牛气管抗菌肽的原核表达、体外抗菌活性及其组织分布分析

本文旨在对牛气管抗菌肽(tracheal antimicrobial peptide,TAP)基因进行原核表达,以及研究重组蛋白的抗菌活性,并分析TAP在各组织的表达分布情况,为开展转TAP基因抗乳腺炎奶牛研究提供技术资料。作者采用RT-PCR从荷斯坦奶牛气管扩增TAP基因,分析其序列后根据大肠杆菌表达系统对密码子的偏好性,人工合成牛TAP基因,构建pET32a(+)-TAP重组质粒;然后转化大肠杆菌BL21,IPTG诱导表达并纯化。对表达蛋白进行SDS-PAGE、Western Blot鉴定;在线软件分析目的蛋白表达量。采用滤纸片法测定重组牛TAP的体外抗菌活性,并用RT-PCR法分析牛各组织中TAP表达情况。测序结果表明,扩增到114bp目的基因片段,可编码含38个氨基酸残基的成熟蛋白。SDS-PAGE分析表达的融合蛋白相对分子质量为24ku,重组蛋白以可溶性方式存在,表达量占菌体总蛋白的52.1%。Western Blot检测只出现单一条带。抗菌效价分析表明0.115μg·μL-1重组蛋白对牛源金黄色葡萄球菌有一定的抗菌活性。TAP基因在奶牛咽喉、肺、气管、小肠、肝、心脏及口腔黏膜中均有表达,在鼻黏膜和舌黏膜中微量表达,在皮肤中几乎不表达。作者成功表达了牛TAP基因,重组牛TAP蛋白在大肠杆菌中实现高效表达,并具有一定的抗奶牛乳腺炎致病菌的活性,为未来培育抗乳腺炎转基因奶牛及相关基因工程产品的开发奠定了基础。     

应用AML基因进行牛性别鉴定的研究

本文以染色体牙釉蛋白基因(AML)对牛肌肉基因组进行性别鉴定，为应用此基因进行胚胎鉴定提供理论依据。本研究应用奶牛染色体牙釉蛋白基因的DNA序列，设计了牛染色体特异性引物P1和P2，并通过聚合酶链反应(PCR)扩增牛肌肉组织中的X和Y染色体上的牙釉蛋白基因内第五内含子。最后依据其分子量的差异对电泳后的性染色体所呈现的不同区带进行区分，从而达到性别鉴定的目的，具有很高的应用价值。     

奶牛乳腺炎抗性候选基因多态性研究进展

奶牛的乳腺炎是一种极其复杂的疾病,是影响奶牛产业发展的一个重要因素。对奶牛乳腺炎抗性的研究有助于选育出有高抗性的奶牛群体,提高奶产品的数量和质量,并且可以减少抗生素的使用。近年来,随着生物信息学、分子育种技术和基因组学的发展,为奶牛的乳腺炎抗病育种提供的新的前景。本文主要介绍了牛锌指蛋白313基因、前脑锌指蛋白基因、T...     

PCR扩增妊娠奶牛牙釉蛋白基因进行胚胎性别鉴定的研究

牙釉蛋白（amelogenin,简写为AML）基因是牙齿发育过程中丰富表达的多拷贝基因,AML基因的同源基因分别定位在XY染色体上。本试验利用x—Y同源的牙釉蛋白基因序列设计一对特异性引物（牛AML基因序列的扩增片段长度：雌性为只有467bp的特异性扩增片段：雄性为同时具有341bp和467bp的两条特异性扩增片段）,应用PCR技术同时扩增X和Y染色体上的特异性片段,扩增产物用PAGE电泳分离技术,经硝酸银溶液染色及扫描分析进行妊娠奶牛早期胚胎的性别鉴定。结果显示,从X染色体上扩增出467bp的片段．从Y染色体上扩增出341bp的特异性片段。由此可知,PCR扩增妊娠奶牛牙釉蛋白基因可以进行胚胎的性别鉴定。     

奶牛早孕诊断技术研究进展

高繁殖率是提高奶牛场经济效益的重要保障,而早孕诊断又是提高奶牛繁殖率的重要手段之一。传统的妊娠诊断方法较难在配种后21 d内确定奶牛妊娠状态,而近年研发的新型妊娠诊断技术可以对奶牛进行有效早孕诊断。本文综述了多普勒彩超技术,中红外线光谱技术及妊娠相关蛋白、循环核苷酸、干扰素τ诱导基因检测等技术在奶牛早孕诊断中的研究进展,比较分析了这些技术的实际应用效果及优缺点,并对新的奶牛早孕诊断技术进行了展望,以期为开发和应用新型高效奶牛早孕诊断技术提供思路和借鉴。     

国外家畜基础学科研究进展（续）

八、生长激素自1982年美国首次利用重组DNA技术使牛的生长激素基因在大肠杆菌中表达成功以来,目前生长激素的生产已经工厂化,这就为人类利用生长激素促进家畜生长发育和提高产奶、产肉性能创造了有利条件。迄今,国外先后在牛、羊、猪上进行了生长激素的使用试验,其中在奶牛上应用生长激素的试验最多。下面主要介绍近几年国外在奶牛上使用牛生长激素(BST)提     

奶牛生物钟基因BMAL1真核表达载体的构建及生物信息学分析

本研究旨在克隆奶牛脑肌类芳香烃受体核转位蛋白1基因（Brain and Muscle Arnt-Like Protein 1,BMAL1）的蛋白编码序列（Coding Sequence,CDS）并构建其真核表达载体,运用生物信息学方法解析其CDS序列和编码蛋白特性。以牛肝脏组织为材料,利用PCR技术扩增奶牛BMAL1基因中带有同源臂的CDS片段。采用同源重组法,将目的片段连接至酶切线性化的pc DNA3.1-Puro-N-3HA真核表达载体上,酶切及测序结果均符合预期的质粒命名为pc DNA3.1-3HA-c BMAL1。将空载与重组质粒分别转染至HEK293T细胞,通过实时荧光定量PCR技术（q PCR）和Western Blotting技术检验奶牛BMAL1基因的过表达效果,并利用生物信息学软件分析BMAL1基因CDS区及其编码蛋白的特性和功能。结果显示,pc DNA3.1-3HA-c BMAL1真核表达载体构建成功,将该质粒转染至HEK293T细胞后,成功实现了BMAL1在m RNA和蛋白水平的过表达;牛BAML1基因与绵羊、山羊同源性最高,BMAL1蛋白为不稳定蛋白,其上不存在...     

世界首批基因定位技术繁殖的高产奶牛在芬兰诞生

世界上首批利用基因定位技术繁殖的4头高产奶牛牛犊,今夏在芬兰南部约基奥伊宁芬兰农业和食品研究中心的试验农场诞生。据报道,芬兰农业和食品研究中心动物良种研究小组的专家们,经过多年努力,成功地从奶牛的胚胎中找到并确定了两个能够控制奶牛产奶量的基因。这两个基因不仅能够控制奶牛的产奶量,还能控制牛奶中蛋白和脂肪的含量。由于这些牛犊的产奶基因在胚胎阶段就已被确定,因此可以预先确切地知道这些牛犊长大后的产奶量以及牛奶中脂肪和蛋白的含量。这种奶牛不仅产奶量高,而且牛奶质量好。芬兰研究人员还将继续从事这方面的研究工作,…     

牛主要抗病候选基因的研究进展

牛抗病基因与其自身的抗病性和免疫能力有一定的相关性,因此研究相关抗病基因对提高牛自身免疫能力和选育优良后代有重要意义,文章着重介绍了国内外关于天然抗性相关巨噬细胞蛋白1（natual resistant macrophage protein 1,Nramp1）、主要组织相容性复合物（major histocompatibility complex,MHC）、甘露糖结合凝集素（maflnan binding lectin,MBL）、Toll样受体（Toll-like receptors,TLR）及干扰素基因（interferon,IFN）与牛某些疾病相关性的研究进展,及其是否可用于后代育种的分子标记,如MHC与奶牛的乳房炎密切相关;牛TLR4基因的外显子1760 C>T突变中的CC基因型可作为奶牛乳房炎抗性筛选中的分子标记等。笔者认为研究这些抗病基因可更深层次了解牛的抗病机制,同时借助先进的技术手段如分子标记、基因工程等,可更好的选育出抗病后代,抗病基因在育种中的应用将是未来研究抗病性和免疫能力的主要方向。     

澳科学用基因组测序技术研究提升牛类健康新方法

<正>"高大上"的基因测序技术,在实际生产生活中其实影也可用于畜牧业——譬如说用来增加牛类的产肉和产量奶效率。14日,英国《自然—遗传学》杂志在线发表的一足篇论文中,澳大利亚遗传学家们汇报了234头牛的完整年DNA序列。这些"调查对象"所具有的品种内部遗传多同样性,可以帮助畜牧业更好、更直接地选择健康的牛。对未知基因组序列的物种进行个体的基因组测序,行是一种强有力的工具。科学家可以通过生物信息手段,间来分析不同个体基因组间的结构差异,进而研究与疾禽病相关基因的突变过程、细胞损伤修复路径、基因调控对网络等。此次,澳大利亚维多利亚州环境和初级产业猪部的本·海耶斯和他的研究团队,将这一技术应用到推奶牛和牛肉产业。导调查中,团队人员对黑白花荷尔斯泰因牛(即最常见的奶牛,荷兰黑白花奶牛)、弗莱维赫牛(一种德系肉好     

蛋白质组学技术在人乳与牛乳中的研究进展

乳蛋白是生命最初阶段中最为重要的营养功能成分,并且种类多样、含量丰富.通过一系列的遗传改变、丰富的蛋白质翻译后修饰,使乳蛋白的构成变得非常复杂、数量异常庞大.采用蛋白质组学方法对乳蛋白进行系统、整体地研究,可以认识乳蛋白质在构成以及调控一些生命活动中的规律.这样能够非常全面、深入地表明乳蛋白质的表达水平.本文阐述了蛋白质组学中的主要技术,包括双向电泳技术、高效液相色谱技术、同位素标记相对和绝对定量(isobaric tags for relative and absolute quantitation,iTRAQ)技术、质谱技术以及生物信息学技术.分别讨论了蛋白质组学技术在人乳、牛乳中的应用,并对人乳与牛乳的差异蛋白质组学进行讨论.利用蛋白质组学技术分析人乳与牛乳蛋白,能够更有目的地加强对人乳与牛乳的认识,旨在为婴幼儿食品以及乳制品提供理论依据.     

Production of transgenic livestock: promise fulfilled

The introduction of specific genes into the genome of farm animals and its stable incorporation into the germ line has been a major technological advance in agriculture. Transgenic technology provides a method to rapidly introduce "new" genes into cattle, swine, sheep, and goats without crossbreeding. It is a more extreme methodology, but in essence, not really different from crossbreeding or genetic selection in its result. Methods to produce transgenic animals have been available for more than 20 yr, yet recently lines of transgenic livestock have been developed that have the potential to improve animal agriculture and benefit producers and/or consumers. There are a number of methods that can be used to produce transgenic animals. However, the primary method to date has been the microinjection of genes into the pronuclei of zygotes. This method is one of an array of rapidly developing transgenic methodologies. Another method that has enjoyed recent success is that of nuclear transfer or "cloning." The use of this technique to produce transgenic livestock will profoundly affect the use of transgenic technology in livestock production. Cell-based, nuclear transfer or cloning strategies have several distinct advantages for use in the production of transgenic livestock that cannot be attained using pronuclear injection of DNA. Practical applications of transgenesis in livestock production include enhanced prolificacy and reproductive performance, increased feed utilization and growth rate, improved carcass composition, improved milk production and/or composition, and increased disease resistance. One practical application of transgenics in swine production is to improve milk production and/or composition. To address the problem of low milk production, transgenic swine over-expressing the milk protein bovine alpha-lactalbumin were developed and characterized. The outcomes assessed were milk composition, milk yield, and piglet growth. Our results indicate that transgenic overexpression of milk proteins may provide a means to improve swine lactation performance.     

Mammary expression of new genes to combat mastitis

Continual advances in the ability to produce transgenic animals make it likely that such animals will become important components of animal agriculture. The full benefit of the technology, and justification of its initial cost outlay, will be dependent on the establishment within these animals of new traits not easily achievable by other means. Potential applications include enhanced nutrient digestibility with reduced fecal losses, significantly altered milk composition with superior nutritional properties, and enhanced disease resistance. Our goal is to enhance mastitis resistance of dairy cows by enabling the cells of the mammary gland to secrete additional antibacterial proteins. Proof of concept has been obtained through experimentation with a transgenic mouse model. Three lines of mice were developed that produce varying levels of lysostaphin in their milk. This protein has potent anti-staphylococcal activity and its secretion into milk confers substantial resistance to infection caused by intramammary challenge with Staphylococcus aureus, a major mastitis pathogen. Additional antibacterial proteins are being sought that will complement lysostaphin. A potential benefit of transgenic application of antibacterial proteins is the concomitant sparing in the agricultural use of antibiotics currently used as human therapeutics. Antibacterial proteins, such as lysostaphin, are not typically used as injectable or oral therapeutics because of immune-mediated or digestive destruction of their activity. In contrast, the immune system of transgenic animals will not consider the transgenic protein as being foreign. In addition we are exploring the potential of involution or mastitis responsive promoter elements for use in subsequent transgenic experiments designed to restrict lysostaphin production to these important time points. It is anticipated that genomics will play a role in unveiling candidate genes whose promoter elements will enable desired temporal expression patterns. The transgenic approach to insertion of new genetic material into agriculturally important animals is feasible but requires extensive prior evaluation of the transgene and transgene product in model systems.     

母乳蛋白质组研究进展

母乳是人类及所有哺乳动物生命最初阶段的唯一食物,乳蛋白质是乳中最重要的营养物质,近年来随着蛋白质组学技术的发展及其在乳蛋白质研究中的应用,人类对乳蛋白质的组成、分泌调控及其中的过敏蛋白原等的认识越来越深入.本文对蛋白质组学在乳蛋白质的全面鉴定、乳蛋白质分泌调控以及乳中过敏原分析鉴定方面的研究策略和进展进行综述,以提高人类对乳蛋白质组相关生物学过程的认识.     

茶多酚与牛乳蛋白相互作用对乳制品功能特性和生物学效应影响的研究进展

茶多酚是茶叶的次级代谢产物,因具有抗氧化性、抗癌和预防心血管疾病等作用而被广泛应用于食品和医药行业。茶多酚与牛乳蛋白的相互作用会使牛乳蛋白结构发生变化,进而影响乳制品的功能特性和生物利用度。牛乳蛋白具有特殊的结构,因此其与酚类物质的结合一直受到广泛关注。本文综述了茶多酚与牛乳蛋白的相互作用机理、影响因素、产物特性、机理分析方法的研究进展以及这种相互作用对乳制品功能特性和生物利用度的影响,为后续利用茶多酚和牛乳蛋白联合开发新型产品提供参考。     

乳蛋白基因在转基因动物乳腺细胞中特异性表达的研究进展

乳蛋白是乳的主要营养成分，乳蛋白的种类及其在乳中的浓度都影响乳的品质，而乳中各乳蛋白的含量都受到相应乳蛋白基因的控制。通过转基因技术，可以在转基因动物乳腺细胞中特异性地表达目的蛋白，从而改善乳的品质以及生产具有特殊药用价值的乳产品。本文主要概述乳蛋白多态性及其作用，乳蛋白基因及多态性，并着重介绍乳蛋白基因在转基因动物体内的表达调控；通过介绍大量转基因试验的研究成果，以探寻改变乳成分的分子遗传基础。     

酶法水解牛乳蛋白技术的研究

为获得易消化、低致敏的婴儿配方食品原料,采用胰蛋白酶水解牛乳蛋白,研究酶解过程中酶解时间、酶解温度、加酶量对乳蛋白水解度的影响。通过单因素试验设计方法,确定最佳酶解条件为:酶解时间40min、酶解温度40℃、加酶量1200U/g蛋白质。     

牛奶中不同酪蛋白遗传变异体及其分型鉴定研究

牛奶中存在不同种类的蛋白质,且不同基因型遗传变异体牛奶蛋白质的构型和功效存在差异。酪蛋白分型鉴定方法有基因鉴定、毛细管电泳分离鉴定、高效液相色谱分离鉴定等。本文对比介绍了不同构型κ-酪蛋白和β-酪蛋白的分型鉴定方法,重点验证了高效液相色分离谱鉴定方法的精密度、回收率,同时将该方法应用于生乳和乳制品酪蛋白分型检测鉴定,区分不同牧场牛群的遗传变异体类型和比例。通过该方法可指导牧场快速分群饲养奶牛,生产不同酪蛋白类型的生乳,并加工不同类型的高品质乳制品。     

Identification of allergens responsible for canine cutaneous adverse food reactions to lamb, beef and cow's milk

Lamb, beef and cow's milk are common causes of cutaneous adverse food reactions in dogs. The aim of this study was to identify the proteins responsible for cutaneous adverse reactions to these foods. Ten dogs with allergen-specific serum immunoglobulin (Ig)E to lamb, beef and cow's milk were included in the study. These dogs had been diagnosed with cutaneous adverse food reactions by convincing clinical history and food-elimination diet trials followed by challenge exposure. Sera were analysed by enzyme-linked immunosorbent assay with bovine proteins and SDS-PAGE immunoblots with lamb, beef and cow's milk extracts. All the dogs had specific IgE against bovine IgG, and it was the only protein in the cow's milk extract that bound IgE from the sera studied. In the lamb and beef extracts, the major allergens recognized by the specific IgE of most sera had molecular masses between 51 and 58 kDa, which were identified as phosphoglucomutase and the IgG heavy chain. Other IgE-binding proteins with molecular masses of 27, 31, 33, 37 and 42 kDa were also detected with some sera. Our results indicate that bovine IgG is a major allergen in cow's milk and hence it appears to be a source of cross-reactivity with beef and probably with lamb because of the high homology with ovine immunoglobulins. These results are similar to those found for meat allergy in humans. However, this is the first time that phosphoglucomutase has been identified as an important allergen involved in allergic reactions to lamb and beef.     

金华市奶牛饲养方案的调查研究

本文通过对市郊奶牛场奶牛饲养方案的抽样调查与分析,得出可按奶牛的生理特点和泌乳曲线配制日粮、推广“全混合日粮”、精心管理等一系列技术措施,从而提高奶牛的产奶量和乳品质。