CN116891526A - 一种抗变形假单胞菌卵黄抗体的制备方法与应用 - Google Patents
一种抗变形假单胞菌卵黄抗体的制备方法与应用 Download PDFInfo
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Abstract
本发明公开一种抗变形假单胞菌卵黄抗体,是以变形假单胞菌为抗原制备灭活疫苗对蛋鸡进行免疫,收集鸡蛋后进行分离提纯获得所述变形假单胞菌卵黄抗体,具有特异性好、效价高的优点,用于防治变形假单胞菌引起的疾病和免疫学检测具有良好的效果;且使用健康母鸡所产鸡蛋为载体,安全无毒,成本低,产量高,易于产业化。
Description
技术领域
本发明属于生物医药技术领域,具体涉及一种抗变形假单胞菌卵黄抗体的制备方法与应用。
背景技术
大黄鱼物美味鲜,素有“海水国鱼”的美誉,是目前中国养殖规模最大的海水鱼类,福建省大黄鱼的养殖区主要集中在宁德蕉城、福安和霞浦的三都澳与福鼎的沙埕港等海域。随着大黄鱼养殖量的增加,病害愈加频发,其中内脏白点病是冬春季低温期大黄鱼最主要的细菌性疾病,该病的典型症状为内脏(脾、肾等)有白色结节,累计死亡率达50%以上,严重危害大黄鱼产业的健康发展。变形假单胞菌(Pseudomonas plecoglossicida)为低温季节大黄鱼内脏白点病的主要病原。变形假单胞菌,于1991年首次从患细菌性出血性腹水病的香鱼(Plecoglossus altivelis)中分离出来,隶属于假单胞菌科,假单胞菌属,恶臭假单胞菌组,杀香鱼假单胞菌种,是一种直或微弯、不呈螺旋状的革兰氏阴性杆菌,以单极毛或数根极毛运动,需氧,不生芽孢,为常见的条件致病菌。
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。卵黄抗体具有高产量、低成本、理化性质稳定等特点,目前已在人、畜牧、水产养殖的疾病防治中广泛应用。与传统的抗生素和化学药物相比,应用卵黄抗体具有以下几个优点:(1)特异性强,与广谱的抗生素不同,卵黄抗体只针对相应的致病菌,不会对其它菌群产生影响;(2)易获取,卵黄抗体可从免疫蛋鸡的鸡蛋中获取,无需宰杀大量动物且产生的抗体效价高,稳定性强,能够避免频繁注射;(3)于鸡类与哺乳动物间存在一定系统发育距离,少量抗原就可以引发有效的免疫应答;(4)无残留,由于卵黄抗体属于免疫球蛋白,发挥作用后会被机体作为营养物质消化吸收;(5)研制开发的周期短,成本低,可长期保存,便于运输和应用。近年来,利用IgY的被动免疫保护作用用于疾病的预防和治疗受到了国内外的广泛关注,并取得了良好的效果。
变形假单胞菌对各个生长阶段的大黄鱼均有致病性,但是目前还没有一种有效的治疗方法,因此需要一种快速、准确、特异性和灵敏度高的检测方法,及时对变形假单胞菌进行诊断、监控和预警,以减少经济损失。而卵黄抗体在水产动物致病菌的快速检测中具有广阔的应用前景。
发明内容
本发明提供一种抗变形假单胞菌卵黄抗体的制备方法与应用,采用变形假单胞菌为抗原,免疫健康的产蛋母鸡,制备具有特异性的变形假单胞菌卵黄抗体,在变形假单胞菌的免疫学检测、抗原的纯化,以及变形假单胞菌感染的预防和治疗上具有重要的作用。
本发明的技术方案如下:
本发明提供一种抗变形假单胞菌卵黄抗体,所述变形假单胞菌卵黄抗体是以变形假单胞菌为抗原制备灭活疫苗对蛋鸡进行免疫,收集鸡蛋后进行分离提纯获得所述变形假单胞菌卵黄抗体。
本发明还提供抗变形假单胞菌卵黄抗体的制备方法,包括如下步骤:
(1)抗原的制备:将活化后的变形假单胞菌接种到培养基中扩增培养,然后用无菌PBS洗涤菌体三遍并重悬,最后用甲醛灭活,得到灭活的变形假单胞菌抗原;
(2)免疫蛋鸡:对生长状况良好的的蛋鸡进行免疫,初次免疫将灭活的变形假单胞菌抗原与弗式完全佐剂配合,之后的若干次加强免疫均用灭活的变形假单胞菌抗原与弗式不完全佐剂配合;
(3)提取卵黄抗体水溶性组分:收集第一次免疫后每一天产下的鸡蛋,做好标记后保存,收集鸡蛋擦干去壳后,分离蛋黄蛋清,收集卵黄液,用去离子水稀释10倍,盐酸调pH至5.0,于-20℃中冷冻过夜,次日4℃缓慢解冻,至少进行冻融处理1次,再利用离心收集上清,即为卵黄抗体水溶性组分;
(4)纯化:利用硫酸铵二次沉淀法进行卵黄抗体的进一步纯化,最终得到抗变形假单胞菌卵黄抗体。
进一步的,所述步骤(1)中变形假单胞菌在液体培养基中扩增培养至变形假单胞菌处于对数增长期。
进一步的,所述步骤(2)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;每隔两周进行一次加强免疫。
本发明提供的抗变形假单胞菌卵黄抗体应用于制备预防和治疗灿烂弧菌引起疾病的制剂。
其中,所述制剂为药物、保健品和饲料添加剂。
本发明提供的抗变形假单胞菌卵黄抗体应用于以变形假单胞菌为抗原的亲和纯化试剂或以变形假单胞菌为抗原的免疫检测试剂的制备中。
相较于现有技术,本发明具有如下有益效果:
本发明提供抗变形假单胞菌卵黄抗体由变形假单胞菌免疫产蛋母鸡而得,具有特异性好、效价高的优点,用于防治变形假单胞菌引起的疾病和免疫学检测具有良好的效果;且使用健康母鸡所产鸡蛋为载体,安全无毒,成本低,产量高,易于产业化。
附图说明
图1为实施例2中ELISA法检测抗变形假单胞菌卵黄抗体的效价示意图。
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;
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
一种抗变形假单胞菌卵黄抗体的制备方法,包括如下步骤:
(1)抗原的制备:将活化后的变形假单胞菌接种到培养基中扩增培养至变形假单胞菌处于对数生长期,在4℃8000rpm的条件下离心10min,倒掉上清后用无菌PBS洗涤菌体三遍并重悬,向菌悬液中加入甲醛溶液至终浓度为1%,在28℃恒温摇床灭活24h,得到灭活的变形假单胞菌抗原;
(2)免疫蛋鸡:将健康的蛋鸡随机分为两组,给予充足的饲料和水源饲养一周,对生长状况良好的的蛋鸡进行免疫,初次免疫将灭活的变形假单胞菌抗原与弗式完全佐剂配合,之后的若干次加强免疫均用灭活的变形假单胞菌抗原与弗式不完全佐剂配合,在本实施例中共进行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;每隔两周进行一次加强免疫;
(3)提取卵黄抗体水溶性组分:收集第一次免疫后每一天产下的鸡蛋,做好标记后在4℃保存,收集鸡蛋擦干去壳后,分离蛋黄蛋清,将蛋黄放在滤纸上滚动以出去多余的蛋清,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,至少进行冻融处理1次,卵黄中大部分脂质因冻融而凝集沉淀,放入离心管中,4℃10000rpm离心10min,收集上清,即为卵黄抗体水溶性组分;
(4)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,最后分装保存于-20℃冰箱备用。
实施例2
(1)抗变形假单胞菌卵黄抗体的效价检测
抗变形假单胞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:将灭活的变形假单胞菌作为抗原,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。
取第五次免疫后产下的鸡蛋,进行提取纯化后将提取纯化好的卵黄抗体进行ELISA实验,测定鸡蛋中抗变形假单胞菌卵黄抗体的效价,由图1可知,当稀释倍数为32000时,OD450nm平均值为0.143,是对照组的2.3倍,大于2.1;当稀释倍数为64000时,OD450nm平均值为0.095,是对照组的1.5倍,小于2.1;由此可知提取纯化的抗变形假单胞菌卵黄抗体的效价为1:32000,如表1所示;
表1 ELISA检测变形假单胞菌卵黄抗体的效价
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 (7)
1.一种抗变形假单胞菌卵黄抗体,其特征在于,所述变形假单胞菌卵黄抗体是以变形假单胞菌为抗原制备灭活疫苗对蛋鸡进行免疫,收集鸡蛋后进行分离提纯获得所述变形假单胞菌卵黄抗体。
2.根据权利要求1所述的抗变形假单胞菌卵黄抗体的制备方法,其特征在于,包括如下步骤:
(1)抗原的制备:将活化后的变形假单胞菌接种到培养基中扩增培养,然后用无菌PBS洗涤菌体三遍并重悬,最后用甲醛灭活,得到灭活的变形假单胞菌抗原;
(2)免疫蛋鸡:对生长状况良好的的蛋鸡进行免疫,初次免疫将灭活的变形假单胞菌抗原与弗式完全佐剂配合,之后的若干次加强免疫均用灭活的变形假单胞菌抗原与弗式不完全佐剂配合;
(3)提取卵黄抗体水溶性组分:收集第一次免疫后每一天产下的鸡蛋,做好标记后保存,收集鸡蛋擦干去壳后,分离蛋黄蛋清,收集卵黄液,用去离子水稀释10倍,盐酸调pH至5.0,于-20℃中冷冻过夜,次日4℃缓慢解冻,至少进行冻融处理1次,再利用离心收集上清,即为卵黄抗体水溶性组分;
(4)纯化:利用硫酸铵二次沉淀法进行卵黄抗体的进一步纯化,最终得到抗变形假单胞菌卵黄抗体。
3.根据权利要求2所述的抗变形假单胞菌卵黄抗体的制备方法,其特征在于,所述步骤(1)中变形假单胞菌在液体培养基中扩增培养至变形假单胞菌处于对数增长期。
4.根据权利要求2所述的抗变形假单胞菌卵黄抗体的制备方法,其特征在于,所述步骤(2)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;每隔两周进行一次加强免疫。
5.根据权利要求1所述的抗变形假单胞菌卵黄抗体在制备预防和治疗灿烂弧菌引起疾病的制剂中的应用。
6.根据权利要求5所述的应用,其特征在于,所述制剂为药物、保健品和饲料添加剂。
7.根据权利要求1或2所述的抗变形假单胞菌卵黄抗体在以变形假单胞菌为抗原的亲和纯化试剂或以变形假单胞菌为抗原的免疫检测试剂的制备中的应用。
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