Safety assessment by in vitro digestibility and allergenicity of genetically modified maize with an amaranth 11S globulin

Prospective testing for allergenicity of proteins obtained from sources with no prior history of causing allergy has been difficult to perform. Thus, the objective of this work was to assess the food safety of genetically modified maize with an amaranth globulin protein termed amarantin. Transgenic maize lines evaluated showed, in relation to nontransgenic, 4-35% more protein and 0-44% higher contents of specific essential amino acids. Individual sequence analysis with known amino acid sequences, reported as allergens, showed that none of these IgE elicitors were identified in amarantin. Amarantin was digested within the first 15 min by Simulated Gastric Fluid treatment as observed by Western blot. Expressed amarantin did not induce important levels of specific IgE antibodies in BALB/c mice, as analyzed by ELISA. We conclude that the transgenic maize with amarantin is not an important allergenicity inducer, just as nontransgenic maize.     

抗对硫磷基因工程抗体ScFv的制备及其初步鉴定

用一段45个核苷酸的片段连接抗对硫磷抗体重链和轻链可变区基因片段VH和VL,获得了抗对硫磷单链抗体(single chain variable fragment,scFv)基因,构建了抗对硫磷scFv基因原核表达载体。SDS-PAGE和Western blot分析显示,该单链抗体基因能在大肠杆菌Origami 2中特异性表达,融合蛋白分子量约为28kD。用Ni-NTA金属亲和层析法对可溶性表达产物进行纯化,得到目的蛋白纯度为74.8%;ELISA反应结果证明,该单链抗体可以与对硫磷发生特异性反应。     

Processing stability of squalene in amaranth and antioxidant potential of amaranth extract

The processing stability of squalene in amaranth and the antioxidant capacity of the oil-rich fraction of amaranth were studied. The processes investigated were continuous puffing and roasting. Puffing was carried out using a single screw extruder, while roasting was carried out in a convection oven. High-performance liquid chromatography was used to quantify squalene content before and after processing. The L-ORAC method was used to study the antioxidant activity of pure squalene and lipophilic amaranth extract containing squalene. It was found that squalene was stable during all of the processing operations with a maximum loss of 12% during roasting (150 degrees C, 20 min) and no loss during puffing. The L-ORAC test showed pure squalene to be a weak antioxidant, whereas the lipophilic extract of amaranth showed higher antioxidant activity as compared to pure squalene at the same concentration, suggesting that tocotrienols and other minor ingredients also played a role as antioxidants.     

Impact of Baking on Vitamin E Content of Pseudocereals Amaranth,Quinoa, and Buckwheat

The aim of this study was to analyze the vitamin E composition of amaranth, quinoa, and buckwheat pseudocereals. The method used consisted of a one‐step extraction with hexane followed by normal‐phase high‐performance liquid chromatography (NP‐HPLC) coupled with a fluorescence detector. This method afforded complete separation of all vitamin E compounds present. In addition, vitamin E stability following high‐temperature processing such as breadmaking was also studied. The vitamin E composition differed significantly from grain type to grain type, and highest vitamin E content (expressed as α‐tocopherol equivalents) was found in quinoa grains, followed by amaranth and buckwheat (24.7, 15.4, and 6.3 μg/g respectively). None of the pseudocereal grains contained tocotrienols, which were only detected in wheat grains in minor quantities. Vitamin E recovery following breadbaking was high (70–93%) and gluten‐free breads containing pseudocereal had significantly higher vitamin E content compared with the gluten‐free control. Amaranth, quinoa, and buckwheat grains proved to be good sources of vitamin E and may be used as ingredients in gluten‐free products for improving vitamin E content and thus overall nutritional quality.     

Preparation of unglycosylated human caseinomacropeptide by engineering DAB Escherichia coli

Human caseinomacropeptide (hCMP) is 65 amino acids in length and was originally derived from the C terminus of human milk kappa-casein. As it is highly abundant in both essential amino acids and branched amino acids, it could be developed as a practical food and even as medicinal nutrition for patients. This study was undertaken to prepare recombinant hCMP without glycosylation using recombinant plasmid and prokaryotic expression system. The gene encoding hCMP was chemically synthesized and directly inserted into the pET28a(+) vector and then expressed in Escherichia coli BL21(DE3). The maximum amount of soluble protein was obtained by incubation with 0.5 mM isopropyl-alpha-D-thiogalactopyranoside at 30 degrees C for 4 h and accounted for 40% of the total intracellular protein. Most of the expressed fusion proteins, located in the cell periplasm and cytoplasm, could be adsorbed by nickel affinity chromatography and eluted with buffer containing 300 mM imidazole. The fusion proteins were cleaved by enterokinase to remove the 6-His tag. Gel filtration chromatography with Sephadex G-10 was performed for desalting and purification. A final yield of 25 mg of the mature protein with high purity up to 99% was obtained from 1 L of E. coli culture. The purified protein was confirmed by MALDI-TOF-MS analysis. This study overcame the problem of glycosylation in hCMP and established a novel approach for the preparation of unglycosylated hCMP.     

Bioactive peptides in amaranth (Amaranthus hypochondriacus) seed

Amaranth seeds are rich in protein with a high nutritional value, but little is known about their bioactive compounds that could benefit health. The objectives of this research were to investigate the presence, characterization, and the anticarcinogenic properties of the peptide lunasin in amaranth seeds. Furthermore, to predict and identify other peptides in amaranth seed with potential biological activities. ELISA showed an average concentration of 11.1 microg lunasin equivalent/g total extracted protein in four genotypes of mature amaranth seeds. Glutelin fraction had the highest lunasin concentration (3.0 microg/g). Lunasin was also identified in albumin, prolamin and globulin amaranth protein fractions and even in popped amaranth seeds. Western blot analysis revealed a band at 18.5 kDa, and MALDI-TOF analysis showed that this peptide matched more than 60% of the soybean lunasin peptide sequence. Glutelin extracts digested with trypsin, showed the induction of apoptosis against HeLa cells. Prediction of other bioactive peptides in amaranth globulins and glutelins were mainly antihypertensive. This is the first study that reports the presence of a lunasin-like peptide and other potentially bioactive peptides in amaranth protein fractions.     

Identification of a novel cytotoxic protein, Cry45Aa, from Bacillus thuringiensis A1470 and its selective cytotoxic activity against various mammalian cell lines

Parasporal inclusion proteins produced by Bacillus thuringiensis strain A1470 exhibit strong cytotoxicity against human leukemic T cells when activated by protease treatment. One of the cytotoxic proteins was separated by anion exchange chromatography and gel filtration chromatography and designated Cry45Aa. Its gene was then expressed in recombinant Escherichia coli, in which the Cry45Aa precursor was accumulated in an inclusion body. It was solubilized in sodium carbonate buffer and processed with proteinase K, and cytotoxic activities of the protein against various mammalian cell lines were evaluated using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide assay. The protein exhibited high cytotoxic activity against CACO-2, Sawano, MOLT-4, TCS, and HL60 cells and moderate activity against U-937 DE-4, PC12, and HepG2 cells. On the other hand, the EC50 values against Jurkat, K562, HeLa, A549, Vero, COS-7, NIH3T3, CHO, and four normal tissue cells (human primary hepatocyte cells, UtSMC, MRC-5, and normal T cells) were >2 microg/mL.     

Overexpression of the soluble form of chicken cystatin in Escherichia coli and its purification

A cDNA encoding chicken cystatin was cloned into the pET-23a(+) expression vector and then transformed into Escherichia coli AD494(DE3)pLysS expression host. An active soluble form of cystatin was expressed in the cytoplasm of E. coli induced by isopropyl beta-D-thiogalactopyranoside. The recombinant chicken cystatin was purified to electrophoretic homogeneity by a simple and rapid method involving heat treatment and Sephacryl S-100 gel filtration chromatography. The recombinant cystatin behaved as a thermal-stable protein and exhibited papain-like protease inhibition activity comparable to the natural chicken cystatin.     

Delta1-pyrroline-5-carboxylic acid formed by proline dehydrogenase from the Bacillus subtilis ssp. natto expressed in Escherichia coli as a precursor for 2-acetyl-1-pyrroline

Proline dehydrogenase (PRODH) catalyzes the biosynthesis of Delta1-pyrroline-5-carboxylic acid (P5C). The Bacillus subtilis subsp. natto gene for the proline dehydrogenase (BnPRODH) was cloned and expressed in Escherichia coli. Nucleotide sequence analysis of the clone revealed an open-reading frame that encodes 302 amino acid polypeptide with a calculated molecular mass of 34.5 kDa. The deduced amino acid sequence showed sequence similarity to bacterial PRODH and PutA of E. coli. The BnPRODH gene was cloned into pET21b and was expressed at a high level in E. coli BL21(DE3). The expressed protein was purified by using nickel ion affinity column chromatography to homogeneity before characterization. The purified recombinant BnPRODH was used to produce P5C. Model system composed of P5C and methylglyoxal was set up to study the formation of 2-acetyl-1-pyrroline. Our data showed that P5C, derived from the conversion of l-proline by the purified recombinant PRODH, might react directly with methylglyoxal to form 2-AP. P5C/methylglyoxal pathway represents the first report of a biological mechanism by which 2-AP may be synthesized in vitro by PRODH.     

应用杆状病毒载体表达狂犬病病毒糖蛋白

本试验利用杆状病毒载体成功地表达狂犬病病毒糖 (G) 蛋白。从转染重组了日本动物用狂犬病病毒疫苗株RC HL株G蛋白基因的转移载体和野生型杆状病毒DNA 的Sf 9 细胞中, 分离出2 个重组杆状病毒克隆。应用印渍法和荧光抗体法(IFA) 从重组杆状病毒感染的Sf 9 细胞检出了狂犬病毒G 蛋白。Sf9 细胞表达的G 蛋白与狂犬病毒RC HL株感染NA 细胞的G蛋白的分子量一致。35 个抗RC HL株G蛋白的单克隆抗体均与重组杆状病毒感染的Sf 9 细胞反应, 表明表达的G蛋白的抗原性没有发生变异。表达的G蛋白主要分布在Sf 9 细胞的膜表面, 形成类似环状的荧光,这种荧光与RC HL株感染的NA 细胞的荧光相同。     

Enhanced expression of chicken cystatin as a thioredoxin fusion form in Escherichia coli AD494(DE3)pLysS and its effect on the prevention of surimi gel softening

The DNA encoding chicken lung cystatin was ligated into a thioredoxin-pET 23a+ expression vector and transformed into Escherichia coli AD494(DE3)pLysS. A high level of soluble recombinant thioredoxin-cystatin (trx-cystatin) was expressed in the cytoplasm of the E. coli transformant. As compared with recombinant cystatin (trx-free), a 38.7% increase of inhibitory activity in the soluble fraction was achieved by introducing the trx fusion protein. Trx-cystatin was purified to electrophoretical homogeneity by 3 min of heating at 90 degrees C and Sephacryl S-100 chromatography. The molecular mass of trx-cystatin was 29 kDa, which was the expected size based on its composition of recombinant trx (16 kDa) and chicken cystatin (13 kDa). The purified trx-cystatin behaved as a thermally stable and papain-like proteinase inhibitor comparable to either recombinant or natural chicken cystatins. The inhibitor could inhibit the gel softening of mackerel surimi.     

小鼠抗病毒基因viperin的原核表达及其抗原性探索

Viperin是一种由Ⅰ型和Ⅱ型干扰素诱导的宿主蛋白,在进化上高度保守。本研究以小鼠脑组织为原材料,通过RT-PCR扩增得到包括完整open reading frame（ORF）的1 155 bp的viperin基因序列,成功亚克隆并构建了原核表达载体pET-32a-vip,将其转化到大肠杆菌Rosetta中进行原核表达。结果表明：原核表达的重组蛋白viperin主要以包涵体的形式表达,用镍离子亲和层析法获得纯度较高的viperin蛋白,为探讨原核表达的viperin抗原特性,将纯化的viperin蛋白免疫4周龄昆明小鼠,制备得到抗viperin多克隆抗体。经Western blot和IFA检测,该抗体能与真核细胞自然表达的viperin反应,特异性良好且效价高,表明原核表达的viperin具有良好的抗原性。获得的多克隆抗体为进一步研究viperin的抗病毒生物学功能奠定了良好的基础。     

Expression of soluble form carp (Cyprinus carpio) ovarian cystatin in Escherichia coli and its purification

A DNA encoding thioredoxin-mature carp ovarian cystatin (trx-cystatin) fusion protein was ligated into a pET-23a(+) expression vector and then transformed into Escherichia coli AD494(DE3) expression host. After induction by isopropyl beta-D-thiogalactopyranoside, a high level of the soluble form of recombinant trx-cystatin was expressed in the cytoplasm of E. coli. The recombinant trx-cystatin could be purified by Ni(2+)-NTA agarose affinity chromatography. The molecular mass (M) of the recombinant trx-cystatin was approximately 28 kDa composed of recombinant thioredoxin (16 kDa) and recombinant mature carp ovarian cystatin (12 kDa). Both recombinant trx-fused and mature carp ovarian cystatins were stable at pH 6-11. No obvious decrease in activity was observed even after 5 min of incubation at 60 degrees C. They exhibited papain-like protease inhibition activity comparable to that of the mature carp ovarian cystatin, which could inhibit papain and mackerel cathepsins L and L-like, but not cathepsin B.     

手掌参中赤霉酸诱导的富含半胱氨酸蛋白基因的表达、纯化及鉴定

目的: 构建含赤霉酸诱导的富含半胱氨酸蛋白基因(gcgasa)的原核表达载体,并在E.coli BL21(DE3)中表达。材料：手掌参。方法：设计带有EcoR I和Hind III酶切位点的引物,分别对gcgasa基因编码区全长及信号肽缺失的片段进行PCR扩增,将目标片段克隆入原核表达载体pET-32(a),构建重组质粒。经测序鉴定后,转化大肠杆菌BL21(DE3),IPTG诱导表达融合蛋白,并采用SDS-PAGE、Western-blotting、电镜超薄切片技术检测外源蛋白的表达及定位情况。对于水溶性蛋白,采用Ni2+-NTA亲和层析及凝胶过滤手段纯化。结果: 含有信号肽的外源基因在大肠杆菌周质空间中以包含体形式存在,而信号肽缺失的片段主要以可溶形式表达,经Ni2+-NTA纯化可获得目的蛋白。结论：首次在原核表达系统中高效表达了水溶性的gcgasa蛋白,为其结构和功能的研究奠定了基础。     

犬白细胞介素2在毕赤酵母中的诱导表达及生物活性的测定

以ConA刺激的犬外周血淋巴细胞总RNA为模板，通过RT-PCR方法扩增出犬IL-2成熟蛋白基因，将目的片段连接到pMD18-T载体，测序结果显示，扩增片段与GenBank上发表的序列一致。然后将目的片段连接到酵母表达载体pPICZa-A上，得到重组酵母犬IL-2表达载体pPICZaA-CaIL-2，经SacⅠ酶切线性化后电转化导入毕赤酵母菌株X-33。PCR方法筛选重组酵母菌，甲醇诱导表达，SDS-PAGE结果显示表达上清中有大小约20kDa的目的条带，比实际分子量略大，推测蛋白可能发生糖基化。MTT法测定生物学活性结果表明，重组犬IL-2能够极显著促进犬外周血淋巴细胞增殖。证明酵母表达的犬重组IL-2具有良好的生物学活性。     

Cloning and functional expression of a mungbean defensin VrD1 in Pichia pastoris

It was shown previously that a bacterially expressed mungbean defensin VrCRP exhibited both antifungal and insecticidal activities. To isolate this protein in a large quantity for its characterization, the defensin cDNA was expressed in Pichia pastoris and the recombinant defensin (rVrD1) was purified. The recombinant VrD1 was shown to inhibit the growth of fungi such as Fusarium oxysporum, Pyricularia oryza, Rhizoctonia solani, and Trichophyton rubrum and development of bruchid larva. The protein also inhibits in vitro protein synthesis. These biological activities are similar to that of the bacterially expressed defensin. Functional expression of VrD1 in Pichia pastoris provides a highly feasible system to study the structure-function relationship of VrD1 using the mutagenesis approach.     

Purification and characterization of an antifungal protein, C-FKBP, from Chinese cabbage

An antifungal protein was isolated from Chinese cabbage (Brassica campestris L. ssp. pekinensis) by buffer-soluble extraction and two chromatographic procedures. The results of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry revealed that the isolated Chinese cabbage protein was identical to human FK506-binding protein (FKBP). A cDNA encoding FKBP was isolated from a Chinese cabbage leaf cDNA library and named C-FKBP. The open reading frame of the gene encoded a 154-amino acid polypeptide. The amino acid sequence of C-FKBP exhibits striking degrees of identity with the corresponding mouse (61%), human (60%), and yeast (56%) proteins. Genomic Southern blot analyses using the full-length C-FKBP cDNA probe revealed a multigene family in the Chinese cabbage genome. The C-FKBP mRNA was highly expressed in vegetative tissues. We also analyzed the antifungal and peptidyl-prolyl cis-trans isomerase activity of recombinant C-FKBP protein expressed in Escherichia coli. This protein inhibited pathogenic fungal strains, including Candida albicans, Botrytis cinerea, Rhizoctonia solani, and Trichoderma viride, whereas it exhibited no activity against E. coli and Staphylococcus aureus. These results suggest that recombinant C-FKBP is an excellent candidate as a lead compound for the development of antifungal agents.     

牛乳铁蛋白肽基因LfcinB的合成及其在大肠杆菌中的表达

根据APD抗菌肽数据库报道的牛乳铁蛋白肽LfcinB氨基酸序列,合成编码LfcinB基因的两条互补的寡核苷酸链,退火后在5’端和3’端分别形成BamHI和XhoI位点的粘性末端。合成的LfcinB基因定向克隆到pET-32a原核表达载体,阳性克隆菌用TB培养基进行扩大培养,然后用终浓度1.0 mmol/L的IPTG进行诱导表达。SDS-PAGE结果显示,LfcinB基因在大肠杆菌中获得了大量表达,表达蛋白以包涵体形式存在,利用TGE透析液对纯化的LfcinB重组蛋白包涵体进行了复性,复性的LfcinB重组蛋白的纯度为96.6%。     

Gas-liquid chromatographic-mass spectrometric determination of alpha- and beta-naphthylamines in FD&C Red No. 2 (amaranth)

A method is described for the simultaneous quantitation of trace amounts of alpha- (alpha-NA) and beta-naphthylamines (beta-NA) with detectability in the 0.1 ppb range and sensitivity of 50 picomoles in certified food grade amaranth (FD&C Red No. 2; C.I. Food Red 9; CI 16185). The amaranth sample is extracted with benzene, and the evoporated residue is derivatized with perfluorooctanoic anhydride. The resulting derivatives are separated by gas-liquid chromatography and identified and quantitated by mass spectrometric monitoring of the m/e at 539.04. The method was used for quantitation of alpha-NA and beta-NA in randomly chosen samples of amaranth. Of 11 samples from different manufacturers, 5 were free of the beta-isomer; the remaining samples contained up to 1.2 ppb beta-NA. The concentration of alpha-NA ranged from no detectable amount to 970 ppb; the majority of the samples contained less than 7 ppb.     

亚非马蜂溶血肽基因与增强型绿色荧光蛋白基因融合在昆虫细胞中的表达

构建亚非马蜂(Polisteshebraeus)溶血肽基因重组转移载体pBacHT-GFPTPhMT,转化含穿梭载体Bacmid的受体菌Escherichiacoli(DH10Bac)中,得重组穿梭载体Bacmid-GFPTPhM,再用Lipofectin介导其DNA转染粉纹夜蛾(Trichoplusiani)细胞系Tn。SDS-PAGE分析表明,感染Bacmid-GFPTPhM的Tn-5B1-4细胞的表达产物在约为34kD处出现特异性条带,表达量约占细胞总蛋白的3%。Westernblot显示表达产物具有良好的免疫活性。感染Bacmid-GFPTPhM的细胞表达时相动态分析进一步表明,与增强型绿色荧光基因融合的亚非马蜂溶血肽基因的重组病毒已在昆虫细胞中进行了成功地表达,感染后72h表达量可达最高水平。