牛初乳乳清制备工艺研究

为优化乳清制备工艺,以脱脂牛初乳为原料,研究通过酸沉酪蛋白方法制备高质量乳清。综合考虑酸的种类、强弱和乳清中IgG的活性变化,确定了酸沉酪蛋白的最佳工艺条件为：0．8mol／L乳酸调脱脂乳pH值至4．3,加水至干物质终质量分数为10％,45℃水浴15min,4000r／min离心5min。经检测所制备的乳清中IgG活性含量仅损失3．05％。     

牛支原体灭活疫苗免疫程序及母源抗体消长规律研究

为评价不同剂量的自主研发牛支原体灭活氢氧化铝佐剂疫苗对怀孕母牛免疫后抗体消长规律和免疫效果,本试验选择产前30～15d围产母牛70头,随机分为7组,每组10头牛,间隔15d,采用不同剂量免疫,定期采集初乳和犊牛血清,采用已建立的牛支原体间接ELISA检测方法检测初乳乳清和犊牛血清中的抗体滴度,统计分析其抗体消长规律。结果表明,使用自主研发牛支原体灭活氢氧化铝佐剂疫苗免疫怀孕母牛无红肿、发热等症状,是安全可靠的;用不同剂量疫苗免疫怀孕母牛,初乳乳清抗体中牛支原体Ig G抗体水平在奶牛分娩后3h达到一个峰值,随着奶牛产后时间的延长抗体水平呈逐步下降,在奶牛产后96h,抗体值均在0.3以下;A组的抗体最低,B组、C组、D组抗体均低于E组和F组;E组和F组抗体滴度较高,两者无差异。不同剂量疫苗免疫母牛所产犊牛哺食初乳后,犊牛血清中牛支原体Ig G抗体在犊牛出生后24h达到最高,随着犊牛日龄的增加,抗体滴度呈现逐步下降趋势;在A组、B组和C组抗体呈现层次不齐的状况,D组抗体水平高于A组、B组、C组,低于E组和F组;E组和F组抗体水平较高,且维持时间较长。研究结果为牛支原体疫苗的免疫程序制定提供了...     

基于免疫磁珠技术快速富集牛支原体方法的建立

为建立牛呼吸系统疾病主要病原-牛支原体的快速富集方法,试验采用PCR方法对牛支原体P48基因扩增,构建重组质粒pET32a(+)-P48。将重组质粒进行原核表达并进行纯化。将纯化的牛支原体P48蛋白作为免疫原与弗氏佐剂混合乳化免疫家兔,获得抗牛支原体P48蛋白的高免血清,同时对高免血清进行纯化。将纯化后多克隆抗体与NHS磁性微球进行耦联,制备免疫磁珠。对耦联缓冲液、抗体浓度进行优化。从富集牛支原体的时间、敏感性和特异性三方面对免疫磁珠进行评价。结果表明:成功地扩增出大小约1 407 bp的P48基因并表达出大小约62 ku的可溶性重组蛋白。制备的多克隆抗体的效价高达1∶64 000,且具有很高的特异性;抗体与磁珠耦联的最佳介质为2-吗啉乙磺酸;抗体质量浓度为200μg/mL与磁珠的耦联率最高;免疫磁珠在15 min之内就能够富集到牛支原体,且富集到浓度为1×10-3ccu/mL,具有良好的特异性。说明免疫磁珠技术能够快速富集牛支原体,为分离鉴定牛支原体提供一种新的可行性的方法。     

牛支原体灭活疫苗免疫小鼠最小免疫剂量及抗体消长规律研究

[目的]对小白鼠进行最小免疫剂量及抗体消长规律初步研究。[方法]采用课题组制备的牛支原体灭活氢氧化铝佐剂疫苗,分别以0.1 mL/只·天、0.2 mL/只·天、0.3 mL/只·天、0.4 mL/只·天的不同剂量,注射小白鼠腿部肌肉。[结果]最小免疫剂量为0.2 mL/只;免疫3周后抗体滴度达到最高峰,随后抗体滴度开始逐步下降,但仍保持相对稳定,抗体滴度维持在0.319的较高水平,5周后抗体滴度表现明显下降趋势。[结论]本试验为后期牛支原体灭活疫苗在牛群中进行试验研究奠定了基础。     

乳清浓缩蛋白改善脱脂发酵乳饮料的稳定性

研究乳清浓缩蛋白WPC80对脱脂发酵乳饮料稳定性的影响.脱脂发酵乳饮料中添加不同比例的乳清浓缩蛋白,通过粒径分析、离心分析以及稳定性扫描等分析手段对发酵乳饮料进行稳定性评价.结果表明:当乳清浓缩蛋白添加量为30％时,发酵乳饮料的平均粒径最小、离心沉淀率最小;稳定性扫描结果表明:乳清浓缩蛋白添加量为30％时,产生沉淀的趋势明显小于全部脱脂乳粉制作的发酵乳饮料.     

牛支原体P48蛋白卵黄抗体的制备

为了研究牛支原体P48抗原蛋白的免疫学功能及相应卵黄抗体的生物学特性,探究其抗体效价随免疫时间的变化规律,试验采用牛支原体P48蛋白疫苗免疫健康产蛋母鸡,定时采血并收集鸡蛋,用水稀释法结合硫酸铵盐析法制备特异性卵黄抗体,利用间接ELISA法检测血清抗体(IgG)和卵黄抗体(IgY)效价。结果表明,低、中、高剂量组均能诱导蛋鸡产生有效免疫应答,血清抗体效价与卵黄抗体效价的变化趋势基本一致,但卵黄抗体的产生滞后于血清抗体,在首次免疫后8~10周Ig Y效价达到峰值,其中中剂量组的免疫效价最高,最高效价为1:212。经SDS-PAGE检测,制备所得的纯度达86%以上,得率在8.6 mg/m L以上。说明采用牛支原体P48蛋白免疫蛋鸡可制备高效价、高纯度的抗牛支原体P48蛋白卵黄抗体,从而为卵黄抗体在牛支原体的检测与防控方面的研究奠定了基础。     

乳清制备和热处理工艺对初乳IgG含量的影响研究

用单向免疫扩散法检测IgG含量,评价了脱脂、去除酪蛋白等乳清制备工艺和热消毒处理对IgC的影响.结果表明,脱脂和去除酪蛋白可造成IgG损失4.5%～11.5%,平均损失为(6.1±4.8)%;低温巴氏杀菌使乳清IgG损失8.0%～18.8%,平均损失为(11.7±7.8)%;(75±2)℃ 15 s和(80±2)℃10 S处理初乳样均未检出IgG,与相关文献报道不一致;UHT处理常乳,未栓出IgG.酸凝固去除酪蛋白与热处理有交互作用,增强了对IgG的破坏,与凝乳酶凝固去酪相比IgG多损失2.1%～4.3%,平均多损失为(3.5±3.4)%.     

牛初乳品质变化规律及控制技术研究

本文对荷斯坦牛不同泌乳期脱脂初乳的酸度、比重、蛋白质含量、活性免疫球蛋白(Ig G)的变化规律进行了分析,并对初乳粉生产过程的杀菌工艺中如何有效控制微生物数量和保持活性Ig G含量进行了研究。通过建立的回归方程能够较好地预测脱脂初乳的酸度、比重、蛋白和Ig G含量随挤奶次数的变化。实验结果表明,乳牛产犊后前3天内的脱脂初乳具有较高的开发价值,脱脂初乳采用70~72℃、1 min的巴氏杀菌工艺可以满足在控制微生物的前提下,最大限度保证初乳中Ig G的活性。     

抗绿脓杆菌外毒素A酶标抗体的制备及其应用

从病死羊体内分离到绿脓杆菌并提取出外毒素A(PEA),再以此毒素作为抗原加油佐剂制成乳化抗原免疫家兔,获得高免血清并提取免疫球蛋白G(IgG);用过碘酸钠法将过氧化物酶标记抗PEA抗体(IgG),制成酶标抗体,经检验,酶标抗体结合物中的HRP浓度和Ab(IgG)浓度分别是0.0608 mg/mL和0.336 mg/mL;HRP/Ab(IgG)克分子比值为1.724%;酶(HRP)结合率是11.15%.利用该酶标抗体以ELISA夹心法对羊体内抗PEA抗体含量进行了检测,结果证明,用酶标抗体ELISA法比用平板凝集实验法检测的抗体效价平均高出二个滴度,表明制备的PEA酶标抗体具有灵敏度高、特异性强的优点.     

奶酪酸法生产研究

为了研究奶酪酸法生产工艺中盐酸浓度及杀菌温度对产品产出及质量的影响,试验以新鲜牛乳为原料,利用酸单因素试验的方法设置浓度梯度,研究不同杀菌温度、盐酸浓度对奶酪出成率、乳清黏度、乳清折光度、脂肪含量、蛋白质含量及感官品质等的影响。结果表明:酸法奶酪的最佳工艺为:75℃杀菌10 min,滴加盐酸浓度为1.5 mol/L;此时奶酪出成率为37.27%,乳清黏度为2 MPa/s,乳清折光度为1.341 9,脂肪含量为0.43%,蛋白质含量为18.89%。     

两种方法纯化免抗绵羊肺炎支原体IgG的比较

目的:比较两种方法纯化兔抗绵羊肺炎支原体IgG的效果。方法:采用饱和硫酸铵和辛酸-硫酸铵两种方法分离纯化兔抗绵羊肺炎支原体抗体,并用十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)、分光光度计和琼脂扩散(AGP)试验对纯化产物进行相对分子质量、蛋白质含量及免疫活性进行鉴定。结果:纯化后抗体的蛋白含量基本相同;产物纯度和效价以辛酸-硫酸铵法较高。结论:辛酸硫酸铵法是一种简便、快速、高效的抗体纯化方法。     

热处理对牛初乳理化性质和功能性蛋白的影响

科学控制牛初乳的加工工艺参数对初乳产品的开发与加工具有重要意义。本试验旨在探究热处理条件对初乳理化性质和功能性蛋白的影响,本研究设置7 种不同的热处理条件（55 ℃/30 min、55 ℃/60 min、60 ℃/30 min、60 ℃/60 min、63 ℃/30 min、63 ℃/60 min和72 ℃/15 s）,探究热处理后初乳理化指标、微生物菌落数和免疫球蛋白IgG以及乳铁蛋白的变化规律。结果表明,63 ℃条件下处理30 min和60 min,以及在72 ℃条件下处理15 s,出现明显的蛋白凝块;72 ℃处理15 s后显著降低初乳密度、酸度、乳蛋白、非脂固形物（SNF）、总固形物（TS）、柠檬酸、酪蛋白和尿素等理化指标;本研究所有热处理条件均显著降低菌落总数,有效控制大肠菌群和金黄色葡萄球菌数;经本研究设置的热处理后初乳中IgG水平显著降低,乳铁蛋白含量不受影响。综上考虑,建议初乳热加工温度控制在63 ℃以下,并根据生产目的针对性控制热加工参数。本研究可为牛初乳加工工艺参数控制提供详实科学数据支持。     

Separation of ovine IgG1 and IgG2 on protein A-sepharose

A method using protein A-Sepharose chromatography was developed to separate and purify ovine IgG1 and IgG2. The IgG1 eluted from protein A-Sepharose at pH 6.8 and IgG2 eluted at pH 4.5. This method was used to show the specific transfer of IgG1 from the colostrum to newborn lambs. After separation on protein A-Sepharose both IgG1 and IgG2 were pure as analyzed by isoelectric focusing, Western Blotting and SDS-PAGE. The isoelectric points for the immunoglobulins were calculated to be 3.5 for IgG2 and a range from 6.2 to 8.1 for IgG1. The subclass, IgG1, was present in the whey and was the subclass that was found in the serum of lambs after being fed colostrum. The ewe sera had a decrease of both IgG1 and IgG2 at the time of lambing compared to 2 weeks prior to parturition.     

Effect of different systems of colostral nutrition on the level of immunoglobulins in the blood of calves]

New-born calves, artificially fed colostrum or native colostral whey, either dried or preserved by another method, had good health and good weight gains (between 0.05 and 0.60 kg). No greater differences were observed between the groups of calves given three times the colostrum of their mothers, calves given mixed colostrum, and calves fed colostral whey powder. In all groups only individual differences in IgG content in the blood serum were observed after 48 hours from birth. Hypogammaglobulinaemia occurred in individual cases both in calves given small amounts of colostrum or colostral whey and in calves given sufficient quantities. The time that had elapsed from birth to the first drinking did not exert any greater influence upon the IgG level in the blood; the decisive factor was the amount of colostrum taken in by the calf in the first dose. The rate of the absorption of IgG1, IgG2, IgA, and IgM from colostrum and the concentration of the immunoglobulins in the serum depended on the quantity of colostrum in the first dose and were not influenced to any greater degree by the amount of colostrum given to the calves in further doses. The amount of IgG in the blood serum of calves corresponded approximately to the level of colostral antibodies to the virus PI-3. The antibodies to the virus PI-3 and small quantities of IgG were observed also in the serum to new-born calves before drinking colostrum.     

Passive protection against porcine epidemic diarrhea (PED) virus in piglets by colostrum from immunized cows.

The effects of hyperimmune cow colostrum (HCC) on experimentally induced porcine epidemic diarrhea (PED) were investigated in piglets. In experiment 1, four 2-day-old piglets fed HCC containing an antibody titer of 1:512 and another four piglets fed unimmune cow colostrum (UCC) were orally inoculated with 10LD50 of PED virus. The piglets were given colostrum three times a day at 4 hr intervals. Half of the piglets fed HCC showed diarrhea and recovered, and all piglets survived. In contrast, all piglets fed UCC developed diarrhea and three of them died. In experiment 2, 2-day-old piglets fed HCC containing antibody titers of 1:512, 1:128 and 1:32, and UCC were inoculated with PED virus, and survival rates after challenge were 100, 75, 50 and 0 %, respectively. In experiment 3, 1-day-old piglets fed HCC with 1:512 antibody titer or UCC were inoculated and necropsied at 24, 48 and 72 hr after the inoculation for pathological examination. Piglets fed HCC remained healthy and PED virus antigen was not detected in the epithelial cells of the small intestine, and the length of the villi in small intestine was normal. On the other hand, in piglets fed UCC, villous atrophy and PED virus antigen were observed in epithelial cells of the jejunum and ileum from 24 hr. It was concluded that oral administration of HCC to piglets was effective in preventing PED virus infection and reduced their mortality.     

Composition of sow milk during lactation

The composition of sow colostrum and milk was quantitated in 25 sows at 14 time points throughout lactation. All animals belonged to the same experimental herd of German Landrace, farrowed within 4 d, and were of various lactation numbers and various litter sizes. In the first 6 h of lactation colostrum total solids (TS) and protein contents were higher, while fat and lactose contents were lower than in mature milk. Decreased total protein and whey protein contents and concomitantly increased fat and lactose content, with nearly unchanged TS levels, indicate transition from colostrum to mature milk. The high protein content of colostrum was largely due to immunoglobulin (Ig). During the first 6 h, IgG accounts for nearly all the protein in colostrum but plays a decreasing role in sow milk as lactation proceeds. After 2 wk, IgA levels begin to increase and at the end of lactation, IgA constitutes 40% of the total whey protein. No influences of lactation number and litter size on milk composition could be ascertained in this study.     

初乳清粉的货架期预测和贮藏稳定性

为了掌握初乳清粉的贮藏特性,采用货架期预测方程来估计初乳清粉在室温(25℃)、50％相对湿度(RH)条件下PET包装的货架期,并对90d贮藏期过程中其理化指标以及生化指标的变化进行研究.研究结果表明:在PET包装90d贮藏时间内,初乳清粉的水分含量、硫代巴比妥酸(TBA)值和羟甲基糠醛(HMF)含量均随贮藏时间的延长而显著增加(P＜0.05),色差随贮藏时间的延长有所波动,但总体呈现上升趋势,而免疫球蛋白(IgG)的含量在60d内逐渐降低,60d后其下降不显著;在25℃、50％ RH条件下PET包装的初乳清粉的货架期为99.40d.     

两种纯化兔抗酸性红73免疫球蛋白G方法的比较研究

分别采用饱和硫酸铵法和辛酸—硫酸铵法纯化兔抗酸性红73(acid red 73,AR73)免疫球蛋白G(IgG)。通过二喹啉甲酸(BCA)法、十二烷基硫酸钠—聚丙烯酰胺凝胶电泳(SDS-PAGE)试验、间接ELISA法分别测定IgG的浓度、纯度及效价。结果显示,饱和硫酸铵法所提IgG浓度为36.44 mg/mL,最高效价达1.6×10⁵;辛酸—硫酸铵法所提IgG浓度为15.79 mg/mL,最高效价达3.2×10⁵。提示,辛酸—硫酸铵法IgG纯度优于饱和硫酸铵法。     

绵羊肺炎支原体分离株交叉反应性的测定

绵羊肺炎支原体分离株致敏经过鞣酸和戊二醛处理的绵羊红细胞,制备成试验用抗原,与通过攻毒取得的几种血清进行间接血凝试验来检测其交叉反应性。通过检测,绵羊肺炎支原体分离株同无乳支原体、巴氏杆菌等几种可以引起肺炎症状的菌种无交叉反应性。