CN111217906A - 猪消化系统疾病的预防或治疗用卵黄抗体的制造方法以及基于此而制造的卵黄抗体及其用途 - Google Patents
猪消化系统疾病的预防或治疗用卵黄抗体的制造方法以及基于此而制造的卵黄抗体及其用途 Download PDFInfo
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Abstract
本发明涉及利用猪消化系统疾病致病菌或病毒抗原制造异种抗体,然后将结合了上述抗原及异种抗体的复合体接种到产卵鸡,由此制造卵黄抗体的猪消化系统疾病的预防或治疗用卵黄抗体的制造方法以及基于此而制造的卵黄抗体及其用途,根据本发明,本发明的猪消化系统疾病的预防或治疗用卵黄抗体的制造方法能够通过将抗原‑异种抗体复合体注入产卵鸡而大量制造及生产卵黄抗体,由此制造的卵黄抗体针对猪消化系统疾病致病菌或病毒的特异性及结合性高而有效地抑制上述菌及病毒的生长,由此,能够预防或治疗猪消化系统疾病。
Description
技术领域
本发明涉及猪消化系统疾病的预防或治疗用卵黄抗体的制造方法以及基于此而制造的卵黄抗体及其用途,更详细地涉及利用猪消化系统疾病致病菌或病毒抗原制造异种抗体(heteroantibody),然后将结合了上述抗原及异种抗体的复合体接种到产卵鸡,由此制造卵黄抗体的猪消化系统疾病的预防和治疗用卵黄抗体的制造方法以及基于此而制造的卵黄抗体及其用途。
背景技术
抗体(antibody)是基于B淋巴细胞或B细胞形成于淋巴组织的球蛋白系蛋白质,是参与特异性的免疫识别的一种重要的物质。抗体具有彼此不同的两种功能,第一种是特异性识别及结合引起免疫反应的抗原的特定部位而中和抗原的作用,第二种是抗体结合于抗原,由此发挥将用于除去抗原的各种物质和免疫细胞聚集的作用。例如,抗体既能够与毒素结合,使毒素的单纯的化学性组成发生变化而中和毒性,也能够附着于侵入的微生物,由此消除微生物的运动性而阻碍其侵入体细胞。此外,被抗体包围的抗原与补体引起化学性连锁反应,引起侵入的微生物的直接性分解或诱导吞噬细胞。应用像这样的抗原抗体反应的特异性和高度的亲和度及能够区分数千万种的抗原的抗体的多样性,现今出现了包含诊断剂和治疗剂等的多种抗体医药产品(Jim E.Eyles et al.,Vaccine,25,pp73017306,2007)。
另一方面,作为针对疾病的防御机制,不仅是人,各种哺乳类及鸟类也在为了从感染性疾病中保护自己或保护后代而制造的物质中包含抗体。哺乳类与大部分人类类似,会生产约150Kd的IgG。哺乳类除了生产IgG以外,还生产其结构或功能略微不同的IgA、IgD、IgE及IgM。
一直以来,为了制造各种病原性细菌的抗体,广泛使用了下述方法:将兔子、牛、山羊等哺乳动物进行免疫处理而获得血清,从该血清中生产病原性细菌特异性抗体。然而,这样的方法存在必须从小的哺乳动物的少量血清中提取抗体的缺点,抗体的生产量极其微量。因此,作为用于容易地生产病原性细菌的抗体的方法,当前利用将产卵鸡进行免疫处理所生成的抗体转移到产卵鸡的鸡蛋中而提取抗体的方法备受瞩目。特别是存在于产卵鸡的卵黄中的特异性抗体被称为卵黄免疫球蛋白(Immunoglobulin Yolk,IgY)或卵黄抗体,其是存在于产卵鸡的血清中的作为特异性抗体的免疫球蛋白(IgG)向鸡蛋的卵黄移动而生成的抗体,在鸡蛋的卵黄中以高浓度蓄积。IgY能够容易地分离,因此能够利用于多种领域,特别是具有针对家畜疾病的预防及治疗效果的IgY的开发及作为饲料添加剂的有效利用处于增加的趋势。
将疾病性细菌作为抗原而从产卵鸡的鸡蛋中获得卵黄抗体的方法及包含该抗体的组合物或食品等的开发正在活跃进行,利用抗原-抗体复合体的疫苗的制造或用于治疗多种疾病的组合物的开发也在活跃进行。具体而言,韩国公开专利第2009-0088121号中公开了在利用抗原-抗体复合体用作疫苗的佐剂的情况下,能够增强对抗原的免疫反应,从论文Phase I trial of a murine antib ody to MUC1 in patients with metastaticcancer中可知,在为了治疗癌细胞而利用抗原-异种抗体复合体的情况下,能够在使T细胞激活而治疗癌细胞中显示优异的效果(Ann Oncol.2004Dec;15(12)∶1825-33)。然而,为了大量生产卵黄抗体而利用抗原-抗体复合体的方法迄今尚不可知。
因此,本发明人为了克服上述现有技术的问题而进行了深入研究,结果发现,在利用猪消化系统疾病致病菌或病毒抗原制造异种抗体,然后将结合了上述抗原及异种抗体的复合体接种到产卵鸡,由此制造卵黄抗体的情况下,能够大量制造及生产对猪消化系统疾病致病菌或病毒抗原的免疫反应增强了的卵黄抗体,完成了本发明。
现有技术文献
专利文献
专利文献1:KR10-2009-0088121 A
非专利文献
非专利文献1:J.S.de Bono JS et al.,Annals of Oncology.2004Dec;15(12)∶1825-33
发明内容
发明要解决的问题
因此,本发明的主要的目的在于提供能够大量制造及生产卵黄抗体的猪消化系统疾病的预防或治疗用卵黄抗体的制造方法及基于此而制造的对猪消化系统疾病致病菌或病毒抗原的免疫反应增强了的卵黄抗体。
本发明的另一个目的在于提供上述猪消化系统疾病的预防或治疗用卵黄抗体的制造方法及利用基于此而制造的卵黄抗体的猪消化系统疾病的预防或治疗用疫苗组合物或者猪消化系统疾病的预防或治疗用饲料添加剂组合物。
用于解决问题的方案
根据本发明的一个实施方式,本发明提供包含下述阶段的猪消化系统疾病的预防或治疗用卵黄抗体的制造方法:第1阶段,利用猪消化系统疾病致病菌或病毒制造抗原;第2阶段,将上述第1阶段的抗原接种到与鸡异种的动物而制造异种抗体;第3阶段,使上述第1阶段的抗原与上述第2阶段的异种抗体结合,制造抗原-异种抗体复合体;第4阶段,将混合了第1阶段的抗原及第3阶段的复合体的混合物接种到产卵鸡;以及第5阶段,从上述第4阶段的产卵鸡的卵(egg)中分离针对猪消化系统疾病致病菌或病毒的卵黄抗体。
卵黄抗体(Immunoglobulin Yolk,IgY)是存在于产卵鸡的血清中的作为特异性抗体的免疫球蛋白(IgG)向鸡蛋的卵黄移动而生成的抗体,在鸡蛋的卵黄中以高浓度蓄积,易于分离,因此能够利用于多种领域。为了大量制造及生产这样的卵黄抗体进行了深入研究,结果发现,在将抗原-抗体结合体用作佐剂、将异种抗体用作抗体的情况下,能够增强免疫反应,着眼于这一事实,在利用将抗原和异种抗体结合的复合体制造卵黄抗体的情况下,能够大量制造及生产针对抗原的免疫反应增强了的卵黄抗体,完成了本发明。
本发明的卵黄抗体的制造方法的特征在于,上述第1阶段的猪消化系统疾病致病菌能够包含现有的作为诱发猪的消化系统疾病而为人所知的任一种菌,优选为选自大肠杆菌(E.coli)、鼠伤寒沙门氏菌(Salmonella Typhimurium)、猪霍乱沙门氏菌(Salmonellacholeraesuis)、产气荚膜梭菌A(Clostridium perfringens A)及产气荚膜梭菌C(Clostridium perfringens C)中的一种以上致病菌,更优选为大肠杆菌(E.coli)。
本发明的卵黄抗体的制造方法的特征在于,上述第1阶段的猪消化系统疾病致病病毒能够包含现有的作为诱发猪的消化系统疾病而为人所知的任一种病毒,优选为选自PEDV(猪流行性腹泻病毒,porcine epidemic diarrhea virus)、轮状病毒(Rotavirus)、TGEV(猪传染性胃肠炎冠状病毒,Transmissible gastroe nteritis coronavirus)、圆环病毒(circovirus)及格塔病毒(Getah virus)中的一种以上病毒,更优选为PEDV(porcineepidemic diarrhea virus)。
本发明的卵黄抗体的制造方法的特征在于,上述第2阶段中,异种的动物能够使用现有的用于制造抗体而使用的任一种哺乳类,优选为鼠或兔子。
在本发明的卵黄抗体的制造方法中,优选上述第4阶段中抗原与复合体的比为1∶0.5~1∶0.1。在复合体的比大于该比的情况下,存在由过量的免疫细胞对复合体识别导致的问题,在以更小的比包含的情况下,不易发挥基于复合体应用的充分效果。
本发明的卵黄抗体的制造方法的特征在于,上述第4阶段中,混合物进一步包含佐剂(adjuvant)。上述佐剂能够使用完全弗氏佐剂(complete Freund's adjuvant)、不完全弗氏佐剂、脂质体(lipofectin)、lipotaxi、CaPO4、25至30%蔗糖、DEAE右旋糖苷、聚凝胺(polybrene)、皂苷、像氢氧化铝这样的无机质凝胶、溶血卵磷脂、复合多元醇(pluronicpolyols)、聚阴离子(polyanions)、肽类(peptides)、像油或烃乳液这样的表面活性物质、二硝基酚等,对此没有限制。本发明的实施例中使用ISA70不完全佐剂(incompleteadjuvant)作为佐剂。
本发明的卵黄抗体的制造方法的特征在于,上述混合物与佐剂(adjuvant)的混合比可以为2~3∶7~9,优选混合比为3∶7。混合物的混合比越高,佐剂作为乳化剂的功能越降低,就会产生与抗原混合时发生水溶性层和脂溶性层的分离现象的问题,因此维持上述比很重要。
本发明的卵黄抗体的制造方法的特征在于,上述第4阶段中,接种能够以0.1ml至3ml进行2次至5次,优选以0.5至1ml进行3次。
本发明的卵黄抗体的制造方法的特征在于,上述卵黄抗体的制造方法增加卵黄抗体的生产收率。
根据本发明的一个实施例,确认了根据本发明的制造方法的卵黄抗体的生产收率,结果能够确认:与仅将抗原注入产卵鸡而制造卵黄抗体的例子(比较例)相比,在将抗原-异种抗体复合体注入产卵鸡而制造卵黄抗体的情况下,卵黄内抗体含量增加。这样的结果启示了:鉴于抗体的整体生产量显著增加困难这一事实,在本发明的制造方法中,关于卵黄抗体的生产增加,抗原-异种抗体复合体作为抗体生产的佐剂赋予协同效应,从而能够大量制造及生产卵黄抗体(参照实施例3及表5)。
根据本发明的另一个方式,本发明提供根据上述卵黄抗体的制造方法而制造的猪消化系统疾病的预防或治疗用卵黄抗体。
本发明的卵黄抗体的特征在于,上述卵黄抗体对猪消化系统疾病致病菌或病毒抗原的结合力增强。
根据本发明的一个实施例,确认了根据本发明的制造方法而生产的卵黄抗体的抗原结合力,结果确认了:与仅将抗原注入产卵鸡而生产的卵黄抗体(比较例)相比,在将抗原-异种抗体复合体注入产卵鸡而生产的卵黄抗体(实施例)的情况下,对抗原的结合力优异。这样的结果启示了:在根据本发明的制造方法制造卵黄抗体的情况下,能够增加对特定抗原的特异性卵黄抗体的生成,就结论而言,对抗原的优异的结合力能够增加对猪消化系统疾病致病菌或病毒抗原的免疫反应。此外,上述结合力启示了:将注入到产卵鸡的抗原-异种抗体复合体作为初始抗原,即,将猪消化系统疾病致病菌或病毒作为抗原进行识别,生成了针对上述抗原的IgY(参照实施例5、表6及图6)。
根据本发明的另一个方式,本发明提供包含卵黄抗体作为有效成分的猪消化系统疾病的预防或治疗用疫苗组合物。
本发明的疫苗能够与药剂学上能够容许的载体、佐剂或赋形剂一同使用。
用于本发明的疫苗的载体能够使用包含水解的蛋白质、乳糖等一个以上稳定化剂的生理学上均衡的培养基,也能够使用0.01至0.1M的磷酸缓冲液或0.8%的生理食盐水。此外,药学上能够容许的载体能使用溶液或非溶液、悬浊液、乳液的形态。作为非溶液溶剂的例子,有丙二醇、聚乙二醇、像橄榄油这样的植物性油及像油酸乙酯这样的注射用有机酯。溶液性载体能够使用水、醇溶液、乳液或生理食盐水及像缓冲培养液这样的悬浊液。
用于本发明的疫苗的佐剂能够使用完全费氏佐剂(complete Freund'sadjuvant)、不完全费氏佐剂、脂质体(lipofectin)、lipotaxi、CaPO4、25至30%蔗糖、DEAE右旋糖苷、聚凝胺(polybrene)、皂苷、像氢氧化铝这样的无机质凝胶、溶血卵磷脂、复合多元醇(pluronic polyols)、聚阴离子(polyanions)、肽类(peptides)、像油或烃乳液这样的表面活性物质、二硝基酚等,对此没有限制。
作为用于本发明的疫苗的赋形剂及稀释剂,能够举出乳糖、右旋葡萄糖、蔗糖、山梨醇、甘露醇、木糖醇、赤藓糖醇、麦芽糖醇、淀粉、阿拉伯胶、藻酸盐、明胶、磷酸钙、硅酸钙、纤维素、甲基纤维素、微晶纤维素、聚乙烯吡咯烷酮、水、羟基苯甲酸甲酯、羟基苯甲酸丙酯、滑石、硬脂酸镁、鲸蜡醇、硬脂醇、液体石蜡、山梨醇酐单硬脂酸酯、聚山梨醇酯60、羟苯甲酯、羟苯丙酯及矿物油。
本发明的疫苗能够以通常的方式通过经口、直肠、局部、静脉内、腹腔内、肌肉内、动脉内、经皮、鼻侧内、吸入、眼球内或皮内通路给予。
非经口给予的含义是包含静脉内、肌肉内、腹腔内、胸骨内、经皮及动脉内注射及注入的给予方式。本发明的疫苗的非经口给予优选:在优选的纯度下将药剂学上能够容许的载体,即,在使用的浓度和给予量下对受体非毒性且能够与其他的制剂成分化合的载体混合,以单位给予量的剂型制备。
此外,本发明的疫苗的剂型能够以任一种剂型应用,制造的剂型能够以经口用、注射用、涂抹用的方式使用。上述剂型能够以片剂、胶囊剂、软质胶囊剂、水液剂、颗粒剂、丸剂或注射用形态(溶液、悬浊液或乳浊液)制备,最优选以注射用形态制备。
根据本发明的另一个方式,本发明提供包含卵黄抗体作为有效成分的猪消化系统疾病的预防或治疗用饲料添加剂组合物。
本发明的上述饲料添加剂能够直接使用或进一步添加对家畜容许的谷类及其副产物等公知的载体、稳定剂等,也能够根据需要添加柠檬酸、富马酸、己二酸、乳酸、苹果酸等有机酸或磷酸钠、磷酸钾、酸性焦磷酸盐、聚磷酸盐(聚合磷酸盐)等磷酸盐或多酚、儿茶素、α-生育酚、迷迭香萃取物、维生素C、绿茶萃取物、甘草萃取物、壳聚糖、单宁酸、植酸等天然抗氧化剂、抗生素、抗菌剂及其他的添加剂等,作为其形状可以是粉体、颗粒、药丸、悬浊液等合适的状态。
根据本发明的一个实施例,确认了在根据本发明的制造方法而制造的卵黄抗体的情况下,与阳性对照组及比较例相比,有效地抑制细菌的生长。这样的结果启示了:本发明的卵黄抗体能够容易地用作用于预防或治疗猪消化系统疾病的疫苗或饲料添加剂(参照实施例4及图5)。
发明效果
如上所述,本发明的猪消化系统疾病的预防或治疗用卵黄抗体的制造方法能够通过将抗原-异种抗体复合体注入产卵鸡而大量制造及生产卵黄抗体,由此制造的卵黄抗体针对猪消化系统疾病致病菌或病毒的特异性及结合性高而有效地抑制上述菌及病毒的生长,由此,能够预防或治疗猪消化系统疾病。
附图说明
图1及图2为表示抗体效价测定结果的图。
图3及图4为表示确认卵黄抗体的结果的图((图3;SDS-PAGE(12%凝胶染色(gelstaining)),图4;蛋白质免疫印迹法(Western-blot),1.天然鸡(Natural chicken)-卵黄抗体蛋白(IgY protein)(cat#ab119138),2.负载(loading)10μg IgY(比较例),3.负载(loading)10μg IgY(实施例))
图5为表示确认细菌生长抑制的结果的图。
图6为表示确认抗原结合力的结果的图。
具体实施方式
以下,通过实施例对本发明更详细地说明。这些实施例只是为了示例本发明,不能解释为本发明的范围被这些实施例所限定。
制造例1:抗原的生产
1)E.coli(09∶K35,K99,F41)KVCC-BA0000540-020800491
使用血平板(Blood agar)培养基在37℃培养器(incubator)中进行好氧培养。确认了菌落(Colony)后接种于1000ml BHI broth(脑心浸液肉汤),然后振荡培养24小时至36小时。结束培养后,用0.3%福尔马林(Formalin)在室温处理48小时使其失活。将失活后的抗原进行6000rpm离心并回收。用PBS(磷酸盐缓冲溶液,pH7.2)将回收的抗原分散后,添加0.1%福尔马林(Formalin)在室温放置1天后,在4℃的冰箱中保存。对抗原进行计算,调整浓度,以使制造的抗原利用分光光度计(spectrophotometer)在410nm处的OD(光密度)成为1.0。
2)猪流行性腹泻(Porcine epidemic Diarrhea,PED SM98)病毒
PED病毒以下述方式进行接种:用细胞维持培养基(α-MEM,抗生素(antibiotics),酵母(yeast),FBS 5%)培养Vero细胞,在形成80%单层(monolayer)时,使用添加了非血清培养基α-MEM(alpha-Minimal Essential Medium)、胰蛋白酶(Trypisn,5μg/ml)的培养基,清洗(washing)3次后,以PED病毒稀释液浓度成为100TCID50(组织培养感染剂量50,Tissue culture infectious dose 50)的方式进行接种。致敏90分钟后添加含有5%FBS的α-MEM培养48小时。经过48小时后确认细胞病变效应(Cytopathic Effect,CPE),回收病毒。用0.2M BEI(2-溴乙胺氢溴酸盐,2-Bromoethylamine Hydrobromide)使回收的病毒失活(inactiv ation)。
制造例2:鼠抗体的制造
将上述制造例1中生产的各种抗原失活,抑制毒性后,冷藏保管后使用。在使用鼠抗体制作用疫苗的情况下,使用完全佐剂(complete adjuvant)制造疫苗。制造与各种抗原分别对应的各种疫苗,根据通常的鼠抗体制造方法制造抗体。利用蛋白质-A柱(protein-Acolumn)从生产结束后回收的血清(Serum)中纯化鼠抗体。
制造例3:与家猪抗原结合的样品的制造(mAb complex)
为了使上述制造例1中生产的抗原和上述制造例2中生成的鼠抗体结合,进行如下试验。在失活了的混合疫苗中混合对各种抗原生成的鼠抗体100μg,在4℃过夜(Overnight),诱导抗原抗体结合。将结束结合的样品进行离心分离而回收后,用作产卵鸡接种用样品。
制造例4:产卵鸡接种用家猪混合疫苗的制造
为了制造家猪混合疫苗,将制造例1中准备的各种抗原及mAb复合物(complex)与佐剂(adjuvant)以3∶7左右进行混合后,利用均质器(Homogenizer)制造灭活疫苗(KilledVaccine),然后经过无菌检查和失活确认试验,准备用于特异性卵黄抗体生产的混合疫苗。
实施例1:产卵鸡接种用家猪混合疫苗的制造
产卵鸡接种是将制造的疫苗接种到25周龄的Hyine-brown系列的产卵鸡而实现的。接种以下述方式进行:每次对胸肌接种1ml,以3周的间隔接种3次。接种组如下述表1所示。
表1
具体而言,接种组如下所述组成而制造疫苗。比较例中将针对引起家猪消化系统疾病的病毒和细菌失活的抗原(制造例1)等量准备。实施例中制造结合于比较例的抗原的mAb,将抗原和抗体结合的复合体(制造例4)追加混合于疫苗中而准备,其组成如下述表2所述。
表2
组 | 疫苗组成 | 比 |
对照组 | - | |
比较例 | 猪流行性腹泻病毒+大肠杆菌 | 1∶1 |
实施例 | 猪流行性腹泻病毒+大肠杆菌+mAb-复合物<sup>1)</sup> | 1∶1∶0.5 |
1):mAb复合物(complex)=(猪流行性腹泻病毒(PEDV)+鼠抗猪流行性腹泻病毒免疫球蛋白G(mouse anti-PEDV-IgG))0.25+(大肠杆菌(E.coli)+鼠抗大肠杆菌免疫球蛋白G(mouse anti-E.coli-IgG))0.25
实施例1:抗体效价测定
1)ELISA包被抗原制造
将制造例1中生产的抗原以8000rpm离心分离50分钟。离心分离后弃去上层液,用HEPES缓冲液(buffer)使团块(pellet)再悬浮(suspension),然后进行超声波破碎(sonication)而溶解(Lysis)。将溶解的上层液以8000rpm于4℃离心30分钟,得到上层液。以最终浓度成为0.01%的方式向得到的上层液添加1%N-月桂酰肌氨酸(SIGMA,L-9150),然后在室温处理10分钟。处理后以15000rpm于4℃进行50分钟离心,除去N-月桂酰肌氨酸(SIGMA,L-9150),用50ml HEPES缓冲液(buffer)使其再次悬浮(suspension),以15000rpm于4℃进行50分钟离心分离,回收OMP。回收的抗原用BCA法进行蛋白质定量后,以成为200ng/ml进行包被,用于抗体效价测定。
2)利用ELISA的抗体效价测定
卵黄中的特异性卵黄抗体的效价利用间接酶联免疫吸附测定法(Indirect ELISAmethod)。首先将抗原在包被缓冲液(coating buffer)中稀释,在96孔聚苯乙烯板(wellpolystyrene plate)进行包被,其中,在每孔(well)包被100μl,之后在4℃过夜(或,在37℃放置1小时)。用PBS-T(phosphate buffer saline,0.05%Tween 20、pH7.4)清洗后,用含有BSA的PBS缓冲液在37℃封闭(blocking)1小时,用如上所述的方法进行清洗。将阴性对照组、阳性对照组及样品稀释2X,在每孔加入100μl,在37℃静置1小时。1小时后清洗3次,将2次抗体(anti-chicken∶Sigma,U.S.A)在PBS中适量稀释,在每孔分配100μl,然后在37℃反应1小时。然后进行3次清洗,在每孔分配100μl底物(substrate,OPD,sigma),然后在室温反应约10分钟,分配50μl终止液(Stop solution)使反应终止,用ELISA reader在450nm的波长处测定每孔的吸光度,表示为ELISA值(value)。通过对OD值为与阴性试样(negativecontrol)的OD值相比2倍以上的稀释倍数进行逆运算,计算出各试样的阳性/阴性值(P/Nvalue),将其结果示于下述表3、4及图1、2中。
表3
表4
结果,如上述表3、4及图1、2中能够确认的那样,在将异种抗体与抗原结合,与追加抗原一起对产卵鸡进行接种的实施例的情况下,在1、2次接种中,与比较例相比形成了高的效价,从3次接种开始,抗体的效价的最高值与比较例相比大幅提高,特别是针对于病毒的抗原,更有助于卵黄抗体的效价形成。
实施例2:卵黄抗体的确认
利用硫酸铵(Ammonium sulfate)从接种的卵黄中分离抗体后,为了确认分离纯度进行了试验。
具体而言,在各组别(对照组、比较例及实施例)生产的卵黄中收集显示最高抗体效价的2-3周次鸡蛋,从卵黄中分离IgY。将卵黄和卵白进行分离,仅收集卵黄,然后用纯化水(D.W)稀释回收的卵黄,搅拌30分钟。将与D.W搅拌的样品反复进行冷冻和解冻,分离脂质,回收水溶性蛋白质样品。在回收的样品中添加硫酸铵(Ammonium sulfate)后,在4℃冷藏保存,使卵黄抗体沉淀。回收在冷藏中沉淀18小时左右的样品,在4℃以6000rpm离心分离并回收,然后,将用PBS使回收的抗体样品再悬浮而得到的样品使用PBS进行透析,获得最终样品。获得的样品用BCA蛋白质定量试剂盒(kit)定量后,在12%SDS-PAGE凝胶(gel)中确认分离纯度,将其结果示于图3、4中。
结果,如图3、4中能够确认的那样,分离与市售的经过纯化的卵黄抗体没有大的差异,通过蛋白质印迹(Western-blot)试验确认的结果,均为抗体蛋白质,由此可以判断抗体的纯度分离良好。
实施例3:根据产卵鸡接种的抗体含量的测定
卵黄抗体定量试验利用HPLC进行试验。用于定量的IgY标准物质使用Abcam公司的normal chicken IgY(Abcam Cat No ad119138)。取作为标准溶液的normal IgY,以12000rpm离心分离20秒,使用上层液。用蒸馏水将离心分离的标准溶液稀释为5种浓度,制作标准曲线。标准溶液的稀释是以1、0.5、0.25、0.125、0.0625mg/ml进行稀释的。将卵黄液稀释为0.2mg/ml浓度,然后利用振荡机溶解5分钟。将上述溶液以3000rpm离心分离5分钟,取上层液,用0.45um的针头式过滤器(syringe filter)过滤,用HPLC测定过滤物。将标准溶液按照浓度(1mg/mL、0.5mg/mL、0.25mg/mL、0.125mg/mL、0.0625mg/mL)利用HPLC进行测定,根据标准溶液的浓度制作线形图,求出线性式,导入在HPLC中测定的试验溶液的值进行计算,将其结果示于下述表5中。
表5
结果,如表5中能够确认的那样,在按照组别确认总平均数时,显示出基于疫苗接种,卵黄抗体的含量多少有所增加,与比较例相比,实施例显示出卵黄内抗体含量多少有所增加。
实施例4:细菌生长抑制确认试验
对用作抗原菌的大肠杆菌(E.coli)进行生长抑制试验。各种菌是在LB Broth中以1×107cfu/ml浓度进行接种而准备的,将分离的卵黄抗体以相同的浓度即以约10mg/ml浓度进行统一处理。试验期间,在约接种后12小时为止,以1小时的间隔测定broth,然后以2小时的间隔进行测定,将其结果示于图5中。
结果,如图5中能够确认的那样,试验结果为在大肠杆菌的情况下,直到初始4小时为止,所有组的卵黄抗体与所有阳性对照组相比,没有产生大的差异。特别是,在比较例的卵黄抗体的情况下,能够确认接种后从5小时以后开始显示出与阳性对照组同样的抑制效果,但是9小时以后细菌的生长急剧增加而抑制效果下降。与此相对,在本发明的实施例的情况下,确认了持续抑制抗原菌的生长。
实施例5:抗原结合力确认试验
利用流式细胞仪(Flow cytometer)对分离的抗体确认与抗原的结合力及抗原特异性卵黄抗体的生成比。培养用作抗原的大肠杆菌(E.coli)后,用0.1%福尔马林进行失活处理。对回收的抗原进行下述处理:在光密度(OD)600nm处测定吸光度,稀释为1.0。将分离的卵黄抗体从浓度50mg/ml到500mg/ml进行准备,然后,与抗原混合,在4℃与抗原结合18小时。
将与抗原结合的样品以3000rpm离心分离10分钟,弃去上层液,弃去没有结合的抗体。作为二次抗体,将Anti-chicken-IgG-PE抗体以1∶2000进行稀释而使用,然后用PBS清洗两次后,在流式细胞仪(Flow cytometer)中确认结果,在表6及图6(1.E.coli+Anti-chicken-IgG-PE;2.E.coli+G1-IgY+Anti-chicken-IgG-PE;3.E.coli+G2-IgY+Anti-chicken-IgG-PE;4.E.coli+G3-IgY+Anti-chicken-IgG-PE)中确认其结果。
表6
组 | 亲和力(%) |
对照组 | 17.5% |
比较例 | 46.7% |
实施例 | 79.5% |
结果,如表6及图6中能够确认的那样,结合于抗原菌的抗体的比显示为对照组17.5%、比较例46.7%、实施例79.5%。即使是对于对照组的抗原不致敏的通常的IgY也显示出17.5%的结合力,这表示对于大肠杆菌的母体所具有的抗体基本上以一定程度存在着,可知通过3次接种,结合力进一步增加。在本发明的实施例的情况下,以79.5%的比结合,认为在抗体的全部的量中,由于具有产卵鸡基本能生产的抗体的量,所以不会有大的增加,但其中对抗原的特异性卵黄抗体的生成增加。
实施例6:中和抗体试验方法
将从卵黄中分离的抗体进行二进制稀释(从2倍到1024倍)而准备后,以PEDV成为200TCID50/50ul的方式进行稀释,进行等量分配。于37℃在5%CO2培养器(incubator)中反应1小时,然后,将Vero细胞以2×105细胞数/100ul左右进行悬浮,在所有孔中等量分配后,于37℃在5%CO2培养器中培养5~7天,然后观察CPE,进行判定。在添加了病毒的孔中,CPE被100%阻碍,该孔的单层(monolayer)在形态上与对照组的孔基本一致时,将该孔的最高血清稀释倍数的倒数表示为中和抗体效价,将其结果示于表7中。
表7
猪流行性腹泻病毒SM98 100TCID | 猪流行性腹泻病毒SM98 400TCID | |
对照组 | 2 | 2 |
比较例 | 16 | 16 |
实施例 | 64 | 64 |
结果,如上述表7中能够确认的那样,对照组显示为2,比较例显示为16,实施例显示为64。与比较例相比,在实施例的情况下显示出4倍高的中和抗体效价,中和抗体效价作为能够中和病毒的指标,抗体效价越高越能够有效地中和病毒。
Claims (10)
1.一种猪消化系统疾病的预防或治疗用卵黄抗体的制造方法,包含以下阶段:
第1阶段,利用猪消化系统疾病致病菌或病毒制造抗原;
第2阶段,将所述第1阶段的抗原接种到与鸡异种的动物而制造异种抗体;
第3阶段,使所述第1阶段的抗原与所述第2阶段的异种抗体结合,制造抗原-异种抗体复合体;
第4阶段,将混合了第1阶段的抗原及第3阶段的复合体的混合物接种到产卵鸡;以及
第5阶段,从所述第4阶段的产卵鸡的卵即egg中分离针对猪消化系统疾病致病菌或病毒的卵黄抗体。
2.根据权利要求1所述的猪消化系统疾病的预防或治疗用卵黄抗体的制造方法,其特征在于,
所述第1阶段的猪消化系统疾病致病菌为选自大肠杆菌即E.coli、鼠伤寒沙门氏菌即Salmonella Typhimurium、猪霍乱沙门氏菌即Salmonella choleraesuis、产气荚膜梭菌A即Clostridium perfringens A及产气荚膜梭菌C即Clostridium pe rfringens C中的一种以上致病菌。
3.根据权利要求1所述的猪消化系统疾病的预防或治疗用卵黄抗体的制造方法,其特征在于,
所述第1阶段的猪消化系统疾病致病病毒为选自猪流行性腹泻病毒PEDV即porcineepidemic diarrhea virus、轮状病毒即Rotavirus、猪传染性胃肠炎冠状病毒TGEV即Transmissible gastroenteritis coronavirus、圆环病毒即circovirus及格塔病毒即Getah virus中的一种以上病毒。
4.根据权利要求1所述的猪消化系统疾病的预防或治疗用卵黄抗体的制造方法,其特征在于,
所述第2阶段中,异种的动物为鼠或兔子。
5.根据权利要求1所述的猪消化系统疾病的预防或治疗用卵黄抗体的制造方法,其特征在于,
所述第4阶段中,混合物进一步包含佐剂即adjuvant。
6.根据权利要求5所述的猪消化系统疾病的预防或治疗用卵黄抗体的制造方法,其特征在于,
所述混合物与佐剂即adjuvant的混合比为2~3∶7~9。
7.根据权利要求1所述的猪消化系统疾病的预防或治疗用卵黄抗体的制造方法,其特征在于,
所述第4阶段中,接种以0.1ml至3ml进行2次至5次。
8.一种猪消化系统疾病的预防或治疗用卵黄抗体,是通过权利要求1的制造方法制造的。
9.一种猪消化系统疾病的预防或治疗用疫苗组合物,包含权利要求8所述的卵黄抗体作为有效成分。
10.一种猪消化系统疾病的预防或治疗用饲料添加剂,包含权利要求8所述的卵黄抗体作为有效成分。
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