High expression of a human lactoferrin in transgenic tobacco cell cultures

Transgenic Nicotiana tabacum cell lines were developed expressing the human lactoferrin gene driven by the oxidative stress-inducible peroxidase (SWPA2) promoter. Western blot analysis showed the accumulation of both the full-length human lactoferrin protein as well as a immuno-reactive truncated fragment. Accumulation of human lactoferrin as monitored by ELISA increased proportionally to cell growth and reached a maximal (up to 4.3% of total soluble proteins) at the stationary phase of growth. Protein extracts from transgenic tobacco cells exhibited antibacterial activity.     

重组人乳铁蛋白和重组人溶菌酶对小鼠溃疡性结肠炎的改善作用研究

溃疡性结肠炎（ulcerative colitis,UC）是一种发生于直肠和结肠的慢性非特异性炎症疾病,近年来发病率明显上升。为探究转基因牛乳中提取的重组人乳铁蛋白和重组人溶菌酶对改善UC的作用,采用葡聚糖硫酸钠（dextran sulfate sodium salt,DSS）构建小鼠UC模型,30只雄性C57BL/6N小鼠随机分为空白对照组（CON组）、模型对照组（DSS组）、低浓度乳铁蛋白组（L-rLF组,50 mg·kg^-1·BW^-1）、高浓度乳铁蛋白组（H-rLF组,100 mg·kg^-1·BW^-1）、低浓度溶菌酶组（L-rLZM组,50 mg·kg^-1·BW^-1）和高浓度溶菌酶组（H-rLZM组,100 mg·kg^-1·BW^-1）。造模后用重组乳铁蛋白和溶菌酶分别灌胃1周,取小鼠血清及结肠,观察器官病理变化,测定炎症因子以及肠道菌群等相关指标。研究结果显示,与模型组相比,低浓度乳铁蛋白组和低浓度溶菌酶组小鼠疾病活动指数（disease activity index,DAI）、结肠缩短量、组织病理学评分均显著降低,且结肠组织中促炎因子（IL-6、lL-1β、TNF-α）表达量、血清和肝脏中脂多糖（lipopolysaccharide,LPS）浓度显著降低,高浓度乳铁蛋白处理组小鼠肠道菌群结构显著改善。表明重组人乳铁蛋白和重组人溶菌酶均可以不同程度地改善小鼠UC,为重组人乳铁蛋白和重组人溶菌酶的未来应用提供了新的思路和理论支持。     

Recombinant human lactoferrin: A valuable protein for pharmaceutical products and functional foods

Lactoferrin, the main iron-binding protein of milk, has biological activities that are essential for the newborn and are beneficial for adults. Given this beneficial effect, there is broad interest in exogenous sources of lactoferrin in human nutrition. Consequently, several transgenic approaches to produce lactoferrin have been achieved. However, the activity of heterologous lactoferrin cannot be assumed to identically mimic that of the homologous protein. Human lactoferrin obtained from yeast, transgenic cows, and rice has met the criteria of structural similarity, high yield, and ease of protein isolation. Human lactoferrin from Aspergillus awamori has been mainly directed to therapeutic uses with advanced phases of clinical trials currently in progress. In contrast, human lactoferrin produced in transgenic cows and rice brings the clear advantage of origins compatible with use in foods, although the approval for these applications is still in process.     

Expression,purification, and breast cancer cell inhibiting effect of recombinant human lactoferrin C-lobe

Lactoferrin (LTF), a multifunctional glycoprotein of the transferrin family mainly found in exotic secretions in mammals, is an important defense molecule against not only microbial invasion but also tumors. It folds into two globular domains (N- and C-lobes) each containing an iron-binding site. The cationic antimicrobial peptide in N-lobe is known to exert anti-tumor effect via a non-receptor-mediated pathway. However, whether LTF C-lobe also contributes to its anti-tumor activity remains to be investigated. In this study, a human LTF fragment (amino acid residues 343–682) covering the C-lobe was expressed with a histidine tag in E. coli and the purified polypeptide refolded through a series of buffer changing procedure. The resultant recombinant protein caused significant growth arrest of breast carcinoma cells MDA-MB-231 in a dose- and time-dependent manner, evidently via induction of apoptosis of the cell. Our data suggest a positive role for the C-lobe of human LTF in controlling tumors in vitro.     

N-glycosylation potential of maize: the human lactoferrin used as a model

In order to determine the N-glycosylation potential of maize, a monocotyledon expression system for the production of recombinant glycoproteins, human lactoferrin was used as a model. The human lactoferrin coding sequence was inserted into the pUC18 plasmid under control of the wheat glutenin promoter. Maize was stably transformed and recombinant lactoferrin was purified from the fourth generation seeds. Glycosylation was analysed by gas chromatography, lectin detection, glycosidase digestions and mass spectrometry. The results indicated that both N-glycosylation sites of recombinant lactoferrin are mainly substituted by typical plant paucimannose-type glycans, with 1,2-xylose and 1,3-linked fucose at the proximal N-acetylglucosamine, and that complex-type glycans with Lewis^a determinants are not present in maize recombinant lactoferrin.     

乳腺生物反应器表达的重组人乳铁蛋白的分离纯化及生物活性鉴定

以国产高交联度的快流速琼脂糖为基质,合成了不同配基密度的SP(Sulfopropyl,磺酸基)离子交换介质,建立了乳腺生物反应器表达重组人乳铁蛋白(Recombinant Human Lactoferrin,rHLF)的纯化方法。以溶菌酶为模型蛋白考察了不同配基密度离子交换介质的静态和动态吸附行为,结果表明介质具有良好的吸附性能。不同配基密度离子交换介质均可纯化得到rHLF,其中,高配基密度(0.24mol/L)的离子交换介质每毫升可以处理50mL rHLF牛乳,rHLF收率为86.5%,纯度为98.5%。圆二色谱的测定结果表明纯化的rHLF二级结构与天然人乳铁蛋白一致。生物学功能实验结果表明,rHLF的铁结合与释放活性与天然人乳铁蛋白相似,浓度为5g/L的rHLF对大肠杆菌的生长具有明显的抑制作用。     

Structure of a complex of human lactoferrin N-lobe with pneumococcal surface protein a provides insight into microbial defense mechanism

Human lactoferrin, a component of the innate immune system, kills a wide variety of microorganisms including the Gram positive bacteria Streptococcus pneumoniae. Pneumococcal surface protein A (PspA) efficiently inhibits this bactericidal action. The crystal structure of a complex of the lactoferrin-binding domain of PspA with the N-lobe of human lactoferrin reveals direct and specific interactions between the negatively charged surface of PspA helices and the highly cationic lactoferricin moiety of lactoferrin. Binding of PspA blocks surface accessibility of this bactericidal peptide preventing it from penetrating the bacterial membrane. Results of site-directed mutagenesis, in vitro protein binding assays and isothermal titration calorimetry measurements corroborate that the specific electrostatic interactions observed in the crystal structure represent major associations between PspA and lactoferrin. The structure provides a snapshot of the protective mechanism utilized by pathogens against the host's first line of defense. PspA represents a major virulence factor and a promising vaccine candidate. Insights from the structure of the complex have implications for designing therapeutic strategies for treatment and prevention of pneumococcal diseases that remain a major public health problem worldwide.     

Helicobacter felis infection causes an acute iron deficiency in nonpregnant and pregnant mice

BACKGROUND: The role of Helicobacter pylori infection in iron deficiency during pregnancy is limited. The aim of the present study was to assess the relationship between Helicobacter infection and levels of iron stores in pregnant mice. MATERIALS AND METHODS: Female C57BL/6 mice were either inoculated with 10(8) H. pylori, Helicobacter felis or water. In the nonpregnant study, 15 mice from each group were sacrificed after 4 and 20 weeks of infection. In the pregnancy study, after 6 weeks of infection all female mice were mated and approximately 2 weeks after mating, half of the pregnant mice (n = 9/group) from each group were sacrificed. The remaining mice were allowed to give birth, and approximately 4 weeks after birth, mice were asphyxiated with CO2, followed by heart puncture, and killed by cervical dislocation. Serum ferritin and iron were determined with a micro-particle enzyme immunoassay method and by a timed-endpoint method. RESULTS: Serum iron levels in mice infected with H. felis were significantly (p < .05) lowered compared to control (24%) and H. pylori (27%)-infected mice at 4 weeks of infection. Serum iron in the control, H. pylori and H. felis groups were significantly (p < .05) elevated at 20 weeks by 39, 26 and 77%, respectively, compared to 4 weeks of infection. H. felis-infected mice had a significantly (p < .05) decreased serum ferritin level during pregnancy (61%) compared to H. pylori-infected mice. CONCLUSION: These results suggest that H. felis but not H. pylori infection causes an acute iron deficiency in normal and pregnant mice.     

DNA and oligosaccharides stimulate oligomerization of human milk lactoferrin

Lactoferrin (LF) is a Fe³⁺-transferring glycoprotein and is contained in human barrier fluids, blood, and milk. LF is an acute phase protein, is involved in nonspecific defense, and displays a unique set of biological functions. Small-angle X-ray scattering and light scattering experiments demonstrated that DNA and oligosaccharides added to LF with various levels of initial oligomerization increased the oligomerization rate. Almost complete dissociation into monomers was observed when 1 M NaCl was added to LF oligomers obtained in the presence of DNA, oligosaccharides, and nucleotides, previously identified as oligomerization effectors. LF complexes obtained with different oligomerization effectors differed in stability. Incubation with 50 mM MgCl₂ completely destructed LF complexes formed in the presence of ATP and oligosaccharides but only partly destructed AMP- and d(pT)₁₀-dependent complexes, which was followed by the formation of new complexes with a higher salt stability. A possible role of oligomerization in various LF functions is discussed.     

Helicobacter pylori-related iron deficiency anemia: a review

Several clinical reports have demonstrated that Helicobacter pylori gastric infection has emerged as a new cause of refractory iron deficiency anemia, unresponsive to iron therapy, and not attributable to usual causes such as intestinal losses or poor intake, malabsorption or diversion of iron in the reticulo-endothelial system. Although the interaction between infection and iron metabolism is now well consolidated, our understanding of the pathogenetic mechanism underlying the anemia is still wanting. Microbiological and ferrokinetic studies seem to suggest that Helicobacter pylori infected antrum could act as a sequestering focus for serum iron by means of outer membrane receptors of the bacterium, that in vitro are able to capture and utilize for growth iron from human lactoferrin. The proposed hypothesis does not answer why this complication is such a rare disease outcome in a common human infection but it may be used as a template for further controlled studies to determine the mechanisms of this atypical, medically important putative sequelae of H. pylori infection.     

Nuclear transfer of goat somatic cells transgenic for human lactoferrin gene

Transgenic animal mammary gland bioreactors are used to produce recombinant proteins with appropriate post-translational modifications. The nuclear transfer of transgenic somatic cells is a powerful method to produce mammary gland bioreactors. We established an efficient gene transfer and nuclear transfer approach in goat somatic cells. Gene targeting vector pGBC2LF was constructed by cloning human lactoferrin (LF) gene cDNA into exon 2 of the milk goat beta-casein gene and the endogenous start codon was replaced by that of human LF gene. Goat fetal fibroblasts were transfected with linearized pGBC2LF and 14 cell lines were positive according to PCR and Southern blot. The transgenic cells were used as donor cells of nuclear transfer and some of reconstructed embryos could develop into blastocyst in vitro. __________ Translated from Hereditas (Beijing), 2006, 28(12): 1513–1519 [译自: 遗传]     

硫酸亚铁对缺铁性贫血小鼠肠道健康的影响

【目的】旨在探究硫酸亚铁(FeSO4)对缺铁性贫血(iron deficiency anemia, IDA)小鼠肠道健康的影响。【方法】选取45只24日龄体重(16.0±1.2) g的雄性昆明小鼠,随机分成3组,每组15只,即对照组(正常饲料,饮蒸馏水)、IDA组(低铁饲料造模2周,饮去离子水)、IDA-Fe²⁺组(造模结束后灌胃FeSO₄ 3周,饮去离子水),实验结束时采样。【结果】试验结束时,与IDA组相比,IDA-Fe²⁺组小鼠的红细胞、血红蛋白、红细胞压积、平均红细胞体积、平均红细胞血红蛋白量、平均红细胞血红蛋白浓度等贫血指标均恢复至正常水平(P>0.05),表明FeSO4具有改善IDA的功能;与对照组相比,IDA和IDA-Fe²⁺组小鼠的氧化应激指标和肿瘤坏死因子α均显著升高(P<0.01)、紧密连接蛋白Occludin显著降低(P<0.001),IDA-Fe²⁺组与IDA组除结肠总抗氧化能力(TAC)外均无差异显著性(P>0.05)。结肠组织...     

Nuclear transfer of goat somatic cells transgenic for human lactoferrin gene

Transgenic animal mammary gland bioreactors are used to produce recombinant proteins with appropri-ate post-translational modifications.The nuclear transfer of transgenic somatic cells is a powerful method to pro-duce mammary gland bioreactors.We established an effi-cient gene transfer and nuclear transfer approach in goat somatic cells.Gene targeting vector pGBC2LF was con-structed by cloning human lactoferrin (LF) gene cDNA into exon 2 of the milk goat beta-casein gene and the endogenous start codon was replaced by that of human LF gene.Goat fetal fibroblasts were transfected with lin-earized pGBC2LF and 14 cell lines were positive accord-ing to PCR and Southern blot.The transgenic cells were used as donor cells of nuclear transfer and some of recon-structed embryos could develop into blastocyst in vitro.     

Production of stable isotope enriched antimicrobial peptides in Escherichia coli: An application to the production of a 15N-enriched fragment of lactoferrin

A method is described for the production of recombinant isotopically enriched peptides in E. coli. Peptides are produced in high yield as fusion proteins with ketosteroid isomerase which form insoluble inclusion bodies. This insoluble form allows easy purification, stabilizes the peptide against degradation and prevents bactericidal activity of the peptide. Cyanogen bromide cleavage released peptide which was conjugated with alkylamines to form lipopeptide. An important advantage of this system is that it allows production of peptides that are toxic to bacteria, which we have demonstrated on a dodecapeptide based on residues 21–31 of human bactericidal protein lactoferrin.     

人乳铁蛋白cDNA 基因乳腺表达载体的构建与鉴定

为了构建人乳铁蛋白基因 (hLF) 的乳腺表达载体并验证其在乳腺细胞中的表达情况,本载体以山羊β-casein基因上游包括启动子、外显子1、内含子1、部分外显子2作为5′端调控序列,下游包括部分外显子7、内含子7、外显子8、内含子8、外显子9及3′部分基因组片段作为3′端调控序列,长度分别为6.2 kb和7.1 kb,将hLF基因 (目的基因) 和Neo基因 (筛选标记) 分别插入到5′端调控序列和3′端调控序列的下游,构建成pBC1-hLF-Neo载体,其全长为25.348 kb。为了检测该载体的生物学     

Influence of dietary iron deficiency on acute metal intoxication

The influence of dietary iron deficiency on acute nickel, lead or cadmium toxicity as reflected by the induction of hepatic, renal and intestinal metallothionein (MT), disposition of the metals, and alterations in hematological parameters was investigated in rats. The administration of cadmium induced the hepatic, renal and intestinal MT while that of nickel or lead induced hepatic MT only. However, dietary iron deficiency did not influence the cadmium induced tissue MT but enhanced the ability of nickel or lead to restore the normal synthesis of renal and intestinal MT lowered under the influence of reduced body iron status. The accumulation of lead in liver and kidney and that of cadmium enhanced in liver only, while tissue deposition of nickel remained unaffected by iron deficiency. The induction of hepatic MT by three metals appears related to the concomitant rise in the hepatic zinc, calcium and iron levels in normal rats. However, dietary iron deficiency increased the hepatic zinc in response to nickel or cadmium and that of heptic calcium in response to lead.     

The effect of Helicobacter pylori infection and dietary iron deficiency on host iron homeostasis: a study in mice

BACKGROUND: Helicobacter pylori, which requires iron to survive, may cause host iron deficiency by directly competing with the host for available iron or by impairing iron uptake as a consequence of atrophy-associated gastric hypochlorhydria. The aim of this study was to examine the effect of H. pylori infection and dietary iron deficiency on host iron homeostasis in a mouse model. MATERIALS AND METHODS: H. pylori SS1-infected and uninfected C57BL/6 mice, fed either a normal diet or an iron-deficient diet, were assessed for iron status and infection-associated gastritis over a 30-week period. RESULTS: After 10 weeks, serum ferritin values were higher in H. pylori-infected mice than in uninfected controls, irrespective of dietary iron intake (p = .04). The infection-related increase in body iron stores persisted in the iron-replete mice but diminished over time in mice with restricted dietary iron intake (p < .0001). At 30 weeks serum ferritin levels were lower in these animals (p = .063). No significant difference in bacterial numbers was detected at the 30-week time point (p > .05) and the histological changes observed were consistently associated with infection (p < .01) and not with the iron status of the mice (p = .771). CONCLUSIONS: Infection with H. pylori did not cause iron deficiency in iron-replete mice. However, diminished iron stores in mice as a result of limited dietary iron intake were further lowered by concurrent infection, thus indicating that H. pylori competes successfully with the host for available iron.     

DNA,oligosaccharides, and mononucleotides stimulate oligomerization of human lactoferrin

Using small‐angle X‐ray scattering (SAXS), light scattering (LS), and soft laser ablation we have shown that lactoferrin (LF) in solution at neutral pH is oligomerized in the absence of salt or at physiological salt concentrations. The level of oligomerization depends on the concentration of LF, KCl or NaCl, and on the duration of the protein storage in solution. At the concentrations comparable with those in human milk (1 ÷ 6 mg/ml), the average radius of gyration (R_g) values of LF can attain 400 ÷ 480 Å´, while fresh solution of previously lyophylized LF demonstrate a lower average R_g (50 ÷ 100 Å´), and R_g value characterizing the LF monomer formed at 1 M NaCl is 26.7 Å´. The addition of oligonucleotides, oligosaccharides, or mononucleotides to LF in the presence or in the absence of KCl with different level of initial oligomerization accelerates the oligomerization rate and increases the R_g values up to ～600 ÷ 700 Å´, which correspond to associates containing ten or more protein molecules. During gel filtration on Sepharose 4B, high‐degree LF oligomers dissociate nearly completely forming different degraded complexes, but in some cases it is possible to reveal small amount of a decamer. A possible role for oligomerization of LF, a highly polyfunctional protein, for its different biological activities is discussed. Copyright © 2009 John Wiley & Sons, Ltd.