利用重组基因工程菌制备降血压肽的工艺研究

本文利用正交实验方法,对重组降血压肽基因工程菌细胞破碎所得上清液中的融合蛋白GST-AHP,进行亲和层析吸附上柱条件研究.在此基础上,借助高效液相色谱对融合蛋白凝血酶酶切条件进行了研究,并对此分离条件下所得的降血压肽段的体外生物学活性进行初步确定.实验表明,基因工程菌细胞破碎上清液中的融合蛋白GST-AHP进行亲和吸附的最佳上柱条件为上柱量为4 mg/mL,上柱流速为1.5 mL/min,上柱1次,此条件下融合蛋白的吸附量达3.86 mg/mL,洗脱后其回收率达96.5%;凝血酶酶切的最佳工艺条件为1 mg GST-AHP融合蛋白添加凝血酶15 U,22℃酶切8 h,此条件下可得降血压肽段102 μg.分离纯化所得降血压肽段的体外生物学活性良好.     

cA-GFP融合蛋白在大肠杆菌中的高效功能性表达

为了实现乳酸乳球菌锚定蛋白cA在大肠杆菌中可溶表达以及直观检测其生物学活性,以乳酸乳球菌MG1363为模板,扩增N-乙酰葡萄糖胺糖苷酶AcmA基因,将其C末端序列与GFP基因融合,连接至大肠杆菌表达载体pET28a,构建重组质粒pET28a-cA-GFP,转化至大肠杆菌表达菌株BL21(DE3),通过低温诱导表达重组蛋白cA-GFP。工程菌超声破碎后的上清液与经热酸处理的乳酸乳球菌常温孵育,经SDS-PAGE和荧光显微镜检测。结果表明,工程菌可以表达分子量约53ku的目的融合蛋白质,与预期大小相符。cA-GFP通过锚定蛋白cA的引导回向锚定,成功将GFP展示在乳酸乳球菌表面,目的蛋白cA-GFP在乳酸乳球菌表面展示量为121mg/g干重菌体。GFP锚定至乳酸乳球菌后于4℃保存,连续6d测定其荧光强度,荧光强度仍可达82.2%,证明其稳定性较好。成功获得了具有生物学功能的cA-GFP可溶性蛋白,为进一步展示功能性外源蛋白奠定了坚实的基础。     

乳酸菌胞外蛋白水解酶基因的克隆及在大肠杆菌中的表达

目的：构建连接乳酸菌胞外蛋白水解酶基因的非抗性重组质粒。方法：以瑞士乳杆菌基因组DNA 为模板,克隆胞外蛋白水解酶基因,连接到以thyA 为选择压力的非抗性穿梭表达载体pW425et 上,构建重组质粒pW425et-R,转化入thyA 基因缺陷型E.coli X13 感受态细胞,SDS-PAGE 电泳检测显示该水解酶得到了表达。结果：成功构建了重组质粒pW425et-R,胞外蛋白水解酶基因在E.coli X13 中得到正确表达。结论：成功地表达了乳酸菌胞外蛋白水解酶基因,可为进一步制备具降血压功能基因工程乳酸菌提供参考。     

绿色木霉纤维二糖水解酶基因的克隆及其在乳酸菌中的表达与鉴定

采用乳酸菌表达系统进行绿色木霉纤维二糖水解酶基因cbhⅡ 的表达研究。参照GenBank上发表的绿色木霉（Trichoderma viride）cbhⅡ 基因（GenBank登录号：M55080）设计引物并通过聚合酶链式反应克隆获得其cDNA序列长1 441 bp。为了达到表达分泌该酶的目的,在其基因上游序列前添加了大小为80 bp的信号肽序列。进一步通过生物信息学方法将密码子按照乳酸菌的偏好性进行了优化,通过全基因合成获得了优化后的cbhⅡ 基因。将该基因与穿梭表达载体pMG36e连接, 构建原核表达载体pMG36e-S-cbhⅡ ;利用电转化方法将其转入乳酸乳球菌NZ3900感受态细胞中构建重组乳酸菌,纯化的重组菌蛋白通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳检测,得到一条约为53 kD的目的蛋白条带,与预期大小相符;利用3,5-二硝基水杨酸法测定培养基上清液中水解纤维素酶的活力达到16.7 U/mL,菌体裂解液上清液和菌体沉淀几乎没有酶活力。结果表明,纤维二糖水解酶基因信号肽序列被乳酸乳球菌正确识别,并成功实现了胞外表达。     

重组融合人血清白蛋白-人白介素-2 C125A突变体在毕赤酵母中的表达

构建编码人血清白蛋白-人白介素-2 C125A的重组表达质粒,在Pichia pastoris GS115中表达,获得具有较高IL-2活性的融合蛋白.设计并合成符合P. pastoris密码子偏好,且含有C125A突变的IL-2编码基因IL-2m,通过酶切连接方法将其与人血清白蛋白(HSA)的编码基因连接为融合蛋白HSA-IL2m的编码基因.克隆到表达质粒pPIC9K中,电击转化P. pastoris GS115感受态细胞,获得重组P. pastoris GS115/pPHIm基因工程菌.摇瓶发酵获得分泌表达产物.结果: Western blot鉴定结果显示该融合蛋白与IL-2、HSA的抗体都能发生免疫反应.经脱盐、冻干制备的粗蛋白的IL-2生物学活性为1.51×106 IU/mg.结论:在P. pastoris GS115中成功表达了具有人白介素-2生物学活性的HSA-IL2m融合蛋白.     

乳酸菌红色荧光蛋白融合表达系统的构建

目的:构建以红色荧光蛋白基因(dsred2)为标记的乳酸菌融合表达系统p MG36e-dsred2,并以α-淀粉酶(amy)为报告基因实现融合基因dsred2-amy在乳酸菌内的融合表达。方法 :通过PCR的方法以质粒p PIC9kdsred2为模板扩增得到dsred2基因的全部编码区序列,并将其连接到克隆型载体p MG36e上,得到重组载体p MG36e-dsred2。以地衣芽孢杆菌基因组为模板,扩增只带有起始密码子而不带有终止密码子的α-淀粉酶基因(amy),将其连接到克隆型载体p MG36e-dsred2上,获得融合表达载体p MG36e-dsred2-amy。最后将该载体电转化干酪乳杆菌,利用荧光显微镜观察红色荧光基因表达情况,用淀粉平板检测淀粉酶活性。结果:通过PCR的方法分别扩增到大小为694bp的红色荧光蛋白基因(dsred2)和大小为1 554 bp的淀粉酶基因(amy)。将获得的两个基因片段进行测序,结果与NCBI数据库进行对比,dsred2基因的匹配率为100%,amy基因同源性为99.54%。成功构建了乳酸菌表达系统p MG36e-dsred2及融合表达载体p MG36e-dsred2-amy。在荧光显微镜下观察菌体有荧光且淀粉平板上有明显的透明圈。结论:融合表达载体的构建成功为乳酸菌在生物体内的定植、分布、存活等研究提供了方法,也为研究外源蛋白在乳酸菌内的表达的分泌(细胞内、细胞壁、细胞外表达)、活性检测等奠定了基础。     

植物乳杆菌细菌素基因plnEF的克隆与异源表达

目的:构建目的基因plnEF原核表达系统,诱导工程菌BL21-p ET28a-PL-plnEF表达目的融合蛋白并纯化,以期获得大量纯度较高的植物乳杆菌PlnEF细菌素,开发新的食品防腐剂。方法:构建含plnEF基因的重组质粒,将其转入大肠杆菌BL21中,经过IPTG诱导大量表达目标蛋白,融合蛋白PlnEF经纯化后进行分子量大小、抑菌活性等的检测。结果:重组质粒pET28a-PL-plnEF在大肠杆菌BL21中成功表达,合成PlnEF蛋白,其分子量为15.6 k Da,且该融合蛋白对大肠杆菌JM109具有良好的抑菌活性。结论:plnEF基因片段能够在原核细胞中正确表达且具有活性,本文为进一步研究开发该细菌素作为生物防腐剂奠定了基础。     

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。     

一株乳酸菌细菌素的检测及其产生的最佳条件的研究

以一株分离自牙鲆肠道的乳酸菌L15为材料,用打孔法检测其发酵上清液对枯草杆菌、大肠杆菌、金黄色葡萄球菌的抑制作用。排除有机酸和过氧化氢的干扰,证明上清液中含有抗菌的活性成分;用蛋白酶处理后,抑菌活性明显降低,表明起抑菌作用的是蛋白类物质的细菌素。研究表明,培养温度30℃、时间48h、pH值为7时是乳酸菌细菌素的最佳产生条件。对乳酸菌发酵上清液中的细菌素进行分离纯化,SDS-PAGE电泳表明乳酸菌的细菌素是一种具有抗菌活性的多肽,蛋白或蛋白复合物。     

大豆球蛋白A1a酸性多肽的原核表达及纯化

为建立大豆球蛋白免疫检测方法及致敏机理的深入研究提供基础材料,人工合成了大豆球蛋白A1a酸性多肽基因的cDNA序列,通过聚合酶链式反应(PCR)和Bam HI、Kpn I双酶切构建了原核表达载体pET30a-A1a,将重组质粒转化大肠杆菌(E.coli)BL21后用异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达。SDS-PAGE分析表明,A1a酸性多肽融合蛋白能以可溶蛋白形式进行分泌表达,并且诱导5h后,A1a酸性多肽融合蛋白表达量达到最高。经His亲和层析纯化,获得纯度达90%的融合A1a蛋白,分子质量大小约为38kD。Western blotting显示,所获得的A1a融合蛋白能与对豆粕过敏的仔猪血清发生免疫反应,表明所获得融合蛋白具有免疫活性。     

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.     

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.     

Review of techniques to manufacture micro-hydrogel particles for the food industry and their applications

Microgels are ‘soft’ microscopic cross-linked polymeric particles that are being increasingly exploited in a variety of industries for rheology control, encapsulation and targeted delivery. They are valued because of the ability to tune their functionality to address specific applications in oil recovery, coatings, drug delivery, cosmetics, personal care and foods. Food microgels are typically biopolymer hydrogels in the form of microspheres, nanospheres (also called nanogels), spheroids and fibres. The utilisation of engineered microgels in foods has so far been limited, despite their great potential to address several needs in the food industry, including: satiety control, encapsulation of phytonutrients and prebiotics, texture control for healthier food formulations (e.g. reduced fat products), and targeting delivery to specific areas in the digestive tract. We review the scientific and patent literature on the utilisation and manufacturing methods for producing microgels with an emphasis on micro-hydrogels for food applications.     

12—A CRITIQUE OF SOME HYPOTHESES RELATING TO THE HETEROGENEITY OF THE HIGH-SULPHUR PROTEINS OF WOOL

Joubert and Burns prepared a large number of fractions from the high-sulphur proteins of wool and estimated their molecular weights and amino-acid compositions. Their data have been re-examined in order to look for statistically significant interrelations between amino acids and between the proportion of various amino acids and molecular weight. Statistical analysis of the data is also used to examine the credibility of some hypotheses concerning the mechanism of keratin biosynthesis and to provide further evidence for the existence of families of proteins within the high-sulphur fractions of wool.     

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.     

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 Latest Data about Development of Chinese Printing Industry in 2013

In the 2014 China（Shanghai）International Printing Week,Director Wang Yanbin released the latest data about development of Chinese printing industry in 2013.According to statistics,in 2013,the total output value of Chinese printing industry exceeded 1trillion Yuan for the first time,reaching 1.03985 trillion Yuan.There were 105,000 printing enterprises in China,employees were 3.415 million.The total asset was 1.06247 trillion Yuan;     

The State Council Issued "Directory of Government Approved Investment Projects(2013 Edition)"

正On December 2nd,2013,the State Council issued the notification of"Directory of Government Approved Investment Projects(2013 Edition)"(hereafter referred to as"notification").It is pointed out in the"notification"that in order to further deepen reforms in investment systems and administrative examination and approval systems,simplify administrative procedures and delegate powers to lower levels,earnestly     

China textile machinery: taking off in progress

正Among the 1600 exhibitors who take apart in the ITMA ASIA+CITME2014 2/3 are Chinese manufactures.If the numerous figures failed to attract your attention,the increase of quality should draw your focus.To adopt the demand of developing textile machine market,domestic textile machinery enterprises now follow the slogan of"technology drives development"to enhance product competitiveness.Our domestic sellers will showcase product ranging from spinning,weaving,dyeing and printing,     

Top Ten News of China's Paper Industry 2013

On December 24th, 2013, the meeting on the selection of top 10 news of China＇s paper industry 2013 sponsored by 〈China Paper Newsletters〉 was held in Beijing. The yearly selection of the top l0 news, which began in 2000, has become a brand activity widely recognized in the industry thanks to the support from the authorities at all levels and public participation.