抗肿瘤19肽在大肠杆菌中的融合表达与纯化

将重组载体pet32a-19肽转化到表达宿主菌大肠杆菌中,对其进行摇瓶发酵实验来优化重组蛋白在大肠杆菌中的诱导表达条件。通过Ni Sepharose 6 Fast Flow对重组蛋白进行纯化,用梯度透析的方法进行复性。结果表明:筛选出了pET32a-19肽-BL21(DE3)高效表达菌株,在发酵条件为培养基的pH7.0、接种量3%、IPTG浓度0.1mmol/L、IPTG添加时间OD600为0.7、诱导温度41℃和诱导时间5h时蛋白表达量最高,为54.57mg/L,得到纯度为95%以上的融合抗肿瘤19肽,并成功复性出融合蛋白。     

植物甜蛋白monellin基因工程菌表达条件的研究

将带有monellin基因的重组分泌型表达载体pETMO转入大肠杆菌BL21(DE3),得到重组工程菌株BMC33。经IPTG诱导,其所含有的甜蛋白基因可高效表达。研究不同的表达条件对甜蛋白表达水平的影响,得到优化发酵条件:装液量为50 mL/250 mL三角瓶,当培养液OD值达0.8时,添加诱导剂IPTG至终浓为0.9mmol/L,32℃诱导5 h。此时,甜蛋白monellin表达量可高达细菌可溶性蛋白的44.8%。     

抗克伦特罗单链抗体基因工程菌株发醇条件的研究

重组大肠杆菌BL21携带抗克伦特罗单链抗体(seFv)基因,研究了不同培养基、诱导时期、诱导时间、初始pH以及装液量对重组抗体表达量的影响,并对重组质粒稳定性进行研究.结果表明,在初始pH为7.2、装液量为25mL/250mL的LB培养基中,30℃培养菌体生长至A60onm为0.8时,42℃诱导表达6h,scFv表达量可达33.7%.工程菌在无选择压力条件下连续传代120代,保持稳定,100%的菌株携带重组质粒且经诱导后均有重组抗体的表达.     

单核细胞增多性李斯特菌p60蛋白表达条件的优化

在成功构建重组表达载体pET-28a-p60 和获得重组宿主菌E.coli BL21(DE3)的基础上,对重组宿主菌E.coliBL21(DE3)进行表达p60 蛋白条件的优化研究。结果表明：将重组大肠杆菌培养至OD600nm 达0.7～0.8 左右时,加入浓度为1mmol/L 的异丙基-β-D- 硫代吡喃半乳糖苷(IPTG),在摇床温度为25℃,转速为150r/min 条件下诱导表达6h,可得到重组表达的p60 蛋白占总蛋白的比例为42.32%,其中可溶性蛋白占总p60 蛋白的比例为85.36% 左右,为p60 蛋白诱导表达的最优条件。     

重组人基质金属蛋白酶2(MMP-2)血红素样蛋白表达优化

为确定重组大肠杆菌(BL21(DE3)/PET42a-PEX)最佳表达条件。在单因素实验的基础之上,以目的蛋白PEX表达量为响应值,应用Box-Behnken中心组合设计建立数学模型,进行响应面分析。响应面分析确定的最优因素水平组合为:LB培养基pH为7.50,诱导时菌液OD600为0.60,IPTG诱导浓度为0.60mmol/L,诱导培养时间为5.0h。对此工艺条件进行验证得到目的蛋白PEX表达量为20.98mg/L。结果显示,优化了基因重组大肠杆菌表达人PEX的条件,为实现基因重组原核工程菌发酵法制备重组PEX奠定了基础。     

金黄色葡萄球菌SaeP蛋白表达条件的优化

对金黄色葡萄球菌SaeP蛋白在大肠杆菌中的表达工艺进行优化,提高SaeP蛋白的表达量,为深入研究Sae系统对金黄色葡萄球菌产生毒力因子的机制提供支持。通过单因素法以及全面实验法确定最佳的宿主菌、诱导剂浓度、诱导温度以及诱导时间。结果表明:将通过无限克隆法(Restriction Free Cloning, RF)构建的重组质粒,转入大肠杆菌BL21(DE3)表达宿主中,用浓度为0.5 mmol/L的IPTG,在16℃条件下诱导8 h,金黄色葡萄球菌SaeP蛋白的相对表达量最高金黄色葡萄球菌SaeP蛋白的最高表达量占总蛋白的35.14%,较原诱导条件提高25.49%。     

乳糖诱导葡萄球菌肠毒素A基因在大肠杆菌中的表达

以乳糖作为诱导剂代替传统方法中的IPTG,分别从菌龄、诱导剂浓度、诱导时间及诱导剂的添加方式等方面,对乳糖诱导金黄色葡萄球菌肠毒素A(staphylococcal enterotoxins A,SEA)基因在大肠杆菌BL21(DE3)中的表达进行了研究。结果表明,重组大肠杆菌表达SEA的优化条件为:在对数生长期(OD600约为0.6),一次性添加终浓度为0.5 mmol/L的乳糖诱导6 h。目标蛋白的表达量占菌体总蛋白质的36.9%,略低于以IPTG为诱导剂时38.1%的表达量。乳糖价格仅为IPTG的1%左右,因此,在SEA的大规模制备中,使用乳糖作为诱导剂可以大大节约成本。     

新型冠状病毒N蛋白的原核表达与纯化

采用生物信息学方法对新型冠状病毒(SARS-CoV-2)核衣壳蛋白(N蛋白)进行亲疏水性、抗原表位预测及多序列对比分析,构建重组质粒pET28a/N,在大肠杆菌原核表达体系下通过调整诱导温度和时间,提升蛋白溶解度和表达量,并对表达出的重组N蛋白进行纯化和鉴定。结果表明：SARS-CoV-2 N蛋白编码419个氨基酸,等电点(PI)为10.10,无跨膜区,无信号肽序列,局部亲水性较强,全长蛋白抗原指数较高,高度保守,与SARS冠状病毒(SARS-CoV)N蛋白同源性为90.5%;采用大肠杆菌原核表达体系发酵,工程菌BL21(DE3)/pET28a/N在IPTG终浓度为0.2 mmol/L、16℃低温条件下诱导20 h,蛋白呈可溶性表达,且此时蛋白的表达量最高,占总蛋白表达量的70%;通过Ni-NTA亲和层析、凝胶过滤层析纯化后的目的蛋白纯度达90%,分子质量为55 kDa,具有特异性。     

基于DO-stat流加培养控制的海藻糖合成酶发酵条件研究

溶氧浓度对重组大肠杆菌的生长和外源蛋白的高效表达影响较大,为进一步增加菌体量和重组蛋白表达量,该研究采用DO-stat流加培养进行控制,分别研究了溶氧浓度20%、30%、35%和40%条件下大肠杆菌的生长情况以及海藻糖合成酶的表达情况。结果表明,当发酵罐中溶氧浓度控制在30%时,发酵40 h,菌体干质量可达53.9 g/L,是分批发酵时的6倍,海藻糖转化率达到80.9%,是分批发酵时的2.25倍。     

18型人乳头瘤病毒E6蛋白截短体(HPV18 E6*)的基因克隆与表达

目的 构建HPV18 E6*原核重组表达质粒,并优化其蛋白表达条件.方法 以人宫颈癌HeLa细胞cDNA为模版,PCR扩增HPV18 E6*基因,构建重组表达载体HPV18 E6*-pET28a,在大肠杆菌BL21 (DE3)中诱导表达重组蛋白,并优化其表达条件,SDS-PAGE检测重组蛋白表达状况.结果 成功构建了重组表达载体;37℃表达,HPV18 E6*以不溶包涵体为主,表达量占菌体总蛋白的20％左右;15℃主要为可溶形式;包涵体变复性获得纯蛋白.结论 HPV18 E6*蛋白的高效表达为研究其蛋白作用机制及为研究预防和治疗子宫癌奠定基础.     

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.     

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