卵黄抗体三种提取方法的比较研究

运用硫酸铵法、聚乙二醇(PEG)法、冷乙醇法对卵黄抗体进行提取,并使用间接竞争酶联免疫吸附试验(ELISA)方法测定所得卵黄抗体的特异性,用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)测其纯度,用Nanodrop2000测其蛋白浓度并计算提取量,比较三种方法所得抗体的特异性、纯度和提取量。结果显示,三种提取方法所得卵黄抗体特异性高低依次为硫酸铵法、冷乙醇法、PEG法;纯度高低依次为硫酸铵法、冷乙醇法、PEG法;三种方法的提取量高低依次为PEG法、硫酸铵法、冷乙醇法。经综合分析与评价,硫酸铵法的抗体特异性较好、纯度较高、提取量也较高,且其操作方法相对简便,是比较具有优势的提取方法。     

两种卵黄抗体不同提取方法的比较试验

卵黄抗体(Ig Y)具有许多其他类型抗体所没有的优点,其分离纯化的方法有有机物抽提法、盐析法、水稀释法等。试验分别用聚乙二醇(PEG)6000和氯仿来提取卵黄抗体,对其收集的抗体量和抗体效价进行比较分析。结果表明:100 g的卵黄用PEG6000法比氯仿法多收集71 m L卵黄抗体溶液,且PEG6000法的成本远远低于氯仿法,有很好的经济效益,而PEG6000法提取的卵黄抗体效价比氯仿法略低一个滴度。     

抗新型鸭细小病毒卵黄抗体的制备、纯化及效价测定

为了制备抗新型鸭细小病毒卵黄抗体并改良其制备工艺,试验采用新型鸭细小病毒SD株尿囊液制备的油乳剂灭活苗免疫产蛋鸡,收取高免蛋分别用氯仿法、辛酸法和水稀释-硫酸铵二次沉淀法对其中的卵黄抗体进行提纯,比较提纯效果,然后采用SDS-PAGE和Western-blot法对制备效果进行评价,采用鸭细小病毒抗体酶联免疫检测试剂盒进行效价测定,分析卵黄抗体的消长规律。结果表明:氯仿法提取的卵黄抗体浓度为4.512 mg/mL,透明,呈浅黄色;辛酸法提取的卵黄抗体液浓度为3.422 mg/mL,半透明,呈浅白色;水稀释-硫酸铵二次沉淀法提取的卵黄抗体浓度为3.837 mg/mL,透明,澄清。SDS-PAGE结果显示卵黄抗体有两条特异性条带,一条重链(约为65 ku)和一条轻链(约为33 ku)。制备的卵黄抗体具有特异性。试剂盒测定卵黄抗体效价,氯仿法为1∶6 000;辛酸法为1∶8 000;水稀释-硫酸铵二次沉淀法最高,为1∶10 000,其中水稀释-硫酸铵二次沉淀法效果较优。说明本试验成功制备了新型鸭细小病毒的卵黄抗体。     

鸡传染性法氏囊高免卵黄IgY抗体不同提取方法的比较

本试验旨在提高鸡传染性法氏囊卵黄抗体防制鸡传染性法氏囊的效果,比较不同提纯方法对鸡传染性法氏囊高免卵黄抗体效价的影响。制备高免蛋并测定其效价,在实验室通过直接离心法、酸化水提取法、氯仿提取法、聚乙二醇法4种方法分别对高免卵黄抗体提取及抗体效价检测。试验结果表明,自制卵黄抗体效价为1:64;直接离心法的抗体效价为1:128,酸化水提取法的抗体效价为1:16,氯仿提取法抗体效价为1:256,聚乙二醇法的抗体效价是1:32。试验结果显示,使用氯仿提取法后检测的抗体效价最高,是临床提取抗体的常用方法。     

不同提纯方法对鸡传染性法氏囊高免卵黄抗体效价影响的研究

为提高鸡传染性法氏囊卵黄抗体防治鸡传誊性法氏囊病的效果,比较不同提纯方法对鸡传染性法氏囊高免卵黄抗体效价的影响。通过直接离心法、酸化水提取法、氯仿提取法、聚乙二醇法四种方法分别提取高免蛋高免卵黄抗体并测定其提取前后抗体效价。试验结果表明,自制卵黄抗体效价为1：64;直接离心法的抗体效价为1：128,酸化水提取法的抗体效...     

不同方法同步测定抗NDV的HI抗体和抗IBDV的琼扩抗体的结果

用血清、卵黄氯仿提取液和卵黄生理盐水稀释液同步测定了抗新城疫病毒的抗体和抗传染性法氏囊病毒的琼扩抗体。结果认为用卵黄生理盐水稀释液和卵黄氯仿提取液可代替血清用于HI抗体的检测,测定IBD琼扩抗体以卵黄氯仿提取液效果最好。     

卵黄抗体冻干制剂及其制备方法

本发明提供了卵黄抗体的冻干制剂，包括二联或多价卵黄抗体冻干制剂。所述卵黄抗体冻干制剂的制备方法是：制备细菌或病毒免疫原，用于免疫非免疫产蛋鸡，收集该被免疫鸡所产鸡蛋，从其卵黄中提取出针对该免疫原的卵黄抗体；利用酸化蒸馏水去除上述卵黄中的脂类，再用少量氯仿去除残余不溶性物质，     

ELISA法检测种蛋卵黄中禽网状内皮组织增生症病毒抗体的研究

为研究种蛋卵黄中禽网状内皮组织增生症病毒(REV)抗体的检测方法,运用IDEXX公司的REV抗体ELISA检测试剂盒,比较了不同提取方法、不同稀释倍数、不同洗液对卵黄中REV抗体检测结果的影响。结果显示:当用直接提取的卵黄液做300倍稀释检测,用含0.05%吐温-20的蒸馏水洗涤时,卵黄抗体的S/P比值与相应鸡群的血清抗体的S/P比值吻合度最高。应用本方法分别对50枚SPF种蛋和50枚普通鸡蛋进行检测,SPF种蛋均为REV抗体阴性;普通鸡蛋有4枚为REV抗体阳性,阳性率为8%。实验表明,种蛋卵黄ELISA抗体检测法可以代替传统血清抗体检测,为SPF鸡群的REV感染监测奠定了基础。     

商品蛋鸡新城疫和禽流感疫苗免疫后卵黄抗体代替血清抗体检测的相关性分析

为了评价商品蛋鸡免疫新城疫和禽流感疫苗后卵黄抗体替代血清抗体检测时卵黄的最适处理条件,试验采用经新城疫和禽流感疫苗免疫后40天产蛋率在90%以上的健康商品蛋鸡(海兰褐)56只,采血取血清,每只鸡取1枚鸡蛋分离卵黄,采用完全随机分组试验设计,随机分4组,分别为试验Ⅰ、Ⅱ、Ⅲ组及对照组,每组56个重复,每个重复1份血清和1份相应卵黄。对照组为血清抗体检测,试验Ⅰ、Ⅱ和Ⅲ组分别用生理盐水1∶1倍稀释卵黄、生理盐水1∶3倍稀释卵黄和三氯甲烷1∶3倍稀释卵黄,利用血凝抑制(HI)试验进行血清和处理后卵黄抗体检测。结果表明:检测卵黄中新城疫病毒和禽流感病毒H9抗体时使用生理盐水1∶3稀释卵黄,检测卵黄中禽流感病毒H7、禽流感病毒H5-11和禽流感病毒H5-12抗体时使用生理盐水1∶1稀释卵黄,处理后卵黄抗体效价与血清抗体效价均差异不显著(P>0.05),与血清抗体效价相关系数分别为0.837,0.853,0.888,0.888,0.839。说明在对卵黄进行适宜条件处理后,可以用卵黄抗体代替血清抗体评价商品蛋鸡抗体水平。     

免疫复合物法从病料中提取小鹅瘟病毒核酸的研究

本报道应用免疫复合物法从病料中提取小鹅瘟病毒核酸。将病死雏鹅肠及内容物研磨成乳状，冻融，氯仿抽提，按10：1加入小鹅瘟特异高免卵黄抗体，并于37℃反应2h后，离心沉淀病毒抗体复合物，再经蛋白酶K消化，酚、酚：氯仿：异戊醇、氯仿：异戊醇抽提，乙醇沉积。TE溶解核酸后电泳鉴定。提取核酸为一条带，与标准毒株核酸迁移率一致。     

苦参碱的提取纯化工艺研究

为了优化苦参碱的提取工艺,以浸膏收率、苦参碱含量为评价指标,选择用水量、提取时间及提取次数为考察因素,利用L9（3^4）正交试验法优选出水煎煮法提取苦参碱的最佳工艺条件为用8倍量水提取3次,每次提取2h。采用乙醇沉淀及三氯甲烷萃取相结合的方法对苦参粗提物进行纯化,浓缩药液浓度为1．0g生药／mL时进行醇沉,醇沉后调节药液的pH值至9～11用三氯甲烷萃取3次。优选出的提取工艺稳定、可行,可用于苦参碱的工业化提取。     

A comparison of duck and chicken egg yolk for cryopreservation of stallion sperm

OBJECTIVE: Duck and chicken egg yolk were compared for their protective effects against cold shock during the cryopreservation of stallion sperm in a lactose-EDTA-glycerol cryodiluent. DESIGN: A completely randomised design was used. Procedure Ejaculates from five stallions (n = 14 ejaculates) were split and diluted to either 20 or 200 x 10(6) sperm/mL in a lactose-EDTA extender containing either duck or chicken egg yolk. The extended semen was then frozen in liquid nitrogen. The percentage of sperm total motility and forward progressive motility were assessed before freezing and at 0 and 1 hr after thawing. Morphology data were also collected at 0 and 1 hr post thaw. RESULTS: Total and forward progressive motility were higher when the sperm were frozen in the presence of duck rather than chicken egg yolk. Furthermore, the total and forward progressive motility and percentage of morphologically normal sperm were higher when frozen at a concentration of 200 than 20 x 10(6)/mL. CONCLUSION: The results of this study demonstrate that the motility parameters of stallion sperm are improved when the semen is frozen in lactose EDTA extender supplemented with duck egg yolk rather than chicken egg yolk. Moreover, sperm motility and the percentage of morphologically normal sperm were higher after freezing at a concentration of 200 x 10(6)/ml rather than 20 x 10(6)/ml.     

北极狐、乌苏里貉精液细管冷冻技术研究

为了对濒危珍稀动物种质资源的进行保护,完善精液冷冻技术方案,对北极狐37只次、乌苏里貉32只次的精液进行细管冷冻技术研究,结果表明,北极狐精液用3%柠檬酸三钠—卵黄—甘油作冷冻稀释液,乌苏里貉精液用11%蔗糖—卵黄—甘油作冷冻稀释液,在5℃冰箱内平衡1 h后,进行细管冷冻(液氮-196℃),冻溶粒子活力达4~5级。速冻法优于缓冻法。细管冻精在50℃温水中快速溶化,北极狐精液用3%柠檬酸三钠作解冻液,乌苏里貉精液用7%葡萄糖液作解冻液效果较佳。     

瑞香狼毒粗提取比较与成分分析

主要采用了不同提取液、不同提取液倍数、不同提取方法对瑞香狼毒进行了提取率的研究．同时对不同提取方法所得提取物的成分进行了分析。结果表明：以6倍95％乙醇作为提取液．采用75℃浸泡5d的提取方法提取率高,成分含量多,特别是针对其杀虫效果而言,采用乙醇作为提取液效果更佳。     

中药复方宫炎净中生物碱的提取工艺研究

本试验旨在通过优化提取中药复方宫炎净中的有效成分生物碱,得到最佳提取工艺。试验通过探究乙醇体积分数、超声提取时间、超声提取次数及料液比这4个单因素对提取效果的影响,以盐酸水苏碱为标准品,采用L₉(3⁴)正交设计法优选中药复方宫炎净中总生物碱的提取工艺。结果显示中药复方宫炎净中总生物碱的最佳提取工艺为乙醇体积分数为70%、超声提取时间为25 min、超声提取3次、料液比为1:10,且在影响中药复方宫炎净中生物碱提取的因素中,影响大小依次为料液比、乙醇体积分数、超声提取次数、超声提取时间。     

Canine semen cryoconservation: Emerging data over the last 20 years

Canine semen cryoconservation was used since 1969, and this process is still nowadays in progress. This review aims to have an overview of two factors leading to a successful freezing–thawing semen. The success and efficiency of freezing process can be measured by the post-thawing sperm mobility. The first factor is the best extender used as a cryoprotectant to have a similar osmolarity and pH compared to the seminal plasma to enable sperm survival. Historically, chicken egg yolk was used since 1940, but due to microbial risks and to the presence of granules (which interfere with counting dead spermatozoa and inhibits a spermatozoal respiration), despite these disadvantages, egg yolk is considered an excellent cryoprotectant for sperm of different animal species. The low-density lipoproteins (LDL), contained in EY, when used at a concentration of 6% in a freezing medium associated with 20 mM of glutamine, show a mobility up to 54.5%, which is the best combination found. However, the sperm protection mechanism by LDL during freezing–thawing process only begins to be decrypted. But extraction protocols of LDL are not efficient for an industrial use. Therefore, egg yolk plasma is used within liquid or lyophilized state, and offering similar efficiency as the 6% LDL middle. The equilibration step, in which the diluted sperm is placed for a variable period of time at a temperature of +4°C, before freezing it. The studies show that 6 hr is the optimal duration for the canine sperm equilibration. The future of canine sperm cryopreservation is expected in liposome use and synthetic substances, which mimics LDL role.     

The Effect of Removal of Seminal Plasma, Egg Yolk Level and Season on Sperm Freezability of Canary Buck (Capra hircus)

Goat semen is different from that of other domestic species in its limited tolerance to the inclusion of egg yolk in the freezing medium, and this tolerance depends on the presence of enzymes in the seminal plasma that react with egg yolk, producing toxic compounds to the spermatozoa. Moreover, the goat is a seasonal breeder that shows variations in semen quality throughout the year, and those variations may affect semen freezability; hence in freezing protocols, for instance, removal of seminal plasma (washing) yields varying results. This work was designed to study this problem in Canary goats: semen from six males was collected in spring, autumn or winter, washed or non-washed, diluted in a freezing extender with 1.5, 6 or 12% egg yolk, frozen, and thawed after 2 days, 2 or 6 months of cryopreservation. The effect of egg yolk concentration in the freezing extender was far more important than the effect of washing or season on sperm cryosurvival. The quality of frozen-thawed semen tended to improve as egg yolk concentration increased regardless of the effects of season, washing or period of cryopreservation. Washing produced a positive effect on frozen-thawed semen collected during spring or autumn, but the difference decreased as the concentration of yolk increased. However, washing produced a negative effect on frozen-thawed semen collected during winter, diluted with either 6 or 12% egg yolk. There was no apparent seasonal effect on gross measures of sperm production but the seasonal effect was ever present and was reinforced by freezing.     

Study on the Optimum Extraction Process of Mulberry Leaf Polysaccharide

The experiment was conducted to study the best extract process of mulberry leaf polysaccharide,three factors and three levels orthogonal test was designed to optimize the extraction process.Three factors were considered,including ratio of solid to liquid,decoction time,the time of extracting and the yield of mulberry leaf polysaccharide was determined with phenol-sulfurie acid colorimetry as the evaluate index.Meanwhile,the influence of ethanol concentrations on mulberry leaf polysaccharide extract was studied.The results showed that decoction times could greatest affect the extraction percent of the polysaccharides.The extraction percent of polysaccharide was the highest (4.94%) when the rate of solid to liquid was 25,the time of extracting was 2 h and decoction times was 2.While the most appropriate process was 25 times for the rate of sdid to liquid,2 h for extracting and decoction times was 3,considering with practical situations.The concentration of ethanol would significantly affect polysaccharides yield and purity.When the concentration of ethanol was 70%,the yield of mulberry leaf polysaccharid was highest.When the concentration of ethanol was 40%,the content of mulberry leaf polysaccharide was highest (41.5%),significantly higher than other ethanol concentration (P < 0.05).It suggested that the higher the ethanol concentration,the lower the polysaccharide content,but the higher the yield.The optimum extraction of mulberry leaf polysaccharides were that the rate of solid to liquid was 25,extractign for 2 h,decoction 3 times and 70% ethanol precipitation.