重组花生过敏原Ara h 2生物合成

为获得重组花生过敏原Ara h 2。通过RT-PCR 合成cDNA,并以此为模板进行PCR 扩增目的基因Ara h 2,扩增产物经纯化后克隆至pMD19-T Simple 载体中,构建重组质粒pMD19-T-Ara h 2。上述重组质粒经酶切纯化后定向克隆到pGEX-4T-1 表达载体中,构建原核表达载体pGEX-4T-1-Ara h 2,并转化表达宿主菌BL21-codonPlus(DE3)-RIPL 中,经IPTG 诱导表达。SDS-PAGE 电泳结果表明,该表达蛋白大小约为46kD,与理论值相符。通过Glutathione Sepharose 4B 凝胶亲和层析方法纯化融合蛋白GST-Ara h 2,获得融合蛋白纯度约为90%。Western blotting 分析表明,经纯化的融合蛋白能与抗Ara h 2 兔血清发生特异性反应,说明该蛋白具有良好的免疫原性。     

Streptoverticillium mobaraense谷氨酰胺转胺酶的表达、纯化和复性

以基因组DNA为模板 ,利用PCR技术从茂原轮链丝菌 (Streptoverticilliummobaraense)中扩增得到产成熟谷氨酰胺转胺酶MTG的结构基因mtg ,克隆于表达载体pQE 3 0T ,构建成lac启动子控制下的His6融合表达质粒pMTG ,将此重组质粒转化到受体菌E .coliM1 5中。对质粒稳定性的研究表明 ,E .coliM1 5在无选择压的情况下 ,于 3 7℃连续转接 5次 ,质粒丢失率仅有 2 4%,说明质粒基本稳定。重组菌经IPTG诱导 ,表达的重组谷氨酰胺转胺酶占菌体总蛋白的 1 8%,经SDS PAGE分析 ,表达的蛋白质的分子质量为 3 8ku ,与预期分子质量相符。表达产物主要以包涵体的形式存在。细胞经超声破碎 ,离心取包涵体溶于 8mol/L的尿素中 ,然后通过Ni NTA亲和柱分离纯化和稀释法复性。目的蛋白的纯度可达 95 %以上。比酶活为 1 0 3U/mg。     

乳酸片球菌素PA-1在大肠杆菌中的表达与纯化

为了实现该细菌素的外源表达,本实验首先利用聚合酶链式反应从乳酸片球菌PAF中扩增出乳酸片球菌素PA-1的结构和免疫基因,然后克隆到表达载体pGEX-6p-1,构建了N端含有GST-His-DDDDK标签的重组质粒pGEX/his-pedAB,然后转化进入大肠杆菌Rosetta（DE3）感受态细胞,经异丙基硫代半乳糖苷诱导,重组乳酸片球菌素PA-1在大肠杆菌胞内成功表达。表达的融合蛋白先经过镍亲合层析柱纯化,然后注入谷胱甘肽S-转移酶亲和色谱柱用肠激酶处理,释放出成熟的乳酸片球菌素PA-1。利用高效液相色谱和质谱技术检测乳酸片球菌素PA-1纯度。以单核细胞增生李斯特氏菌CMCC54004为指示菌,利用琼脂扩散法检验乳酸片球菌素PA-1活性。结果表明,携带GST-His-DDDDK标签的融合蛋白无活性,标签切除后其抑菌活性恢复,且其纯度达90%以上。     

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。     

人胰岛素原基因在大肠杆菌中的可溶性表达及分离纯化研究

根据NCBI中的人胰岛素原基因序列及大肠杆菌密码子偏爱性设计特异引物,PCR扩增得到人胰岛素原基因,构建该基因的原核表达质粒p ET32-PI,重组质粒转化大肠杆菌BL21(DE3)。对重组菌进行温度和IPTG浓度优化,发现重组菌在30℃和终浓度为0.6 mmol/L的IPTG条件下表达效果最好且没有包涵体,经SDS-PAGE和Westem blot检测,表达蛋白的相对分子质量与理论相对分子质量一致且具有胰岛素的免疫原性,证明胰岛素原基因得到了正确表达。对重组菌进行流加发酵,发酵液离心收集菌体,破菌后上清液过亲和层析柱和离子交换柱分离纯化目的蛋白,透析后的样品用肠激酶和羧肽酶酶切,利用亲和柱去除融合蛋白与His标签,最终分离胰岛素样品,利用免疫酶标法,l m L样品测得活性92μIU,证明实验所得样品具有人胰岛素活性。     

丝胶抗冻肽在大肠杆菌中的重组表达及其抗冻活性初探

为了高效制备抗冻肽,研究一种丝胶抗冻肽目的基因SerD在大肠杆菌BL21菌株中的重组表达,并分析表达产物的抗冻活性。首先合成SerD基因片段,经KpnI和XhoI双酶切后定向插入质粒载体Pet32a中,构建表达质粒Pet32a-SerD,然后电转化入大肠杆菌BL21(DE3),在IPTG诱导下进行目的基因表达,利用镍琼脂糖亲和层析对目的重组蛋白进行纯化,并对其进行抗冻活性分析。结果表明,最佳表达条件为重组菌在20℃诱导16 h;通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳(Sodium Dodecyl Sulphate-PolyAcrylamide Gel Electrophoresis,SDS-PAGE)和蛋白质免疫印迹试验(Western-Blot)鉴定重组蛋白表达成功且His-SerD融合蛋白表达的分子量在25~35 kDa之间;胞内表达His-SerD融合蛋白的大肠杆菌BL21-SerD复苏后的生长活性明显高于PBL空载菌;添加His-SerD融合蛋白可明显降低溶液中冰晶颗粒大小,具有较好的重结晶抑制效果。本文通过基因工程方法在大肠杆菌中成功构建丝胶肽抗冻肽的重组表达系统,并最终获得具有抗冷冻胁迫保护作用的His-SerD融合蛋白。     

洋葱伯克霍尔德氏菌L68邻苯二酚2,3-双加氧酶基因克隆、表达和纯化

采用自行设计的引物,从洋葱伯克霍尔德氏菌L68中PCR扩增得到phnE（邻苯二酚2,3-双加氧酶）基因,亚克隆到高表达载体pET 32a上,转入表达菌株中.经双向测序,证实构建过程中未出现突变.重组子经IPTG诱导后,可以表达有活性的重组双加氧酶.选择26 ℃进行重组蛋白诱导.薄层扫描显示,表达重组蛋白总量最高可占总蛋白的64.92%.利用金属螯合层析对重组蛋白进行了一步纯化,SDS-PAGE结果显示,重组蛋白纯度达到95%.     

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。     

人血清白蛋白-白介素-2融合蛋白在CHO细胞中的表达

白细胞介素-2在人体免疫应答中具有重要作用,常用于治疗肿瘤、肝炎和免疫缺陷性疾病等多种疾病,但因其体内半衰期短而限制了其临床应用。文中构建了含有融合蛋白基因HSA-IL-2的p MH3质粒,并通过电转染的方法转入中国仓鼠卵巢细胞(Chinese hamster ovary cell,CHO细胞)中,利用p MH3质粒所携带的neo基因进行筛选,经96孔板培养并筛选得到稳定表达目的蛋白质的重组细胞,融合蛋白HSA-IL-2表达量达到2.26 mg/L。利用反转录PCR和Western blot对重组细胞鉴定后发现,融合蛋白编码基因整合入重组CHO细胞基因组中,而且融合蛋白同时具有HSA和IL-2双重免疫原性。利用对IL-2具有依赖性的CTLL-2细胞进行活性分析后发现,筛选得到的表达细胞分泌得到的融合蛋白具有较高的生物活性,表明成功构建具有表达IL-2生物活性能力的CHO细胞。     

仿生鲶鱼抗菌肽编码序列的设计及克隆

人工合成的仿生鲶鱼抗菌肽DNA序列，经聚合酶链式反应(PCR)扩增后得到带有不同限制性酶切位点的序列XH1、XH2、XH3．将此基因克隆至载体pGEX-5X-3，成功构建了三串联的重组质粒．测序验证后转化大肠杆菌BL21(DE3)，37℃IPTG诱导，获得高效表达，SDS—PAGE扫描分析表明，融合蛋白可达细菌全蛋白总量的30％，融合蛋白以包含体的形式存在于大肠杆菌细胞中．     

脱酚及pH偏移处理对菜籽蛋白体外模拟消化的影响

研究了脱酚及pH偏移处理对菜籽蛋白(CPI)及乳液模拟体外消化的影响。采用蛋白凝胶电泳、显微镜观察、粒度分析等方法进行表征。结果表明,在体外消化的过程中,蛋白水解度(DH)整体呈上升趋势,经脱酚、pH偏移及二者协同处理后的DH分别增大了17.1%、2.6%和22.9%。仅脱酚处理使消化产物可溶性蛋白浓度提高了22.3%。SDS-PAGE的结果显示,cruciferin(12S)比napin(2S)更易于水解,并且脱酚导致蛋白在12 kDa以下的条带消失。pH偏移处理显著提高了CPI的自由基清除能力,并改善了由酚的脱除引起的自由基清除能力的降低。对于改性后的蛋白乳液,脱酚导致胃消化阶段更显著的乳液液滴聚集现象,同时降低了肠消化阶段的油滴大小。总的来说,脱酚处理提高了蛋白及乳液的消化率,pH偏移提高了蛋白的自由基清除能力。     

Large-scale preparation of recombinant human calcitonin from a multimeric fusion protein produced in Escherichia coli

In order to develop a large-scale, high-yield production process for human calcitonin (hCT) in Escherichia coli, a stable expression plasmid was constructed and the expressed protein was modified for efficient cleavage by protease. Multiple copies of a synthetic gene encoding hCT-Leu-Arg, a substrate for C-terminal amidation by carboxypeptidase Y (CPY), were inserted into the stable expression plasmid. Using this plasmid, the expression of a multimeric fusion protein was induced by shifting the temperature from 34 to 38 degrees C. The multimeric fusion protein was accumulated as inclusion bodies. After the fermentation, the cells were harvested and disrupted by a homogenizer. The insoluble multimeric fusion protein was suspended in 6.6 M urea solution. At this time, however, the protein could not be solubilized and thus the efficiency of cleavage by protease was low. To solubilize the protein and protect Lys residues against digestion by trypsin, the protein was citraconilated with citraconic anhydride. Following S-sulfonation with Na2SO3-CuSO4, almost all the protein was solubilized. The solubilized, citraconilated, and S-sulfonated protein was digested with trypsin efficiently. By treatment with trypsin, the multimeric fusion protein was cleaved into monomers of citraconilated and S-sulfonated hCT-Leu-Arg (S-hCT-Leu-Arg). The subsequent decitraconilation was performed at low pH. S-hCT-Leu-Arg, isolated by preparative HPLC, was directly converted into S-hCT-HN2 by CPY without removal of the Arg residue. Finally, S-hCT-NH2 was desulfonated and converted into mature hCT. In this way, a large amount of recombinant mature hCT was obtained in a tank fermentation. To our knowledge, this is the first report of industrial-scale, human calcitonin production using a multimeric fusion protein expression system.     

脱酚棉籽分离蛋白功能特性研究

为拓宽棉籽蛋白在食品领域应用范围,对棉籽分离蛋白基本功能性质进行分析,结果显示,本试验条件下所得棉籽分离蛋白(CPI)属酸溶性蛋白质,在酸性条件下(pH1～4)溶解性较好(30%～50%),在近中性溶液中溶解性较差,仅20%左右;乳化性受pH值、离子强度和CPI浓度影响不显著;乳化能力较低,但所得乳状液非常稳定;起泡性受pH值、离子强度和CPI浓度等因素影响很大,在中性pH下最高;添加NaCl对CPI溶液起泡能力影响不大,但会显著影响CPI溶液泡沫稳定性,随NaCl浓度增加,溶液泡沫稳定性急剧降低。粘度分析表明,CPI溶液属典型非牛顿流体,有"剪切变稀"和"热变稀"特征。     

Effect of high hydrostatic pressure on the structure,physicochemical and functional properties of protein isolates from cumin (Cuminum cyminum) seeds

The effect of high hydrostatic pressure (HHP) treatment on the structure, physicochemical and functional properties of cumin protein isolate (CPI) was investigated. More aggregates, pores, irregular conformations and bigger particle size were observed for HHP-treated CPI. HHP resulted in an increase in α-helix, a decrease in β-strand and fluorescence intensity of CPI. Surface hydrophobicity (H_o) of CPI significantly increased after HHP treatment, from 343.35 for native CPI to 906.22 at 600 MPa (P < 0.05). HHP treatment at 200 MPa reduced zeta-potential and solubility of CPI, while had little effect at 400 and 600 MPa. Emulsifying activity and stability of CPI decreased after HHP treatment, of which droplet size of emulsions significantly increased (P < 0.05). HHP-treated CPI could form heat-induced gelation at lower temperature (68.5 °C) and improved storage modulus (G′) comparing to native one (80.6 °C), suggesting that CPI might be potential protein resources as gelation substitute in food system.     

FRACTIONATION AND CHARACTERIZATION OF CYSTEINE PROTEINASE INHIBITOR FROM CHICKEN PLASMA

The fractionation of cysteine proteinase inhibitor (CPI) from chicken blood plasma was carried out using polyethylene glycol‐4000 or ammonium sulfate (AS) precipitation. The addition of PEG at the level of 400 g/L, on the basis of the original volume of plasma protein, was more effective to fractionate CPI than using AS. CPI in the PEG fraction had a molecular weight of about 46 kDa with intramolecular disulfide bond. CPI containing fraction was colorless and had no absorbance in the range of 700–360 nm. The fraction was stable in the temperature range of 40–90C for 10 min and still retained high inhibitory activity toward papain after incubation at 90C for 60 min. NaCl, at 0–3.0% concentration, did not affect the inhibitory activity of the CPI containing fraction. The fraction was stable at pH 8.0, and the minimal inhibitory activity against papain was found at pH 5–6. Therefore, PEG fractionation effectively isolated the CPI from chicken plasma.     

Acid-induced aggregation and gelation of heat-treated chia proteins

This work studied for the first time the acid-induced aggregation and gelation of heat-treated chia protein isolates obtained by extraction at pH 10 or 12 (CPI10 and CPI12, respectively). The aggregation state of proteins was modified during acidification. The size of the aggregates was reduced for both samples when the pH decreased but below pH 4.5 further protein aggregation took place for CPI12. Gelation of CPI12 was completed after about 30 min of acidification with glucone-δ-lactone. By contrast, this period was not enough to reach a constant value in G′ for CPI10. When gelation was ensured, confocal laser scanning micrographs from those gels revealed a coarse and irregular structure with large pores (median size of diameters: 30 μm). Instead, micrographs from CPI12 cold gels showed a more regular and interconnected network, with smaller pores (median size of diameters: 9 μm). These differences are consistent with a higher elastic behaviour ( = 13.6 ± 0.1 Pa).     

Cloning and sequencing of cDNA and genomic DNA encoding serine carboxypeptidase of Fusarium moniliforme that was copurified with phosphatase

A previous study [Yoshida, H. et al., J. Biochem., 140, 813-823 (2006)] revealed that a protein of unknown nature was copurified with PDM phosphatase of Fusarium moniliforme. In this study, the identity of this protein was investigated. The results of homology search for the tryptic peptides derived from the purified preparation of PDM phosphatase strongly suggested that it might be serine carboxypeptidase. In fact, carboxypeptidase activity was demonstrated in the preparation and partial separation of carboxypeptidase from PDM phosphatase was achieved by gel filtration high-performance liquid chromatography. Cloning and sequencing of the full-length cDNA encoding the carboxypeptidase was successfully conducted. The cDNA possessed an open reading frame for a protein of 575 amino acid residues with a molecular mass of 64,650 Da, which was highly homologous to certain fungal serine carboxypeptidases. Comparison of the deduced amino acid sequence with the N-terminal sequence of the separated carboxypeptidase revealed that the mature enzyme starts at serine 56 of the precursor and has a molecular mass of 58,487 Da. Cloning and sequencing of the genomic DNA corresponding to the cDNA demonstrated that the gene of carboxypeptidase consists of four exons. A limited number of close homologs of F. moniliforme carboxypeptidase were detected among fungi by homology search and their evolutionary relationship was discussed.     

Control by the embryo axis of the breakdown of storage proteins in cotyledons of germinating seeds of citrus limon

Azocaseolytic (endopeptidase) and carboxypeptidase activities in cotyledons of germinating seeds of Citrus limon (L) Burm f (cv Verna) showed a peak on the fourth day after the onset of imbibition and then decreased. A second peak of activity in both enzymes appeared 16 days after imbibition. The activities of leucine-aminopeptidase and alanine-aminopeptidase increased until 12 days after imbibition and then declined. When cotyledons were detached from seedlings 8 days after imbibition and then incubated in water, protein breakdown was accelerated and α-amino N accumulation increased. In detached and incubated cotyledons, the azocaseolytic activity decreased whereas the carboxypeptidase activity continued to increase more markedly than when the axis remained attached. The effect of the axis on azocaseolytic activity was replaceable by kinetin. On the other hand, kinetin, GA₃ and IAA showed no significant effect on the carboxypeptidase and aminopeptidase activities in detached cotyledons.     

Functional Properties of Protein Isolate from Cowpea (Vigna unguiculata L. Walp.)

ABSTRACT: Functional properties of protein isolates prepared from 3 cowpea varieties and 2 soybean varieties in each of 2 y were determined. Both cowpea protein isolate (CPI) and soy protein isolate (SPI) showed a u-shaped curve for solubility with the minimum solubility occurring at pH 4.5. The CPI had lower emulsifying activity than SPI but was similar in stability. Foaming capacity and foaming stability ranged from 58.6 to 60.2 mL and 63.7 to 64.4 min for CPI and from 31.9 to 33.0 mL and 43.4 to 45.0 min for SPI, respectively. Gels were formed at 70 °C for 40 min and 30 min for CPI (12%) and SPI (10%), respectively. The CPI needs modification to enhance functional properties for potential application in food products.     

酶法改性对鹰嘴豆分离蛋白功能性的影响

研究了Alcalase及转谷氨酰胺酶(TGase)酶法改性对鹰嘴豆分离蛋白(CPI)的溶解性及乳化性的影响。CPI经A1calase水解60min(DH5.91%),低离子强度对溶解性不再有负面影响;CPI水解30min(DH5.80%),无盐或0.1mol/L NaCl条件下,蛋白质的乳化活力分别是未改性前的1.22和2.78倍。TGase对CPI在0.1mol/L NaCl条件下的溶解性及乳化活力不能起到良好的改善作用。