转基因牛乳中重组人乳铁蛋白中试纯化工艺的建立及其活性分析

目的建立转基因牛乳中重组人乳铁蛋白(recombinant human lactoferrin,rh LF)的中试纯化工艺,并分析纯化的rh LF的活性。方法以含rh LF的转基因牛乳为原料,通过陶瓷膜分离技术除菌,一步阳离子交换层析,非离子型去污剂与金属络合剂去除内毒素,以及超滤浓缩脱盐,冷冻干燥制备重组蛋白纯品等步骤纯化rh LF,采用SDSPAGE、凝胶过滤色谱(SEC-HPLC)法和反相色谱(RP-HPLC)法分析rh LF的纯度;ELISA法检测rh LF的含量;鲎试剂法检测rh LF的内毒素含量;圆二色谱技术分析rh LF的二级结构;并检测rh LF的铁结合能力和抑菌活性。结果纯化的rh LF的平均纯度可达96%,内毒素含量小于0.5 EU/mg,整个工艺回收率达75%,且具有与天然人乳铁蛋白(human lactoferrin,h LF)相近的二级结构及正常的铁结合能力和抑菌活性。结论建立的牛乳中rh LF的中试纯化工艺简便高效,适用于rh LF的规模化生产。     

牛乳铁蛋白十肽的基因改造、克隆及表达

目的克隆并表达牛乳铁蛋白十肽及其突变体M1和M2基因。方法参照大肠杆菌偏爱密码子,分别针对牛乳铁蛋白十肽及其突变体M1和M2基因,设计并合成两个具有相同黏性末端的DNA片段,同时在其基因前后分别加上天冬酰胺和甘氨酸的密码子,构成羟胺裂解位点,通过连接获得其二拷贝基因同向串联体。分别将该串联体克隆至载体pUC18上,经双酶切、PCR及测序鉴定后,构建重组表达质粒,转化大肠杆菌BL21(DE3),IPTG诱导表达。表达产物纯化后,用凝血酶切割及羟胺裂解。结果获得了牛乳铁蛋白十肽及其突变体M1和M2二拷贝基因,并在大肠杆菌BL21(DE3)中表达出相对分子质量约29000的目的蛋白条带,纯化后的蛋白经凝血酶切割后,相对分子质量约29000的目的蛋白条带消失。结论已成功克隆并表达了牛乳铁蛋白十肽及其突变体M1和M2基因,为基因工程抗真菌肽的制备奠定了基础。     

SPEC 70 SLS阳离子树脂分离牛乳中乳铁蛋白

建立了乳铁蛋白和乳过氧化物酶的反相液相色谱检测法;并采用SPEC 70 SLS离子交换树脂,研究了一步法分离脱脂牛乳中乳铁蛋白和乳过氧化物酶的工艺条件。高效液相条件:波长220 nm,流速1 mL/min,乙腈体积分数在20 min内从25%增加到75%。SPEC 70 SLS树脂静态吸附条件优化:温度15℃,pH7.0,时间3 h。在此条件下,乳铁蛋白标准品的吸附量(质量比)为22.0 mg/g。乳铁蛋白吸附过程符合Langmuir吸附等温线模型,最大吸附量为21.73 mg/g。在静态脱脂牛乳吸附过程中,乳铁蛋白和乳过氧化物酶的吸附量分别为7.64,6.89 mg/g,优于CM Sepharose FF和SP Sepharose FF。     

Box-Behnken设计优化阳离子交换色谱分离牛初乳中乳铁蛋白的工艺研究

以新鲜牛初乳为原料,对阳离子交换色谱法分离牛初乳中乳铁蛋白的工艺条件进行优化。本试验采用阳离子交换色谱CM-Sepharose fast flow分离牛初乳中的乳铁蛋白,研究了起始缓冲液pH、洗脱速度、洗脱液浓度以及上样速度对乳铁蛋白得率的影响,并通过四因素三水平的响应面分析法优化了阳离子交换色谱法的分离工艺。结果表明,乳铁蛋白最佳分离条件为起始缓冲液pH 7.03,洗脱速度1.01 mL·min~(-1),洗脱液浓度1.01 mol·mL~(-1),上样速度0.82 mL·min~(-1)。在上述条件下,乳铁蛋白的得率为84.43%,与模型高度拟合。此方法操作简单,方便快捷,准确可靠,重现性好。     

人乳铁蛋白肽的分子改良及在大肠杆菌中的表达研究

采用SOE-PCR技术,设计合成hLF18-40的编码基因；以融合表达策略构建了pET-43.1a-hLF18-40表达质粒,并获得较高的可溶融合蛋白表达；将融合蛋白Nus-hLF18-40进行Ni2+柱亲和层析、除盐、浓缩后,用肠激酶切割释放抗茵肽hLF18-40,为今后基因工程法表达人乳铁蛋白肽提供了依据.同时,将...     

牛乳中生长因子的提取及应用概况

牛乳中含有多种具有重要生物活性的生长因子.从牛乳和乳清中提取生长因子的多种方法中,阳离子交换、微滤、双层超滤等应用广泛.基因分型微点阵法可成功分离出酪蛋白.生长因子提取物在临床上已经用于伤口愈合和体内器官功能紊乱的治疗,还可用于骨组织的再生和发炎性皮肤病的治疗.     

乳铁蛋白纯化和检测方法研究进展

乳铁蛋白是一种具有抗微生物活性的铁离子结合糖蛋白,属于转铁蛋白家族的一员,具有调节炎症反应和激活免疫系统等功能,因此乳铁蛋白被广泛应用于医学和食品领域。本综述汇集了乳铁蛋白纯化和检测方法的研究进展,并比较了各方法之间的优缺点。     

以脱铁蛋白为模板控制合成CoO纳米颗粒

提出利用生物模拟化学原位合成纳米颗粒的方法.利用马脾铁蛋白作为限制性反应器控制金属Co离子水解、氧化还原等反应组装成单分散的CoO纳米粒子.在碱性条件下脱铁铁蛋白限制性合成磁性CoO纳米颗粒,通过透射电子显微镜(TEM)和元素分析(EDX)对产品的结构和组成分析表征,颗粒尺寸在3～8nm范围.此外,采用紫外、可见吸收光...     

乳铁蛋白介导的肺靶向纳米脂质载体的研究进展

纳米脂质载体属纳米给药体系，是在固体脂质纳米粒基础上发展起来的新型靶向给药系统，以生物相容性好的固态和液态脂质混合作为载体材料，将药物包裹于类脂核中，具有胶体载体如脂质体和纳米乳的优势，可用作抗肿瘤药物的靶向载体，同时，相比而言，纳米脂质载体雾化稳定性更好，在气道中具有良好的耐受性和理想的肺沉积率，适用于雾化吸入给药，该给药方式可使药物直达病灶，雾滴长时间滞留在肿瘤部位，发挥长效作用。为进一步提高药物的生物利用度、增强肺癌靶向性，可用乳铁蛋白对纳米脂质载体进行表面修饰。乳铁蛋白属转铁蛋白家族，正常肺组织中转铁蛋白受体呈阴性或低表达，而在非小细胞肺癌中转铁蛋白的阳性表达率远远高于正常肺组织，存在过度表达现象，可利用乳铁蛋白作为配体修饰纳米脂质载体，通过转铁蛋白介导的方式增加药物在肺癌细胞内的富集，受体介导的内吞可以进一步促进负载药物的纳米脂质载体的细胞摄取。     

乳铁蛋白纯化方法的优化

将纯度为50%左右的乳铁蛋白溶液加入带羧甲基的弱酸性阳离子交换剂CM-Sepharose Fast Flow进行动态吸附,采用质量分数为1.6%和5%的NaCl溶液进行阶跃洗脱;并对层析过程和接样方法进行了优化.结果表明,经过重复层析和优化方法接样,乳铁蛋白纯度可达96.7%.     

Antibacterial and Anti-biofilm Activity of the Human Breast Milk Glycoprotein Lactoferrin against Group B Streptococcus

Group B Streptococcus (GBS) is an encapsulated Gram-positive human pathogen that causes invasive infections in pregnant hosts and neonates, as well as immunocompromised individuals. Colonization of the human host requires the ability to adhere to mucosal surfaces and circumnavigate the nutritional challenges and antimicrobial defenses associated with the innate immune response. Biofilm formation is a critical process to facilitate GBS survival and establishment of a replicative niche in the vertebrate host. Previous work has shown that the host responds to GBS infection by producing the innate antimicrobial glycoprotein lactoferrin, which has been implicated in repressing bacterial growth and biofilm formation. Additionally, lactoferrin is highly abundant in human breast milk and could serve a protective role against invasive microbial pathogens. This study demonstrates that human breast milk lactoferrin has antimicrobial and anti-biofilm activity against GBS and inhibits its adherence to human gestational membranes. Together, these results indicate that human milk lactoferrin could be used as a prebiotic chemotherapeutic strategy to limit the impact of bacterial adherence and biofilm formation on GBS-associated disease outcomes.     

Lactoferrin Directly Scavenges Hydroxyl Radicals and Undergoes Oxidative Self-Degradation: A Possible Role in Protection against Oxidative DNA Damage

In this study, we examined the protective effect of lactoferrin against DNA damage induced by various hydroxyl radical generation systems. Lactoferrin (LF) was examined with regard to its potential role as a scavenger against radical oxygen species using bovine milk LF. Native LF, iron-saturated LF (holo-LF), and apolactoferrin (apo-LF) effectively suppressed strand breaks in plasmid DNA due to hydroxyl radicals produced by the Fenton reaction. In addition, both native LF and holo-LF clearly protected calf thymus DNA from fragmentation due to ultraviolet irradiation in the presence of H₂O₂. We also demonstrated a protective effect of all three LF molecules against 8-hydroxydeoxyguanosine (8-OHdG) formation in calf thymus DNA following ultraviolet (UV) irradiation with H₂O₂. Our results clearly indicate that native LF has reactive oxygen species-scavenging ability, independent of its nature as a masking component for transient metals. We also demonstrated that the protective effect of LF against oxidative DNA damage is due to degradation of LF itself, which is more susceptible to degradation than other bovine milk proteins.     

复合乳酸菌发酵生产天然鲜奶香基的研究

通过吖啶橙诱变提高乳酸菌的脂肪酶和蛋白酶活性,结合感官评定发酵乳风味,筛选得到了具有产香潜力的7株乳酸菌突变菌。组合各株产香菌共发酵,得到了一个能产生鲜奶香基的菌株组合。该组合在以蔗糖-葡萄糖（2∶1）为碳源、添加0.5%柠檬酸铵的牛乳产香培养基中,42℃条件下连续发酵菌株La2、Lc1和St2 12 h,发酵液均质后能作为鲜奶香基直接应用。     

Ionic Liquid-Based Three Phase Partitioning (ILTPP) for Lactoferrin Recovery

A novel technique called ionic liquid-based three phase partitioning (ILTPP) that combines the interesting properties of ionic liquids as extracting solvents and the advantages of interfacial partitioning for protein recovery is presented in this work. The ternary system BmimBF₄/NaH₂PO₄/H₂O is used to accumulate lactoferrin (a bovine whey with important nutraceutical properties) at the liquid-liquid interface. Between 74% and 99% of the lactoferrin is recovered at the interface, depending on the temperature, the ionic liquid content, and, especially, the salt concentration. Consequently, ILTPP can be seen as a promising technique that may overcome the drawbacks of conventional techniques to recover lactoferrin.     

中性氧化铝柱净化-UPLC-MS/MS法测定乳和乳制品中黄曲霉毒素M_1

建立了中性氧化铝固相萃取柱净化,超高效液相色谱-串联质谱法(UPLC-MS/MS)测定乳和乳制品中黄曲霉毒素M1的分析方法。样品经乙腈提取,乙酸锌辅助沉淀蛋白,中性氧化铝SPE柱净化,以乙腈和0.1%甲酸水溶液作为流动相进行梯度洗脱,以电喷雾离子源(ESI)在正离子多反应监测(MRM)模式下进行测定,外标法定量。结果表明,黄曲霉毒素M1在0.1~50μg/L浓度范围内呈良好的线性关系,相关系数(r2)大于0.99;空白样品加标回收率在70.8%~79.2%之间,相对标准偏差小于8%。方法检出限为0.02μg/kg,定量限为0.05μg/kg。该方法实用、准确、灵敏,适用于乳和乳制品中黄曲霉毒素M1的测定。     

Lactoferrin Ameliorates Dry Eye Disease Potentially through Enhancement of Short-Chain Fatty Acid Production by Gut Microbiota in Mice

Lactoferrin is a glycoprotein found at high concentrations within exocrine secretions, including tears. Low levels of lactoferrin have been implicated in the loss of tear secretion and ageing. Furthermore, lactoferrin possesses a range of functionalities, including anti-inflammatory properties and the ability to modulate the gut microbiota. Expanding evidence demonstrates a crucial role of the gut microbiota in immune regulation and development. The specific composition of bacterial species of the gut has a profound influence on local and systemic inflammation, leading to a protective capacity against a number of inflammatory diseases, potentially by the induction of regulatory immune cells. In this study, we demonstrated that oral administration of lactoferrin maintains tear secretion in a restraint and desiccating stress induced mouse model of dry eye disease. Furthermore, we revealed that lactoferrin induces the reduction of inflammatory cytokines, modulates gut microbiota, and induces short-chain fatty acid production. Whereas, the antibiotic vancomycin abrogates the effects of lactoferrin on dry eye disease and significantly reduces short-chain fatty acid concentrations. Therefore, this protective effect of LF against a mice model of DED may be explained by our observations of an altered gut microbiota and an enhanced production of immunomodulatory short-chain fatty acids.     

液相色谱-串联质谱法测定乳制品中的氯霉素

建立了超高效液相色谱-串联质谱/质谱法测定奶粉、凝固型酸奶及液态奶中氯霉素的方法。试样用10%亚铁氰化钾及20%乙酸锌作为沉淀剂,以乙酸乙酯提取,采用电喷雾离子源,负离子扫描,多反应监测(MRM)模式进行检测,外标法定量。结果表明,氯霉素在0.5~100μg/L浓度范围内线性关系良好,空白样品加标回收率均大于80%,相对标准偏差小于6%。氯霉素方法检出限(LOD)均为0.1μg/kg。该方法准确、灵敏、操作相对简便,有效降低基质干扰,适用于奶粉、牛奶中氯霉素的测定。     

石墨炉原子吸收光谱法测定奶粉中的六价铬

基于Cr(Ⅲ)与噻吩甲酰三氟丙酮(TTA)生成的络合物在石墨炉中的挥发性,建立一种前处理简单、灵敏度高的测定奶粉中六价铬的分析方法。对样品的消化方式、Cr(Ⅲ)与TTA络合的酸度、试剂的加入量、超声波水浴的温度和时间、静置时间与石墨炉挥发Cr(Ⅲ)的原子化条件进行探讨。结果表明,Cr(Ⅵ)在0.002~0.010mg·L-1浓度内呈良好线性关系(r=0.9995)。奶粉样品中Cr(Ⅵ)不同加入量的平均回收率为78%~93%,其相对标准偏差为3.8%~9.4%,检出限为0.4 mg·L-1。方法具有分析速度快、灵敏度高、干扰少、操作简便等特点,应用于奶粉中六价铬的测定获得了满意的结果。