CN116891527A - 抗创伤弧菌卵黄抗体的制备方法及应用 - Google Patents
抗创伤弧菌卵黄抗体的制备方法及应用 Download PDFInfo
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Abstract
本发明公开抗创伤弧菌卵黄抗体的制备方法,以创伤弧菌进行灭活来制备免疫源蛋鸡经过五次免疫后,收集高免鸡蛋,经过水稀释、冻融、硫酸铵两步盐析、超滤浓缩等方法对收集的蛋黄液进行提取纯化,通过酶联免疫吸附法来测定抗创伤弧菌卵黄抗体的效价和稳定性以及提取纯化各阶段的纯度变化,为后期考察抗创伤弧菌卵黄抗体作用效果提供一定的基础。
Description
技术领域
本发明属于生物医药技术领域,具体涉及抗创伤弧菌卵黄抗体的制备方法及应用。
背景技术
创伤弧菌(Vibrio vulnificus)是一种嗜温、嗜盐的海洋致病菌,分布较为广泛,大多出现在沿海地区,在海水中、海底沉积物以及海水生物中均能找到。创伤弧菌不仅能够使水产养殖中的多种经济动物患病,如罗非鱼、金鲳鱼、鳗鲡、鲟鱼、石斑鱼、草鱼和南美白对虾等,还能威胁到人的生命健康,给全球水产养殖业带来极为不好的影响。目前创伤弧菌的疫苗还在实验室研发阶段,我国养殖业主要还是以抗生素为主,但抗生素的滥用会引发致病菌耐药性以及药物残留问题,亟需寻找一种绿色、安全的抗生素替代品。
卵黄抗体(Immunoglobulin of yolk,IgY)是一种免疫球蛋白,主要存在于禽类、两栖动物以及爬行动物的血清当中。目前研究最多的是鸡的卵黄抗体,卵黄抗体技术已与基因工程技术、免疫检测技术、磁珠技术、噬菌体展示技术等多种当前主流的抗体技术相结合,应用于病原体检测和诊断,动物疾病治疗,食品工程,新药研发以及快速大量生产科研用抗体等方面。
发明内容
为解决现有技术中存在的问题,本发明提供适用于鱼类饲料添加的抗创伤弧菌卵黄抗体的制备方法,以创伤弧菌为抗原制备灭活疫苗,免疫蛋鸡,收集鸡蛋进行卵黄抗体的提取与纯化,进而获得适用于鱼类饲料添加且对创伤弧菌病害防治具有良好效果的抗创伤弧菌卵黄抗体。
本发明的技术方案如下:
抗创伤弧菌卵黄抗体的制备方法,包括以下步骤:
(1)活化后的创伤弧菌以1%的接种量接种于TSB培养基,28℃180rpm恒温培养至对数生长期后,用TSB培养基将菌液OD595nm的值调为0.68,再在4℃8000rpm离心10min,倒掉上清,用无菌PBS洗涤菌体三遍并重悬;
(2)向菌悬液中加入甲醛溶液至终浓度为1%,在8℃恒温摇床灭活24h后取100μL涂布于胰蛋白胨大豆肉汤固体平板,28℃培养24h,检验灭活是否合格,若合格,将灭活菌液放入4℃5000rpm的离心机中离心10min,倒掉上清,用无菌PBS溶液将沉淀菌体洗涤三遍以除去残留的甲醛,并加入等体积无菌PBS溶液重悬备用;
(3)对生长状况良好的的蛋鸡进行免疫,每隔两周进行一次加强免疫,共进行5次免疫;第一次免疫配合弗氏完全佐剂进行,之后的四次都是用弗氏不完全佐剂;
(4)将蛋黄与蛋清分离开,除去多余的蛋清,用无菌针头刺破卵黄膜,收集卵黄液,用去离子水稀释10倍,0.1M盐酸调pH至5.0,于-20℃中冷冻过夜,次日4℃缓慢解冻后放入离心管中,4℃10000rpm离心10min,取上清,即为卵黄抗体水溶性组分;
(5)利用硫酸铵二次沉淀法进行纯化,最终得到抗创伤弧菌卵黄抗体。
进一步的,所述步骤(3)中蛋鸡的免疫过程采用胸肌和翅膀分点注射免疫,初次免疫时左右胸肌各注射0.1~0.2mL,翅膀皮下注射0.4~0.6mL;加强免疫中,左右胸肌和翅膀皮下各注射0.3mL。
本发明还提供抗创伤弧菌卵黄抗体的制备方法在制备预防创伤弧菌引起疾病的鱼类饲料中的应用。
本发明还提供抗创伤弧菌卵黄抗体的制备方法在制备治疗创伤弧菌引起疾病的试剂中的应用。
相较于现有技术,本发明具有如下有益效果:
1、本发明提供的抗创伤弧菌卵黄抗体通过间接ELISA法测得其具有耐高温、耐酸碱、耐胃蛋白酶消化的特点,能够对创伤弧菌的直接作用;通过细菌凝集实验,证明抗创伤弧菌卵黄抗体能特异性识别并作用于创伤弧菌;通过结晶紫染色定量法检测特异性卵黄抗体对创伤弧菌生物被膜形成能力的影响,结果表明表明浓度高于5mg/mL的特异性卵黄抗体能够阻止创伤弧菌的黏附作用,影响其生物被膜的形成;
2、本发明提供的抗创伤弧菌卵黄抗体对斑马鱼的预防保护效果良好,能够有效抑制创伤弧菌在斑马鱼肠道中生长繁殖,为卵黄抗体抑菌机制的深入研究及创伤弧菌的病害防治提供一定的科学基础。
附图说明
图1为实施例1中ELISA法检测抗创伤弧菌卵黄抗体的效价示意图;
图2为实施例1中不同温度处理对抗创伤弧菌卵黄抗体活性的影响;
图3为实施例1中不同pH处理对抗创伤弧菌卵黄抗体活性的影响;
图4为实施例1中胃蛋白酶消化对抗创伤弧菌卵黄抗体活性的影响;
图5为实施例2中抗创伤弧菌卵黄抗体与创伤弧菌微生物凝集试验的显微照片;其中,(A)为抗创伤弧菌卵黄抗体浓度为1mg/ml;(B)为抗创伤弧菌卵黄抗体2mg/ml;(C)为抗创伤弧菌卵黄抗体4mg/ml;(D)为抗创伤弧菌卵黄抗体6mg/ml;(E)为非特异性卵黄抗体;(F)为空白对照;
图6为实施例2中抗创伤弧菌卵黄抗体对创伤弧菌生物被膜形成的影响;
图7为实施例3中口服抗创伤弧菌卵黄抗体对感染创伤弧菌的斑马鱼的存活率的影响;
图8为实施例4中注射抗创伤弧菌卵黄抗体对感染创伤弧菌的斑马鱼的存活率的影响;
图9为实施例4中斑马鱼感染创伤弧菌的症状;其中,A为未感染创伤弧菌的斑马鱼,B为感染创伤弧菌后的斑马鱼;
图10为实施例5中斑马鱼肠道细菌在门水平的分布情况示意图;
图11为实施例5中斑马鱼肠道细菌在属水平的分布情况示意图。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到;其中,创伤弧菌FJ03-X2是福建省农业科学院生物技术研究所鱼病研究室于2003年在福建省长乐某养殖场分离到的一株鳗源创伤弧菌,保存于-80℃,在现有技术中有记载;
下述实施例中的实验方法,如无特殊说明,均为常规方法。
实施例1
抗创伤弧菌卵黄抗体的制备方法,包括以下步骤:
(1)活化后的创伤弧菌以1%的接种量接种于TSB培养基,28℃180rpm恒温培养至对数生长期后,用TSB培养基将菌液OD595nm的值调为0.68,再在4℃8000rpm离心10min,倒掉上清,用无菌PBS洗涤菌体三遍并重悬;
其中,TSB培养基为胰蛋白胨大豆肉汤培养基,称取6g胰蛋白胨大豆肉汤,倒入干净的500ml锥形瓶中,加入200ml蒸馏水,用玻璃棒搅拌,带其充分溶解后,pH 7.1~7.4,121℃灭菌20min制得;
(2)向菌悬液中加入甲醛溶液至终浓度为1%,在8℃恒温摇床灭活24h后取100μL涂布于胰蛋白胨大豆肉汤固体平板,28℃培养24h,检验灭活是否合格,若合格,将灭活菌液放入4℃5000rpm的离心机中离心10min,倒掉上清,用无菌PBS溶液将沉淀菌体洗涤三遍以除去残留的甲醛,并加入等体积无菌PBS溶液重悬备用;
(3)对生长状况良好的的蛋鸡进行免疫,每隔两周进行一次加强免疫,共进行5次免疫;蛋鸡的免疫过程采用胸肌和翅膀分点注射免疫,初次免疫时左右胸肌各注射0.1~0.2mL,翅膀皮下注射0.4~0.6mL;加强免疫中,左右胸肌和翅膀皮下各注射0.3mL;收集第一次免疫后每一天产下的鸡蛋,并做好标记放入4℃保存;第一次免疫配合弗氏完全佐剂进行,之后的四次都是用弗氏不完全佐剂;
(4)用清水洗净鸡蛋表面的污垢,擦干去壳后,将蛋黄与蛋清分离开,除去多余的蛋清,用无菌针头刺破卵黄膜,收集卵黄液,用去离子水稀释10倍,0.1M盐酸调pH至5.0,于-20℃中冷冻过夜,次日4℃缓慢解冻,此时卵黄中的大部分脂质因冻融而凝集沉淀,放入离心管中,4℃10000rpm离心10min,取上清,即为卵黄抗体水溶性组分;
(5)利用硫酸铵二次沉淀法进行纯化,将卵黄抗体水溶性组分缓慢加入100%饱和硫酸铵溶液当中,至最终饱和度为55%,边缓慢加入边在冰上搅拌混匀,4℃10000rpm离心15min,将上清液倒掉并用少量的PBS溶液将沉淀重悬,再缓慢加入到100%硫酸铵溶液,至最终饱和度为33%,在冰上搅拌混匀后静置于4℃冰箱过夜,次日4℃10000rpm离心15min,去除上清液,加入少量的PBS溶液重悬,重悬液转移至10kDa透析袋中进行透析;将装有上述重悬液的透析袋放入提前预冷好的透析液(灭菌PBS溶液)中,4℃充分透析,每隔12h换一次透析液,透析三天,透析完成后用0.22μm的过滤器过滤除菌,最终得到抗创伤弧菌卵黄抗体;
其中,称取NaCl 8.0g,Na2HPO4 1.42g,KCl 0.2g,KH2PO4 0.27g,溶于1L去离子水,pH 7.4,121℃灭菌20min制得灭菌PBS溶液。
实施例2抗创伤弧菌卵黄抗体检测
(1)抗创伤弧菌卵黄抗体效价检测
上述实施例制得的抗创伤弧菌卵黄抗体的效价由酶联免疫吸附法进行测定,具体步骤如下:
抗原包被:将灭活的创伤弧菌作为抗原,用包被液稀释800倍后,以每孔200μL的量加入酶标板中,4℃包被过夜;
封闭:次日,倒掉孔板中的包被液,用洗涤液荡洗3次,每次3min,再每孔加入200μL封闭液,37℃封闭1h;
加样:将待测卵黄抗体用稀释液进行倍比稀释,稀释梯度为1:100至1:102400,,非特异性卵黄抗体作为阴性对照,每孔加入100μL,设置三个重复孔,37℃孵育1h,甩干后用洗涤液荡洗3次,每次3min;
加酶标二抗:用稀释液将兔抗鸡IgY-HRP按1:10000稀释后,每孔加入100μL,37℃孵育1h,甩干后用洗涤液荡洗3次,每次3min;
显色:避光加入TMB显色液,每孔100μL,轻摇混匀后,37℃避光显色15~20min;
终止:每孔加入50μL的终止液,用酶标仪检测OD450nm;
结果:OD样品/OD阴性≥2.1时,最大稀释倍数为特异性卵黄抗体的效价。
当取第五次免疫后产下的鸡蛋,进行提取纯化后,进行效价检测,从图1中可知,当稀释倍数为51200时,OD450nm平均值为0.053,是对照组的3.5倍,大于2.1;当稀释倍数为102400时,OD450nm平均值为0.019,是对照组的1.5倍,小于2.1。由此可知提取纯化的抗创伤弧菌卵黄抗体的效价为1:51200。
(2)抗创伤弧菌卵黄抗体稳定性检测
热稳定性实验:
取200μL卵黄抗体放入1.5mL EP管中,共9管;分别放入28、37、45、55、65、70、75、80、90℃水浴中处理30min,采用间接ELISA法测定不同温度处理的样品的吸光值;如图2所示,抗创伤弧菌卵黄抗体经过28、37、45、55、65℃加热30min,OD450nm的值都在1.0以上,说明卵黄抗体在28~65℃时,依然能保持较高的活性,并且效价不会受到影响,而70℃时,OD450nm的值有很大幅度的下降,此时卵黄抗体开始受到温度的影响,直到90℃时,OD450nm的值趋近于0,卵黄抗体完全失活;
酸碱性实验:
取200μL卵黄抗体放入1.5mL EP管中,共11管;分别加入等体积pH 1~11的Tris-HCl缓冲液作两倍稀释,37℃水浴3h,之后用Tris碱或HCl将pH调为7.0左右,采用间接ELISA法测定不同pH处理的样品的吸光值;从图3中可知,在pH=1、9、10时,抗创伤弧菌卵黄抗体OD450nm的值有明显的降低,这说明在pH=1、9、10时,抗创伤弧菌卵黄抗体的活性会受到一定的影响,但不至于完全失活;
胃蛋白酶实验:
取100μL卵黄抗体放入1.5mL EP管中,共11管。分别加入等体积pH 1~11的Tris-HCl缓冲液作两倍稀释,再加入用同一pH的Tris-HCl缓冲液配置的胃蛋白酶溶液(40mg/mL),使其终浓度为2mg/mL,37℃水浴3h,最后加入50μL pH8.32的Tris-HCl缓冲液终止反应;从图4中可知,在pH为2~11时,抗创伤弧菌卵黄抗体OD450nm的值在0.4上下波动,在pH=8时,OD450nm值相对来说较高,此时卵黄抗体的活性较好,在pH=9、10时,OD450nm值相对来说低了一点,这就说明在pH=9、10时,蛋白酶处理抗创伤弧菌卵黄抗体,对其活性有一定影响,但不至于完全失活,但在pH为1时,抗创伤弧菌卵黄抗体OD450nm的值直接降为0.012,此时卵黄抗体已完全失活;
由上述稳定性实验可是,本实施例制得的抗创伤弧菌卵黄抗体具有较好的稳定性,在65℃以下,抗创伤弧菌卵黄抗体不会受到温度的影响,依然保持着较高的活性,不会因为温度的升高而变性失活,只有90℃以上的高温才能使它完全的变性失活。在pH=2~8时,抗创伤弧菌卵黄抗体不会受其影响,依然保持着活性,在pH=1、9、10时,抗创伤弧菌卵黄抗体受到一点影响,但不至于完全失活;在pH=2~8时,用胃蛋白酶处理抗创伤弧菌卵黄抗体,活性依旧不会受到影响,只有在pH=1这种极酸条件下,胃蛋白酶才会对抗创伤弧菌卵黄抗体产生很大的影响,使其完全失活。
本实施例中通过微生物凝集验证抗创伤弧菌卵黄抗体能特异性识别并作用于创伤弧菌;将活化好的创伤弧菌以1%的接种量接种至TSB培养基,放入28℃180rpm的恒温摇床培养3~4h;以TSB培养基为零点,将培养好的创伤弧菌菌液用紫外分光光度计测其在595nm的吸光值,并用TSB培养基将OD595nm的值调为0.68(约2×108CFU/mL);取OD595nm为0.68的创伤弧菌菌液,4℃8000rpm离心10min,用PBS溶液洗涤3次,最后用等量PBS溶液将菌体重悬;取重悬好的创伤弧菌菌液加入到无菌96孔板,100μL/孔,再加入特异性卵黄抗体,使其终浓度分别为1mg/mL、2mg/mL、4mg/mL、6mg/mL,以PBS和6mg/mL非特异性卵黄抗体为对照组,放入28℃孵育3h,用倒置显微镜观察凝集情况;结果表明,在所有处理浓度下,特异性卵黄抗体均能与创伤弧菌有效凝集,但凝集程度由特异性卵黄抗体的含量而异,特异性卵黄抗体含量越高,微生物凝集情况越明显,非特异性卵黄抗体和对照组未显示细菌凝集,如图5所示。
本实施例中还通过传统结晶紫染色定量法检测特异性卵黄抗体对创伤弧菌生物被膜形成能力的影响;向96孔板中加入含有1mg/mL、5mg/mL、10mg/mL抗创伤弧菌卵黄抗体和10mg/mL非特异性卵黄抗体的TSB培养基,每孔100μL,每孔再以10%的接种量加入过夜培养的创伤弧菌菌液,28℃培养24h,设置3个重复孔;吸去培养基,用无菌PBS洗涤3次,动作轻柔;每孔加入100μL甲醇固定15min;吸去甲醛,剩余的自然风干,之后再每孔加入1%结晶紫溶液染色5min;将每孔中的结晶紫溶液吸出,用流水冲洗96孔板中剩余的结晶紫溶液,直至没有多余的颜色洗出;将孔板倒置,以去除多余的水,自然风干;每孔加入33%乙酸溶液,37℃孵育30min;用酶标仪测定590nm的吸光值(D值);以TSB培养基空白孔为阴性对照(Dc),阴性值的2倍为界限值;
基于D值可以将菌株分为3类:强生物被膜形成菌株、弱生物被膜形成菌株和无生物被膜形成菌株;
D>2Dc为强生物被膜形成菌株;
2Dc≥D>Dc为弱生物被膜形成菌株;
D≤Dc为无生物被膜形成菌株;
如图6所示,以未接种创伤弧菌的TSB培养基为阴性对照,阴性值(Dc)的2倍为界限值,创伤弧菌菌株属强生物被膜形成菌株,而用10mg/mL和5mg/mL的抗创伤弧菌卵黄抗体处理的菌株,其生物被膜形成量显著低于未添加抗创伤弧菌卵黄抗体组和10mg/mL非特异性卵黄抗体组(p<0.05),表明浓度高于5mg/mL的抗创伤弧菌卵黄抗体能够阻止创伤弧菌的黏附作用,影响其生物被膜的形成。
实施例3
本实施例将上述实施例1制得的抗创伤弧菌卵黄抗体应用于制备预防创伤弧菌引起疾病的鱼类饲料中;
(1)饲料的制备:
将蛋白含量为10mg/mL的抗创伤弧菌卵黄抗体用无菌水稀释2倍和10倍,得到5mg/mL和1mg/mL的抗创伤弧菌卵黄抗体,分别将3mL 10mg/mL、5mg/mL、1mg/mL的抗创伤弧菌卵黄抗体喷涂在3g脱壳丰年虾卵,置于37℃烘箱2h,制成抗体含量分别为1‰、5‰、10‰的饲料,置于4℃冰箱备用。
(2)饲料预防实验:
捞取40条体重约为0.2g的成年斑马鱼分为4组,10条一组,并标记为1~4,其中1~3组依次分别饲喂等量的抗体含量为1‰、5‰、10‰的饲料,第4组饲喂等量的普通丰年虾卵饲料,上午下午各投喂一次,共6天;将菌液浓度为4×106CFU/mL的创伤弧菌菌液,以每尾10μL的剂量进行注射攻毒,置于28℃的培养箱观察,记录一星期的存活情况;
如图7所示,饲喂10‰特异性卵黄抗体饲料组的七天内存活率为80%,显著高于普通饲料组的存活率(30%)(p<0.05),饲喂1‰和5‰特异性卵黄抗体饲料组七天内存活率分别为40%和50%,与普通饲料组无显著差别(p>0.05)。三组的相对保护率如表1所示;这表明饲喂含有10‰特异性卵黄抗体饲料能够增强斑马鱼的被动免疫保护作用,预防创伤弧菌的感染;
表1口服含有特异性卵黄抗体饲料的相对保护率
实施例4
本实施例将上述实施例1制得的抗创伤弧菌卵黄抗体应用于制备治疗创伤弧菌引起疾病的试剂;
选取50条体重约为0.2g的成年斑马鱼分为五组,每组10条,标记为一到五组;第三组和第四组腹腔注射10μL 15mg/mL的抗创伤弧菌卵黄抗体溶液,第五组注射非特异性卵黄抗体,全注射完毕后,将4组斑马鱼放入28℃培养箱培养2h;用无菌PBS溶液将创伤弧菌菌液的浓度调为4×106CFU/mL,第一、四、五组的斑马鱼腹腔注射10μL创伤弧菌菌液,放入28℃培养箱中观察一星期,记录存活情况;如图8所示,对照组斑马鱼腹腔注射感染创伤弧菌(4×106CFU/mL),24h内存活率仅为40%,非特异性卵黄抗体组24h内存活率为50%,第二天至第七天均未出现死亡;感染创伤弧菌的斑马鱼腹部会充血肿胀,如图9所示,并且游动缓慢、身体失衡,未加任何处理的自然实验组和只注射特异性卵黄抗体的实验组存活率都为100%,这表明斑马鱼健康状况良好,培养环境适宜,并且特异性卵黄抗体无毒性,不会令斑马鱼产生不良反应;在攻毒之前2h注射特异性卵黄抗体的实验组,7天内的存活率为90%,显著高于攻毒对照组(p<0.05),其相对保护率为83.33%,这说明注射特异性卵黄抗体能够有效的保护斑马鱼不受创伤弧菌的侵害,因此,将抗创伤弧菌卵黄抗体应用于制备治疗创伤弧菌引起疾病的试剂是完全可行的。
实施例5
本实施例中对经过实施例1制得的抗创伤弧菌卵黄抗体的特性进行分析,将三组分别经过创伤弧菌感染、抗创伤弧菌卵黄抗体、注射抗创伤弧菌卵黄抗体后感染创伤弧菌和一组未经过任何实验处理的斑马鱼的肠道组织样本进行第三代微生物测序,12个样品测序后通过Barcode识别后共获得156406条CCS序列,每个样品至少产生12917条CCS序列,平均产生13034条CCS序列,如表2;
表2样品测序数据处理结果统计
图10是斑马鱼肠道细菌在门水平的分布情况,优势菌群是Proteobacteria(变形菌门)和Fusobacteria(梭杆菌门),空白对照组优势菌群的比例分别为34.11%和65.20%,模型组为57.54%和41.89%,IgY注射组为49.17%和49.02%,IgY预防组为81.39%和17.80%;经过IgY预防保护实验的斑马鱼,其肠道菌群中,Proteobacteria(变形菌门)的比例升高,Fusobacteria(梭杆菌门)的比例下降;
图11是斑马鱼肠道细菌在属水平的分布情况,主要是Cetobacterium、Plesiomonas、Vibrio、Aeromonas、Enterobacter等,IgY预防组弧菌属所占的比例为7.24%,比模型组所占的比例低许多,模型组所占的比例为31.76%,空白对照组和IgY注射组弧菌属所占的比例为0.07%和0.04%,这就说明抗创伤弧菌卵黄抗体能够有效抑制创伤弧菌在斑马鱼肠道中生长繁殖。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围内。
Claims (4)
1.抗创伤弧菌卵黄抗体的制备方法,包括以下步骤:
(1)活化后的创伤弧菌以1%的接种量接种于TSB培养基,28℃180rpm恒温培养至对数生长期后,用TSB培养基将菌液OD595nm的值调为0.68,再在4℃8000rpm离心10min,倒掉上清,用无菌PBS洗涤菌体三遍并重悬;
(2)向菌悬液中加入甲醛溶液至终浓度为1%,在8℃恒温摇床灭活24h后取100μL涂布于胰蛋白胨大豆肉汤固体平板,28℃培养24h,检验灭活是否合格,若合格,将灭活菌液放入4℃5000rpm的离心机中离心10min,倒掉上清,用无菌PBS溶液将沉淀菌体洗涤三遍以除去残留的甲醛,并加入等体积无菌PBS溶液重悬备用;
(3)对生长状况良好的的蛋鸡进行免疫,每隔两周进行一次加强免疫,共进行5次免疫;第一次免疫配合弗氏完全佐剂进行,之后的四次都是用弗氏不完全佐剂;
(4)将蛋黄与蛋清分离开,除去多余的蛋清,用无菌针头刺破卵黄膜,收集卵黄液,用去离子水稀释10倍,0.1M盐酸调pH至5.0,于-20℃中冷冻过夜,次日4℃缓慢解冻后放入离心管中,4℃10000rpm离心10min,取上清,即为卵黄抗体水溶性组分;
(5)利用硫酸铵二次沉淀法进行纯化,最终得到抗创伤弧菌卵黄抗体。
2.根据权利要求1所述的抗创伤弧菌卵黄抗体的制备方法,其特征在于,所述步骤(3)中蛋鸡的免疫过程采用胸肌和翅膀分点注射免疫,初次免疫时左右胸肌各注射0.1~0.2mL,翅膀皮下注射0.4~0.6mL;加强免疫中,左右胸肌和翅膀皮下各注射0.3mL。
3.根据权利要求1所述的抗创伤弧菌卵黄抗体的制备方法在制备预防创伤弧菌引起疾病的鱼类饲料中的应用。
4.根据权利要1所述的抗创伤弧菌卵黄抗体的制备方法在制备治疗创伤弧菌引起疾病的试剂中的应用。
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