Expression and bioactivity of recombinant human lysozyme in the milk of transgenic mice

Human milk lysozyme is an important protein for innate immunity, but human breast milk is a fairly poor source for commercial production of this enzyme. Research on the expression of recombinant human lysozyme (rHlys) is therefore potentially valuable to the dairy industry. In this study, 2 different kinds of transgenic mice, PBC-hLY and PBC-sighLY, were generated and used as system models to express rHlys. Six lines of PBC-hLY transgenic mice with human lysozyme genomic DNA-based constructs were generated, and a maximum expression level of rHlys approaching 0.154 mg/mL was achieved. Antibacterial activity of the whey from PBC-hLY female transgenic mice was determined by a turbidimetric assay. Results showed that antibacterial activity of the whey was strongly enhanced, and confirmed that rHlys retained full activity. For rHlys to be secreted efficiently into the milk of transgenic mice, 5 lines of mice were also generated, in which the signal peptide DNA of bovine β-casein was substituted for that of lysozyme in PBC-hLY transgenic mice. Compared with PBC-hLY transgenic mice, both the expression levels of rHlys and the antibacterial activity of the whey were much higher in the PBC-sighLY transgenic mice. The concentration of rHlys in one of these mice amounted to 1.405 mg/mL—3 times higher than the level in human whey. The antibacterial activity of the whey was also 3 times higher than that of human whey. The rHlys from both PBC-hLY and PBC-sighLY transgenic mice had the same antibacterial activity as human milk lysozyme. The effect of the signal peptide and copy numbers of the transgene on expression of rHlys was also evaluated. This work will certainly permit a better understanding of how mammary gland bioreactor systems can be applied to produce rHlys in other mammals, such as cattle.     

Antimicrobial activity of recombinant human lactoferrin from <Emphasis Type="Italic">Aspergillus awamori</Emphasis>, human milk lactoferrin and their hydrolysates

The antibacterial activity of human lactoferrin from milk (hLF), recombinant human lactoferrin from Aspergillus awamori (rhLF) and their hydrolysates obtained with pepsin was investigated against Escherichia coli O157:H7, Salmonella Enteritidis and Listeria monocytogenes. The minimum inhibitory concentrations (MIC) and the minimum bactericidal concentrations (MBC) were determined for all the bacteria and the proteins assayed. Taking into account the MICs found for both lactoferrins studied, we can say that they behave very similarly, except for L. monocytogenes for which rhLF was more active. We studied the effect that heat treatments exerted on the antibacterial activity of the two types of lactoferrin and the only heat treatment that had a negative effect on that activity was 85 °C for 10 min. The activity of hLF and rhLF in UHT milk and whey against E. coli O157:H7 and L. monocytogenes, was also assayed. Our results showed a reduction in the number of viable cells for both microorganisms when were incubated with rhLF or hLF, but this decrease was lower than in broth media.     

Effect of bovine lactoferrin and human lactoferrin on the proliferative activity of the osteoblast cell line MC3T3-E1 in vitro

We conducted a comparative in vitro study on the proliferative effects of natural human lactoferrin (nhLF) and bovine lactoferrin (bLF) on osteoblasts. We investigated cell proliferation, cell survival, cell cycle, and mRNA and protein expression of proliferating cell nuclear antigen. Results indicated that treatment with 100 μg/mL of bLF or nhLF promoted the proliferation and sustenance of osteoblasts, and increased the length of the G₂/M and S phases compared with the untreated osteoblasts. Results of real-time quantitative PCR and Western blot showed that mRNA and protein expression of proliferating cell nuclear antigen by osteoblasts treated with bLF or nhLF were greater than those of the untreated control. At the same concentration, bLF demonstrated a greater effect on osteoblast proliferation than did nhLF. This study provides insights of significance in the utlization of bLF in healthy food formulas.     

人乳铁蛋白在原核中的融合表达

设计了特异引物，通过PCR扩增人乳铁蛋白（HLF）基因，将扩增的DNA片段重组到原核表达载体pGEX-4T3中，重组体转化大肠杆菌BL21菌株，经IPTG诱导后，得到了高效融合表达。分析结果表明，获得了特异表达的乳铁蛋白。     

市售进口婴幼儿配方奶粉中乳铁蛋白含量的检测

目的 根据国家进口婴幼儿配方奶粉全项目检测的需求,采用酶联免疫方法对市售的多个品牌的进口婴幼儿配方奶粉中的乳铁蛋白含量进行了定量测试,为相关乳品企业、检测部门和消费者提供有益参考.方法 采用实验室建立的酶联免疫方法,方法操作方便、灵敏度高、可同时定性定量检测多个样品.结果 对市售16种进口婴幼儿配方奶粉中乳铁蛋白的含量进行检测,检测出乳铁蛋白的含量从16 ～167 mg/100 g.结论 乳铁蛋白作为高端进口婴幼儿奶粉中添加的一种营养强化剂,在不同品牌的奶粉中的含量有一定的差异,提醒有关部门、乳品企业和消费者关注.     

乳铁蛋白和溶菌酶在婴儿配方奶粉中添加量的计算及应用

从婴儿配方奶粉模拟母乳含量和成分的角度出发，提出了在婴儿配方奶粉中添加乳铁蛋白和溶菌酶(两种功能性活性蛋白质)的依据。文章重点对乳铁蛋白和溶菌酶的添加量进行了精确计算，并与国外的同类产品的添加量进行了对比．最后对添加工序的关键点进行了分析。     

转人乳铁蛋白基因大米的慢性毒性研究

目的 观察转人乳铁蛋白(hLF)基因大米是否具有慢性毒性作用.方法 初断乳的180只SD大鼠按性别、体重随机分为3组:转基因hLF基因大米组、亲本大米对照组和AIN-93对照组,分别饲喂相应饲料12个月.观察大鼠的体重、进食量、血常规、血生化情况,实验末期处死动物,称量脏器重量并对脏器进行病理学检查.结果 转hLF基因大米组血常规(LYM％、GRN％)、血生化(ALT、AST、GLU)在个别时间点上与亲本对照组或AIN-93对照组存在显著差异(P＜0.05);其他观察指标与两个对照组均无显著差异.结论 现有实验结果不能证实转hLF基因大米对大鼠有慢性毒性作用.     

毕赤酵母高效表达人乳铁蛋白的研究

将人乳铁蛋白全长基因克隆至酵母表达载体pPIC9K中，并建立hLF／pPIC9K／SMD1168酵母表达系统，在细胞高密度发酵条件下，乳铁蛋白表达量约1000mg／L，并具有天然蛋白相似的糖基化结构。本研究为商业化生产人乳铁蛋白提供了理论依据。     

乳铁蛋白的功能特性及其在婴儿配方奶粉中的应用

介绍了母乳中乳铁蛋白的功能特性，及其应用在婴儿配方奶粉中对非母乳喂养婴儿营养的重要性，国外有关乳铁蛋白在婴幼儿配方奶粉中的应用情况。探讨了乳铁蛋白在婴儿配方奶粉中应用的研究过程，包括有关配方设计依据、使用的原料、生产工艺流程、检验方法、检验结果等。     

山羊奶与牛奶和人奶营养成分的比较

对山羊奶、牛奶和人奶中蛋白质、脂肪、维生素、矿物质等主要营养成分进行了比较,分析了3种奶中主要营养成分的差别。通过比较发现,山羊奶在总体营养成分方面优于牛奶,更接近人奶。但山羊奶存在铁、叶酸和维生素B12含量较低以及羊奶膻味的问题,应在营养上对这方面加以重视。     

Protective effects of recombinant lactoferrin with different iron saturations on enteritis injury in young mice

Infant intestinal development is immature and, thus, is vulnerable to bacterial and viral infections, which damage intestinal development and even induce acute enteritis. Numerous studies have investigated that lactoferrin (LF) has protective effects on the intestine and may play a role in preventing intestinal inflammation in infants. Lactoferrin is divided into 2 types, namely apo-LF and holo-LF, depending on the degree of iron saturation, which may affect its bioactivities. However, the role of LF iron saturation in protecting infant intestinal inflammation has not been clearly clarified. Therefore, in this study, young mice models with intestinal damage induced by lipopolysaccharides (LPS) in vivo and primary intestinal epithelial cells in vitro were constructed to enteritis injury in infants for investigation. The apo-LF and holo-LF were subsequently applied to the mouse models to investigate and compare their levels of protection in the intestinal inflammatory injury, as well as to identify which LF was most active. Moreover, the specific mechanism of the LF with optimal iron saturation was further investigated through Western blot assay. Results demonstrated that disease activity index, shortened length of colon tissue, and histopathological score were significantly decreased in the apo-LF group compared with those of the LPS group and the holo-LF group. In the apo-LF group, the concentration of LPS in the intestinal tract and the number of gram-negative bacteria colonies decreased significantly and the expression levels of proinflammatory factors in the colon tissue were downregulated, in comparison with those in the LPS group. The findings of this study thus verify that apo-LF can significantly alleviate enteritis injury caused by LPS, through regulating the PPAR-γ/PFKFB3/NF-κB inflammatory pathway.     

Detection of the adulteration in pure cow ghee by electronic nose method (case study: sunflower oil and cow body fat)

Cow ghee is very used in some regions of Iran, such as Kermanshah province. Cow ghee is a natural source that contains high-quality nutrients which are needed for the human body. Adulteration in dairy products is not only a serious threat to human health but also it causes economic losses. Diagnosis of foodstuff cheating and its estimation is one of the key concerns in recent years. The aim of this study was the detection of the adulteration in cow ghee by olfactory machine system. Therefore, an electronic nose system was used for the different levels of sunflower oil and cow body fat mixed with pure cow ghee (10%, 20%, 30%, 40%, and 50%). The principal components analysis (PCA) and artificial neural networks (ANNs) methods were used to achieve this goal. Based on the results, the accuracy of the principal components analysis of sunflower oil and cow body fat were 96% and 97% of the data variance, respectively. According to the results, artificial neural networks identified the adulteration with sunflower oil and cow body fat with an accuracy of 91.3% and 82.5%, respectively.     

Direct sandwich ELISA to detect the adulteration of human breast milk by cow milk

The demand for commercially available human breast milk has significantly increased in recent years. For various reasons, a significant amount of commercially available human breast milk is being adulterated with other types of milk. This fraudulent practice poses a threat to consumers' health due to potential adulterants such as cow milk, which may put the infant at risk due to intolerance or allergy. A direct sandwich anti-bovine IgG ELISA has been developed for the sensitive and specific detection of cow milk in adulterated human breast milk. This assay uses polyclonal anti-bovine IgG antibody as a capture antibody and monoclonal anti-bovine IgG-alkaline phosphatase antibody as a detection antibody. Once optimized, the assay was found to be highly sensitive, and specific to bovine IgG. The assay had no significant cross-reaction with human breast milk, indicating that it was highly specific. The anti-bovine IgG ELISA was able to detect the presence of cow milk in adulterated human breast milk with a detection limit of 0.001% cow milk. The developed assay was highly reproducible (coefficient of variation <10%). The developed direct sandwich anti-bovine IgG ELISA is simple, reliable, and reproducible, making it an ideal test for this purpose.     

乳铁蛋白与肿瘤关系的研究进展

乳铁蛋白是一种天然糖蛋白,主要存在于母乳中,具有多种生物学功能。近年来,一些研究认为,乳铁蛋白具有化学预防和抗肿瘤作用。本文就乳铁蛋白和肿瘤的关系作一综述。     

口服乳铁蛋白生理功效、保健机理及应用于食品的最新进展

乳铁蛋白是一种多功能的铁结合性糖蛋白,对细菌、真菌、病毒等有广谱的防御能力,对感染、炎症、癌症等有明显的宿主保护功能。综述口服乳铁蛋白的生理功效及其独特的营养、抗菌防腐、免疫、抗氧化特性在食品中应用的最新进展。     

Expression and purification of goat lactoferrin from Pichia pastoris expression system

ABSTRACT:  The recombinant goat lactoferrin (rGLF) was expressed in the methylotropic yeast Pichia pastoris using pGAPZαC vector, GAP as promoter, and Zeocin as the selective marker. After transformation of the GLF-pGAPZαC into Pichia pastoris X-33 expression host, the GLF-pGAPZαC vector was integrated into the GAP promoter locus of Pichia pastoris X-33 chromosome. The rGLF was expressed and secreted into the broth using α-factor preprosequence. SDS-PAGE and PAS staining analysis indicated that the rGLF could be purified to electrophoretic homogeneity by heparin-Sepharose 6 Fast Flow affinity chromatography and glycosylated by the expression host. The yield of purified rGLF was approximately 2.0 mg/L of culture broth. The N-terminal sequence was identical to the native goat lactoferrin (nGLF). The iron-binding behavior, papain-inhibiting property, and thermal stability of the purified rGLF were comparable to nGLF. This is the 1st report of intact goat lactoferrin expression using the P. pastoris system.     

干酪乳清中乳铁蛋白分离纯化的研究

采用超滤、阳离子交换层析方法从热钙法处理过的干酪乳清中分离纯化乳铁蛋白。结果表明,经预处理后的干酪乳清加入经过磷酸盐缓冲液(pH值为8.0)平衡的树脂中,分别用浓度为0.3 mol/L和0.8 mol/L的NaCl进行阶跃洗脱,所得乳铁蛋白经SDS-PAGE和免疫印迹法(Western Blotting)检测,纯度达93.6%。     

Peptides from the N-terminal end of bovine lactoferrin induce apoptosis in human leukemic (HL-60) cells

To determine the effects of the multifunctional iron-binding glycoprotein, lactoferrin (LF) and related compounds on the growth of leukemic cells, human myeloid leukemic cells (HL-60) were exposed to bovine lactoferrin (bLF) and proteolytic hydrolysates of bLF. Pepsin hydrolysates of bLF showed a greater growth suppressive effect than tryptic hydrolysates or mature bLF. Four peptides with proliferation inhibition activity were purified from pepsin hydrolysates by ion-exchange chromatography, reverse-phase HPLC, and gel-filtration. All peptides were from the N-terminal end, in a region where lactoferricin B (Lfcin B), an antibacterial peptide, is located. Among the four peptides, peptide 1 (pep1) was found to exhibit highest activity and corresponded to residues 17 to 38 of bLF, with a molecular weight of 2753.88. The IC50 value of this peptide was 6.3 micrograms/ml. Three other peptides were less active and corresponded to sequences 1 to 16 and 45 to 48, linked by disulfide-bridge (pep2, molecular mass of 2430.13), 1 to 15 and 45 to 46 linked by disulfide bridge (pep3, molecular mass of 2017,92) and from residues 1 to 13 (pep4, molecular mass of 1558.73). Cell proliferation inhibition activity of the peptides was thought to be due to induction of apoptosis, which was evaluated by DNA ladder formation, DNA fragmentation, enhanced expression of phosphatidyl serine, and morphological changes. The IC50 values of the three peptides were confirmed using synthetic peptides and were consistent with those of purified peptides.