牡蛎诺如病毒受体合成基因类FUT10的克隆与组织表达

目的 克隆太平洋牡蛎类FUT10基因的cDNA序列,探究类FUT10 mRNA在牡蛎各组织中的表达差异.方法 基于同源比对方法,利用cDNA末端快速扩增(rapid amplification of cDNA ends,RACE)技术克隆完整的类FUT10基因,通过实时荧光定量聚合酶链式反应(real-time qua...     

Nanog蛋白在大肠杆菌中的可溶性表达及发酵优化

为高效制备可溶性人源Nanog蛋白,降低Nanog蛋白的使用成本。该研究通过分子克隆技术构建重组大肠杆菌(pET32a-Nanog),利用IPTG诱导进行可溶性融合表达。采用镍离子柱亲和层析法纯化产物并将纯化后产物进行肠激酶酶切后得到目的蛋白,Western blot鉴定该蛋白抗体结合的特异性并进一步优化发酵条件。结果表明,原核表达载体pET32a-Nanog构建成功,可在大肠杆菌中与硫氧还蛋白融合表达,融合蛋白经肠激酶酶切后得到相对分子质量约为38 kDa的蛋白,Western blot鉴定该蛋白可与Nanog抗体特异性结合。在摇瓶培养条件下,得到最优发酵条件是诱导温度37℃、IPTG浓度0.2 mmol/L、甘油为补料碳源;15 L发酵罐上进一步优化溶氧条件及温度控制方式,最终在溶氧设置在30%条件下,37℃(诱导期间30℃维持30 min)发酵12 h后得到最高Nanog产量1 386.4 mg/L,经蛋白纯化后纯度达到90%,为后续Nanog多克隆抗体的制备和基础医学实验研究奠定了基础。     

人甲胎蛋白AFP在大肠杆菌中表达和鉴定

本研究通过应用基因工程技术,重组构建编码人全长甲胎蛋白AFP基因的原核表达载体pET22b-AFP,并将其转化到大肠杆菌宿主菌BL21（DE3）中进行诱导表达目的蛋白AFP。采用PCR法扩增编码AFP蛋白的cDNA序列片段,并将其克隆到含有His-tag的pET22b原核表达载体上;重组质粒经双酶切及测序鉴定正确后转化到大肠杆菌BL21(DE3)中进行IPTG诱导表达;对诱导过程中不同浓度的IPTG和不同的温度进行优化;选定最佳的优化条件进行诱导表达目的蛋白;SDS-PAGE电泳和Western Blot法鉴定表达产物。结果表明,通过PCR扩增技术获得了编码AFP蛋白的基因片段;重组质粒经双酶切和基因测序等方法鉴定后,确认了重组质粒已经构建成功;表达产物通过利用SDS-PAGE和Western Blot法检测到在66 kDa附近出现条带,与预期值相符,表明重组AFP蛋白已成功表达。     

马槟榔甜蛋白基因(MabinlinⅡ)的密码子优化及其在大肠杆菌中的表达

本研究根据大肠杆菌密码子的偏好性,对马槟榔甜蛋白基因MabinlinⅡ(A链+B链)的密码子进行优化,并将优化后的马槟榔甜蛋白基因命名为MabinlinⅡ(AB)Plus。将MabinlinⅡ(AB)Plus基因与大肠杆菌表达载体pET-28a(+)片段连接,构建重组表达载体pET-28a(+)-MabinlinⅡ(AB)Plus,进而转化到大肠杆菌宿主表达菌株BL21(DE3),IPTG诱导蛋白表达。结果表明,密码子优化后的基因MabinlinⅡ(AB)Plus在大肠杆菌中最佳表达条件为:最适IPTG浓度1.4 mmol/L,最佳诱导剂温度为37℃,最适菌株起始生长量OD₆₀₀为0.7,最佳诱导时间8 h。在最佳诱导表达条件下,目的蛋白表达量占菌体总蛋白约33.0%,与优化前约有提高7%。目的蛋白的上清液经过亲和纯化,在19 kDa处有单一的目的条带。经过感官评定,目的蛋白的甜度约为2%标准蔗糖溶液的400倍。这为研发以马槟榔甜蛋白为基础的新型甜味剂提供了科学的理论依据。     

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。     

人甲胎蛋白 AFP 在大肠杆菌中表达和鉴定

本研究通过应用基因工程技术,重组构建编码人全长甲胎蛋白AFP基因的原核表达载体pET22b-AFP,并将其转化到大肠杆菌宿主菌BL21（DE3）中进行诱导表达目的蛋白AFP。采用PCR法扩增编码AFP蛋白的cDNA序列片段,并将其克隆到含有His-tag的pET22b原核表达载体上;重组质粒经双酶切及测序鉴定正确后转化到大肠杆菌BL21（DE3）中进行IPTG诱导表达;对诱导过程中不同浓度的IPTG和不同的温度进行优化;选定最佳的优化条件进行诱导表达目的蛋白;SDS-PAGE电泳和Western Blot法鉴定表达产物。结果表明,通过PCR扩增技术获得了编码AFP蛋白的基因片段;重组质粒经双酶切和基因测序等方法鉴定后,确认了重组质粒已经构建成功;表达产物通过利用SDS-PAGE和Western Blot法检测到在66 kDa附近出现条带,与预期值相符,表明重组AFP蛋白已成功表达。     

植物乳杆菌亚硝酸盐还原酶基因在大肠杆菌中表达

构建乳杆菌亚硝酸盐还原酶基因的重组质粒并表达。以植物乳杆菌基因组DNA为模板,PCR扩增亚硝酸盐还原酶基因,连接到表达载体pET-32a(+)上,构建的重组载体转入大肠杆菌BL21(DE3)中,经IPTG诱导表达后,菌体总蛋白经SDS-PAGE鉴定显示重组质粒诱导表达产物有特异性蛋白条带。该表达产物经细菌亚硝酸盐还原酶试剂盒测定显示有明显的酶活性。实验结果为深入研究植物乳杆菌亚硝酸盐还原酶提供有益的参考。     

pfs基因原核表达载体的构建、表达及蛋白纯化

以植物乳杆菌KLDS1.0391的基因组DNA为模板,采用PCR方法扩增得到pfs基因,与Gen Bank中的序列对比发现同源性为99%。将目的基因与表达载体p QE-30连接,并将重组质粒p QE-30-pfs转化到E.coli M15中。异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导表达重组蛋白,经SDS-PAGE分析,在全菌体蛋白中含有一条明显的特异性蛋白条带,大小约为27.4ku。经Ni-NTA纯化系统纯化后,在SDS-PAGE凝胶电泳图上获得纯化产物的条带。结果表明,原核表达载体p QE-30-pfs构建成功,且Pfs蛋白在大肠杆菌中成功表达,采用Ni-NTA纯化系统成功纯化了重组蛋白Pfs,为体外合成AI-2(Autoinducer-2)奠定了基础。     

抗黄曲霉毒素B_1纳米抗体的原核表达、纯化及活性分析

表达和纯化抗黄曲霉毒素B1(Aflatoxin B1,AFB1)纳米抗体(G8),并分析纳米抗体的热稳定性及生物学活性,建立基于纳米抗体检测AFB1的ELISA方法。将编码抗AFB1纳米抗体的基因亚克隆至原核表达载体,转化至大肠杆菌BL21(Rosetta,DE3),IPTG诱导表达后,SDS-PAGE分析蛋白表达情况;采用酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA)分析纳米抗体的热稳定性、有机溶剂耐受性、盐离子耐受性及耐酸碱性。结果显示,在大肠杆菌中成功表达可溶性的抗AFB1纳米抗体,表达量为80 mg/L,在25~65℃之间,抗体具有良好的热稳定性。在5%甲醇、p H7.4、10 mmol/L PBS条件下,建立了G8-ELISA检测AFB1的方法,该方法的半抑制浓度(IC50)为4.61 ng/m L,线性范围为0.95~42.45 ng/m L。     

噬菌体基因Ⅴ蛋白基因的合成、重组表达及功能分析

目的:研究丝状噬菌体基因V蛋白(gene V protein,GVP)基因的合成、重组表达及其功能。方法:根据GVP的基因序列,选择大肠杆菌偏爱的密码子,设计合成了8个寡核苷酸片段,利用重叠延伸PCR合成GVP基因序列,将其与原核表达载体pET-28a-c(+)质粒重组,转化大肠杆菌,获得GVP蛋白阳性表达菌株,异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导表达,产物经Ni+-NTA琼脂糖凝胶层析纯化,获得目的蛋白GVP,DNA结合实验检测其功能。结果:成功合成出GVP基因,重组体在大肠杆菌BL21(DE3)中诱导获得高效表达,DNA结合实验表明GVP与单链DNA间解离平衡常数Kd=7.27×10-5mol/L。结论:重组构建并高效表达的GVP蛋白具有较高单链DNA结合能力,可用于食品病原微生物特定单链DNA分子的浓缩和分离。     

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.     

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     

Chinese CTP Market

According to Printing and Printing Equipment Industries Association of China（PEIAC）＇s statistics to the plate manufucturer in China, in 2013, the actual offset plate production has reached 346 million square meters in China. Among them, the CTP production volume was 245 million square meters, up by 11% than that of last year; the total sales of the CTP plate was 239 million square meters, up by 13%.     

Preparatory Work of Print China 2015 is Going Smoothly

正Held at Guangdong Modern International Exhibition Center,Print China 2015 will cover 7exhibition halls,besides the original Hall No.3,4,5,6,7,the newly built F zone of Hall 3 will be used too.The total area will be140,000 square meters.Hall 3:Offset and large printing equipment,package printing equipment,post press