lup I通过高效液相色谱法进行纯度分析,其纯度可达99%以上。     

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。     

玉米黄烷酮-3-羟化酶原核表达条件对香橙素生成量的影响

香橙素是类黄酮代谢途径中重要的中间化合物,黄烷酮-3-羟化酶(flavanone-3-hydroxylase,F3H)能以柚皮素为底物催化C3位接羟基从而生成香橙素.为实现香橙素绿色发展,该研究利用微生物法,对来自玉米的Zmf3h构建大肠杆菌原核表达系统后诱导ZmF3H蛋白表达.通过发酵实验验证ZmF3H在胞内可溶性表...     

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.     

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.     

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.     

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.     

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.     

Analysis of benzo[a]pyrene in spiked fatty foods by second derivative synchronous spectrofluorimetry after microwave-assisted treatment of samples

A simple, rapid and inexpensive method has been developed for the determination of benzo[a     

The development of reference materials for paralytic shellfish poisoning toxins in lyophilized mussel. II: Certification study

This paper describes the second part of a project undertaken to develop certified mussel reference materials for paralytic shellfish poisoning toxins. In the first part two interlaboratory studies were undertaken to investigate the performance of the analytical methodology for several PSP toxins, in particular saxitoxin and decarbamoyl-saxitoxin in lyophilized mussels, and to set criteria for the acceptance of results to be applied during the certification exercise. Fifteen laboratories participated in this certification study and were asked to measure saxitoxin and decarbamoyl-saxitoxin in rehydrated lyophilized mussel material and in a saxitoxin-enriched mussel material. The participants were allowed to use a method of their choice but with an extraction procedure to be strictly followed. The study included extra experiments to verify the detection limits for both saxitoxin and decarbamoyl-saxitoxin. Most participants (13 of 15) were able to meet all the criteria set for the certification study. Results for saxitoxin.2HCl yielded a certified mass fraction of <0.07 mg/kg in the rehydrated lyophilized mussels. Results obtained for decarbamoyl-saxitoxin.2HCl yielded a certified mass fraction of 1.59+/-0.20 mg/kg. The results for saxitoxin.2HCl in enriched blank mussel yielded a certified mass fraction of 0.48 +/- 0.06 mg/kg. These certified reference materials for paralytic shellfish poisoning toxins in lyophilized mussel material are the first available for laboratories to test their method for accuracy and performance.     

Announcing a new international peer-reviewed journal:Food Science and Human Wellness now accepting manuscript submission in English

<正>We are pleased to announce the launch of a new international peer-reviewed journal-Food Science and Human Wellness,ISSN 2213-4530,which is an open access journal,produced and hosted by Elsevier B.V.on behalf of Beijing Academy of Food Sciences.Food Science and Human Wellness is an international peer-reviewed English journal that provides a forum for the dissemination of the