CN107188947B - 经修饰的初乳蛋白质及其应用 - Google Patents
经修饰的初乳蛋白质及其应用 Download PDFInfo
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- CN107188947B CN107188947B CN201710102914.2A CN201710102914A CN107188947B CN 107188947 B CN107188947 B CN 107188947B CN 201710102914 A CN201710102914 A CN 201710102914A CN 107188947 B CN107188947 B CN 107188947B
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- modified colostrum
- colostrum protein
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Abstract
本发明涉及一种经修饰的初乳蛋白质，其氨基酸序列如SEQ ID NO：1所示的氨基酸序列，为自如SEQ ID NO：2所示的野生型初乳蛋白质的氨基酸序列的第33位置的异亮氨酸被丙氨酸取代，第101位置的谷氨酸被半胱氨酸取代，以及第175位置的精氨酸被半胱氨酸取代而产生。
Description
技术领域
本发明涉及一种初乳蛋白，特别是涉及一种经修饰的初乳蛋白质。
背景技术
抗体，亦称免疫球蛋白，为一种主要由浆细胞分泌，并可被免疫系统用来识别以及中和外来物质，例如细菌或病毒等病原体的蛋白质。抗体包括IgA、IgD、IgE、IgG及IgM，其中IgA可在母乳、唾液、眼泪以及支气管中的黏液被发现，对于作为身体抵御外来病原体的第一道防线的黏膜免疫十分重要。详细而言，许多病原体可通过接触于呼吸道、肠道及生殖泌尿道的黏膜表面而感染宿主，而于黏膜的分泌性抗体IgA可通过与病原体的多个抗原决定位(epitope)结合，使得病原体无法与黏膜细胞结合而感染宿主。
畜产业于农业生产中扮演重要的地位，其中养猪育成不佳的主要因素与猪的高死亡率有关。一般而言，绝对性病原普遍被认为是猪只疾病的主因，然而，根据中兴大学兽医病理学系的血清学调查结果显示，高病原性的疾病，例如猪瘟或假性狂犬病并无明显爆发的现象，但相对一些病原性较低的病原体，例如支原体属(mycoplasma)、巴斯德氏菌属(pasteurella)及沙门氏菌属(salmonella)，其于单独感染时可能无明显病害，但在猪只的抗病能力低下加上复合多种病原的原发及继发感染，会使病害加成以致死亡。也就是说，这样的低病原性病原体对猪只均数低下的影响甚大。
雌性哺乳动物于产后2-3天内所初分泌的乳汁称的为初乳(colostrum)，其后再分泌的乳汁则被称为常乳。初乳中含有五种免疫球蛋白，分别是IgA、IgD、IgE、IgG及IgM，其中以IgG的含量最高，其他免疫球蛋白含量都较低。该些免疫球蛋白对于病毒、细菌、寄生虫及酵母菌等病原体皆有良好的防御作用。
然而，目前于初乳中，除了乳铁蛋白已被纯化、利用及培育基因转殖动物外，其余有益成分则鲜少被有效分离及使用。且，由于初乳的分泌时程过短，以及乳中的蛋白不稳定、收集不易及保存困难等因素，使得初乳效益虽多，但于实际应用层面上尚存在诸多困难。
发明内容
因此，本发明的目的即在提供一种经修饰的初乳蛋白质，不仅可提高其于体外的稳定性，还具有预防或抵御外来病原体的功效。
本发明为解决现有技术的问题所采用的技术手段提供一种经修饰的初乳蛋白质，其氨基酸序列如SEQ ID NO：1所示的序列，该经修饰的初乳蛋白质为自如SEQ ID NO：2所示的野生型初乳蛋白质的氨基酸序列的第33位置的异亮氨酸被丙氨酸取代，第101位置的谷氨酸被半胱氨酸取代，以及第175位置的精氨酸被半胱氨酸取代而产生。
在本发明的一实施例中提供一种编码上述的经修饰的初乳蛋白质的氨基酸序列的DNA，具有SEQ ID NO：3所示的碱基序列。
在本发明的一实施例中提供一种口服剂型，包含上述的经修饰的初乳蛋白质。
在本发明的一实施例中提供一种动物饲料组成物，包含上述的经修饰的初乳蛋白质。
在本发明的一实施例中提供一种动物饲料组成物，该经修饰的初乳蛋白质占该动物饲料组成物0.01wt％～0.02wt％。
在本发明的一实施例中提供一种医药组成物，包含医药载剂、疫苗佐剂以及上述的经修饰的初乳蛋白质。
在本发明的一实施例中提供一种如上述的经修饰的初乳蛋白质用于制备增加动物的免疫的饲料的应用。
在本发明的一实施例中提供一种应用，应用中通过增加该动物的免疫球蛋白IgA生成而增加该动物的免疫。
在本发明的一实施例中提供一种如上述的经修饰的初乳蛋白质用于制备投递于动物的饲料的应用。
在本发明的一实施例中提供一种如上述的经修饰的初乳蛋白质用于制备投递于动物的医药组成物的应用。
在本发明的一实施例中提供一种如上述的经修饰的初乳蛋白质用于制备预防或治疗引发黏膜免疫的疾病的饲料的应用。
本发明的一实施例中提供一种如上述的经修饰的初乳蛋白质用于制备预防或治疗引发黏膜免疫的疾病的饲料的应用，该疾病包括猪繁殖和呼吸障碍综合症(porcinereproductive and respiratory syndrome,PRRS)、口蹄疫、猪流行性下痢病毒(porcineepidemic diarrhea,PED)及禽流感。
在本发明的一实施例中提供一种如上述的经修饰的初乳蛋白质用于制备预防或治疗引发黏膜免疫的疾病的医药组成物的应用。
在本发明的一实施例中提供一种如上述的经修饰的初乳蛋白质用于制备预防或治疗引发黏膜免疫的疾病的医药组成物的应用，该疾病包括猪繁殖和呼吸障碍综合症(porcine reproductive and respiratory syndrome,PRRS)、口蹄疫、猪流行性下痢病毒(porcine epidemic diarrhea,PED)、禽流感及人类的流行性感冒。
经由本发明所采用的技术手段，本发明的经修饰的初乳蛋白质的三级结构相较于野生型的初乳蛋白更为稳定。且，经该经修饰的初乳蛋白质可通过提高免疫球蛋白IgA的生成而增强黏膜免疫，进而预防或治疗非特定病原的疾病的感染。
附图说明
图1为显示经分离与菌膜结合的初乳蛋白的结果。
图2为显示于猪的乳清中的PGRP的结果。
图3为显示根据本发明的实施例的经修饰的初乳蛋白质以及野生型初乳蛋白质经于培养液中添加50μg/ml放线菌酮(cycloheximide,CHX)处理的折线图。
图4为显示以酵母菌表现猪病原辨识蛋白的系统。
图5为显示根据本发明的实施例的经修饰的初乳蛋白质的西方墨点图。
图6为显示根据本发明的实施例的经修饰的初乳蛋白质与大肠杆菌混合时的电子显微镜图。
图7为显示根据本发明的实施例的经修饰的初乳蛋白质于管喂小鼠后肠道总菌数的折线图。
图8为显示根据本发明的实施例的经修饰的初乳蛋白质于管喂小鼠后的肠道免疫染色图。
具体实施方式
以下根据图1至图8，而说明本发明的实施方式。该说明并非为限制本发明的实施方式，而为本发明的实施例的一种。
依据本发明的一实施例的一经修饰的初乳蛋白质，其氨基酸序列如SEQ ID NO：1所示的氨基酸序列，该经修饰的初乳蛋白质为自如SEQ ID NO：2所示的野生型初乳蛋白质的氨基酸序列的第33位置的异亮氨酸被丙氨酸取代，第101位置的谷氨酸被半胱氨酸取代，以及第175位置的精氨酸被半胱氨酸取代而产生。而该经修饰的初乳蛋白质的氨基酸序列由具有SEQ ID NO：3所示的碱基序列的DNA予以编码。
详细而言，本发明的经修饰的初乳蛋白质为纯化结合于细菌的细胞壁上的胜肽聚醣的蛋白质，且该蛋白质经修饰其序列而得，并依其特性命名为病原辨识蛋白(Pathological Recognition Protein,PRP)。
进一步而言，本发明的经修饰的初乳蛋白质可制成口服剂型，例如固体、半固体、或液体的口服剂型。详细而言，该固体的口服剂型包括药片、多颗粒、粉末、或胶囊。
进一步而言，本发明的经修饰的初乳蛋白质可经与饲料混合后而制成动物饲料组成物。且，该经修饰的初乳蛋白质占该动物饲料组成物0.01wt％～0.02wt％。当然，本发明不以此为限。在其它实施例中，该经修饰的初乳蛋白质占该动物饲料组成物的比例可根据情况而有所不同。
进一步而言，本发明的经修饰的初乳蛋白质可用于制备增加动物的免疫的饲料的应用，其通过增加该动物的免疫球蛋白IgA生成而达到。详细而言，该动物可为哺乳动物，例如猪或牛。
进一步而言，本发明的经修饰的初乳蛋白质可用于制备投递于动物的医药组成物的应用，并包括医药载剂及疫苗佐剂。
进一步而言，本发明的经修饰的初乳蛋白质可用于制备预防或治疗引发黏膜免疫的疾病的饲料或医药组成物，其中该疾病包括猪繁殖和呼吸障碍综合症(porcinereproductive and respiratory syndrome,PRRS)、口蹄疫、猪流行性下痢病毒(porcineepidemic diarrhea,PED)及禽流感。
概括而言，在本实施例中，以猪(在其它实施例中，亦可采用其他哺乳动物，例如：牛)的初乳为材料，通过钠十二烷基的硫酸盐聚丙烯酰胺凝胶电泳法(SDS-PAGE)分析，以纯化可能会结合于细菌的细胞壁上的胜肽聚醣的初乳蛋白质纯化，并经液相层析串联式质谱仪(LC/MS/MS)鉴定分析及基因库比对后，而初步了解该胜肽聚醣结合蛋白的身分及特性，而将该胜肽聚醣结合蛋白命名为病原辨识蛋白(Pathological Recognition Protein,PRP)。待取得该病原辨识蛋白的氨基酸序列后，选殖出猪的病原辨识蛋白基因，并且构筑于酵母菌表现系统。并将该体外表现的病原辨识蛋白陆续完成表现量评估、发酵环境测试、小鼠管喂后肠道菌相检验、活体大肠杆菌及沙门氏菌抑制试验等确定其蛋白质活性，如下所示。
纯化蛋白质：
革兰氏阳性介质(GEM)颗粒制备：无菌沾取Lactoccous lactis菌液，接种于MRS(DifcoTM Lactobacilli MRS Broth)培养基上并筛选出单一菌落，挑选单一菌株至250ml的MRS培养液，于厌气环境37℃培养18小时。将培养完毕的菌液分装于50ml的离心管中，以13000xg离心10分钟，去除上清液，以原体积1/2的ddH2O悬浮菌块，以13000xg离心10分钟后，去除上清液，加入原体积1/5的酸溶液(0.6M TCA,pH＝1)，松盖，置于沸水隔水加热30分钟，再以13000xg离心10分钟，去除上清液，并以原体积1/2的PBS悬浮菌块，上述步骤重复3次，再以13000xg离心10分钟后，去除上清液。最后分别以原体积1/10的PBS回溶菌块，使用细胞计数器计算评估每ml中所含的GEM颗粒数并保存于-80℃备用。
乳清制备：将所取得初乳分装于50ml的离心管，以10000xg、4℃条件，离心30分钟后留取下层乳汁，并移至另一50ml的离心管，再依体积加入100％的醋酸并使最终醋酸浓度为1％，将其置于37℃恒温箱10分钟，进行酸化作用。其后以其1/10体积的1M醋酸盐中和乳汁，最后以10000xg、4℃条件，离心10分钟后吸取上清液，此即为乳清。将制备好的乳清采Bradford法进行蛋白质定量，保存于-20℃备用。
GEM颗粒与乳清的结合试验：以100μl的GEM颗粒(约5.6×108GEM)与乳清(约7mg)混和后，置于震荡器上在室温下震荡30分钟，再以13000xg离心10分钟，去除上清液。而沉淀物则以1ml的PBS buffer悬浮，并以13000xg离心10分钟。上述步骤重复3次后，再以1ml的elution buffer(含1M NaCl)悬浮，最后以13000xg离心10分钟，去除上清液后用20μl的PBSbuffer回溶，再加入等体积的2倍sample buffer混合均匀后，置于干浴槽以95℃加热10分钟，再以13000xg离心10分钟，吸取上层溶液，进行蛋白质胶体电泳分析。
免疫小鼠：将2.2×109GEM颗粒与初乳乳清(约25mg)混和震荡均匀后，以13000xg离心10分钟去除上清液，沉淀物则以1ml的PBS buffer悬浮，并以13000xg离心10分钟，上述步骤重复3次，再以1ml的1M NaCl悬浮，最后以13000xg离心10分钟，去除上清液后用50μl的PBS buffer回溶，加入等体积的2倍蛋白质Sample buffer混合均匀，置于干浴槽以95℃加热10分钟，再以13000xg离心10分钟，吸取上层部份，以PBS buffer定量至100μl，加入等量的完全佐剂(Freund’s Adjuvant,Complete)，于4℃环境中震荡混和12hr，作为首次免疫小鼠的针剂。之后佐剂则改混和以不完全者(Freund’s Adjuvant,Incomplete)乳化抗原蛋白。免疫试验中抗原注射以三周为一免疫周期，第一周以混和完全佐剂的抗原蛋白进行小鼠腹腔免疫针剂注射，每周以穿刺片采集小鼠脸颊血，制备血清及保存于-20℃备用；至第三次免疫，取得小鼠血清作为西方墨点法的一次抗体，侦测初乳乳清中胜肽聚醣亲合蛋白，若比较于前二次免疫血清的抗体浓度有明显上升，则再给予第四剂混和不完全佐剂的抗原蛋白针剂注射，并于隔周进行小鼠的全血采集。
蛋白质西方墨点试验：聚偏二氟乙烯(PVDF membrane)以无水甲醇润湿15分钟，再浸于transfer buffer中备用。将转渍槽转印夹依序将吸水棉、滤纸、欲转印的蛋白质电泳胶片、PVDF membrane、滤纸及吸水棉迭置，中间避免气泡产生。槽内以transfer buffer注满，外以冰浴降温，转印电压为100伏特时间1小时，转印完成后将PVDF membrane浸泡于含有5％(w/v)脱脂奶粉的TBS buffer中，于4℃摇晃至少二小时，再以TBS buffer浸洗，每次五分钟，共六次，以上述免疫试验所得的小鼠免疫初乳乳清中胜肽聚醣亲合蛋白的抗体为探针，使用时以TBS buffer稀释1000倍作为一次抗体，与PVDF membrane于室温下摇晃反应一小时，同样再以TBS buffer浸洗，每次五分钟，共六次，将带有Alkaline Phosphatase的抗体做为二次抗体，以TBS buffer稀释1500倍作为二次抗体，与PVDF membrane于室温下摇晃反应一小时，再以TBS buffer浸洗，每次五分钟，共六次，最后加入BCIP/NBT液态受质呈色，当呈色效果适当时，尽快以二次清洗终止呈色反应。
基因取得：
液相层析串连质谱仪：LC-MS/MS为液相层析仪串连两组质谱仪进行样本分析。原理为利用液相层析仪的高分析能力，将含有许多胜肽片段的混和物加以分离，再利用质谱仪中离子原将样本气化为离子态，产生分子大小及电荷不同的胜肽，即进入第一阶段质量分析器(mass analyzer)，利用电子或碰撞气体等外力撞击欲分析的离子，以产生更小的离子碎片，再以第二阶段质量分析器进行样本碎片离子的质荷比(mass to charge ratio；m/z)测量，便可利用其所带电荷推算该分析物的质量。通过液相层析仪分离出的胜肽，经过两次离子化及裂解，经过比对分析，便可获得胜肽的氨基酸序列，再利用如Mascot Analysis(Matrix Science,London,UK)等生物信息搜寻服务平台软件，进行DNA序列数据库比对分析以获得相对应的基因。
猪病原辨识蛋白的选殖与活性分析：由LC-MS/MS所获的部分氨基酸序列将用为设计degenerate primers的依据，此引子既与Olgo-d(T)作为掉取猪乳腺cDNA中病原辨识蛋白基因的工具，待RT-PCR后显示于凝胶电泳上所有条带将逐一选殖于TA-vector，经核酸定序及生物信息比对后既可获得猪病原辨识蛋白完整基因，此基因序列如SEQ ID NO：3所示。选殖及修改序列后的猪病原辨识蛋白先转构筑于大肠杆菌表现系统，诱发菌体表现后并加以纯化，纯化后的病原辨识蛋白将测试原与GEM颗粒结合的能力是否存在及稳定，结合后以Western blotting进行分析。
酵母菌表现载体构筑：为避免原使用的大肠杆菌表现系统污染饲养环境，因此改用饲料中既有使用的酵母菌为载体表现病原辨识蛋白，选殖、修改及定序后的猪病原辨识蛋白基因既构筑于pYES2.1V5-His TOPO vector(Invitrogen)，此载体为酵母菌表现型者，因此构筑后转染于酵母菌INVSc1株中，惟此系统原有诱发表现的机转以被修改为对温度及营养性者，在特定温度及营养物质存在时方启动表现病原辨识蛋白，但在尚未申请专利前对于培养条件的信息束无法详细告知。
小鼠肠道微生物试验：肠道菌相的观察：小鼠以管喂方式给予体重的1/100病原辨识蛋白，两天管喂一次历时六天，对照组将仅喂与同体积的二次灭菌水，实验结束时牺牲动物取小肠(胃下1.5～2.5cm)进行分析，小鼠肠道内容物将以灭菌过的PBS稀释成适当倍数，再以培养基培养，24hr后计算其菌落数，菌相以菌落的对数值(log cfu)表示之。另以细菌快速鉴定方法(API 20E)鉴定肠道杆菌科细菌及其他革兰氏阴性菌，其结果将以下列表1判定之。
【表1】
发酵槽发酵条件测试：经构筑染后的酵母菌以连续式发酵槽(WinpactBioreactor and Fermentor)培养，酵母菌先行活菌及悬浮后，按OD值稀释及更换培养液后在诱发环境下培养八小时，期间监控温度、转速、pH值及溶氧量等变因使维持在恒定范围。而每批培养后的酵母菌将取样以western blotting分析病原辨识蛋白的表现。将发酵八小时的发酵液进行酵母菌分离，约一公升发酵液可得约九～十克的重组酵母菌，并将该重组酵母菌存放于干燥环境备用。
初乳中结合菌膜蛋白的纯化与蛋白的定性：异性蛋白结合至微生物细胞膜或细胞壁，在固着方式大致上可分为五种：(1)通过transmembrane protein上疏水性的transmembrane domain而固着于微生物细胞膜上，(2)藉脂蛋白N端上的半胱氨酸氨经乙酰化共价结合于细胞膜的长链脂肪酸，(3)通过LPXTG motif anchor提供蛋白质暂时停留于细胞膜上，经sortase作用将LPXTG motif中的苏氨酸、甘氨酸与胜肽聚醣共价结合，(4)细胞壁与细胞壁结合蛋白的非共价结合；如存在于各种细菌的lysin motif(LysM)，及(5)表层蛋白结合。在实验所制备GEM颗粒经填塞在管柱及灌流初乳后，以SDS-PAGE分析会与菌膜结合的未知蛋白，试验中发现初乳中会与菌膜结的蛋白应不只一个，过程中合计分离及氨基酸定序者有七个(图1，C1～C7)，其中之一为已知的乳铁蛋白，而C7则经NCBI序列比对后似为胜肽聚醣辨识蛋白家族一员(C7:RecName:Peptidoglycan recognition protein；Flags:Precursor，Nominal mass(Mr):21024；Calculated pI value:9.62，Variablemodifications:Carbamidomethyl(C)，Oxidation(M)Cleavage by Trypsin:cuts C-termside of KR unless next residue is P，Sequence Coverage:11％)。
由于市面上缺乏猪PGRP抗体，因此实验先以纯化后的C7蛋白并注射于小鼠腹部，再以诱发免疫的腹水检视猪初乳与常乳中该蛋白的表现模式，结果如下图，猪的乳清在此我们的研究中也被发现具有PGRP，并且猪初乳的乳清(图2，分娩后2～4天，标示*为PGRP约17kDa)中PGRP含量明显高于猪常乳乳清的PGRP含量。然而，如图3所示，实验过程也发现该蛋白极不稳定，同样样品在低温(-20℃)保存两天后该蛋白旋即消失，此种不稳定性会使得该蛋白无法实际应用于产业。因此，于后续实施例中，对其蛋白质进行修饰，使该蛋白三级结构更趋稳定，并依其特性命名为病原辨识蛋白(pathological recognition protein,PRP)，即本发明的经修饰的初乳蛋白质。
猪病原辨识蛋白基因选殖、酵母菌表现及产量监测：根据以往开发商用重组蛋白的前例，虽实验室可以研发具市场价值的重组蛋白，但在蛋白表现量、蛋白稳定度及生产成本上是较难克服的问题，有鉴于此，猪病原辨识蛋白基因将转殖于酵母菌表现系统，虽培养条件较难设定，但由于酵母菌在商用上有诸多优点，因此实验还是决定以酵母菌为表现猪病原辨识蛋白的系统(图4)。经由多次更换培养培养基配方、温度、溶氧率及发酵时间后，现已可稳定表现出猪病原辨识蛋白，月产量约可供配制280吨的哺乳猪饲料，每次发酵馈菌时旋即收取样品1ml，待发酵结束时再取样一次，两次样品将以Western blotting确定病原辨识蛋白的表现与产量。图5为其中一次蛋白表现的监测结果，图中不同时间所收集的发酵样品在抗体呈现下，箭头(约17kDa处)指出在诱导发酵八小时后可获取最佳的表现量，如此的监测将于每批发酵后立即进行，用以确认发酵条件及菌株是否在最佳状态。而发酵后所收集的酵母菌将离心去悬浮液后，固形物将立即冷冻并于-50℃环境干燥，干燥后避免潮湿备用，待配制饲料前先行与饲料基质预混均匀后，再于其他成分混合。
同时参照第6～8图，ICR(Institute for Cancer Research)品系小鼠以管喂方式给予体重的1/100病原辨识蛋白，两天管喂一次历时六天；而对照组将仅喂与同体积的二次灭菌水。实验结束时牺牲动物取小肠(胃下1.5～2.5cm)进行分析。每次实验时对照组与受测组各为25只，试验中若因管喂导致动物死亡则将剔除该数据，在总菌数上经喂予猪病原辨识蛋白者约较对照组下降15～50％(蓝色曲线为给予PRP的测试组，当时间拉长时PRP约可抑制50％的菌数)，而菌相的鉴别上已知减少的菌以格兰氏阳性菌为主。此外，由于病原辨识蛋白具有黏着于菌膜的特性，因此试验将酵母菌表现后纯化的病原辨识蛋白与大肠杆菌混合及更换培养液两次后，以电子显微镜观察菌膜上是否有病原辨识蛋白的结合，圈选处为可明显看见蛋白结合处。
以下为本发明的经修饰的初乳蛋白质与动物饲量混合后，并投予小猪的试验：
同期比较：16头4周龄小猪，8头一组饲养于相邻二栏猪舍，抽血检测PRRS抗体力价(IgG)，并于喂饲添加PRP0.02％饲粮4周后(8周龄)，检测血液中PRRS抗体力价(IgG)。
结果：表二中显示猪只于试验前无论试验组或对照组血液中免疫球蛋白IgG皆呈现阴性反应，然而8周龄时添加PRP处理组仍呈阴性，而对照组全部呈现曾被感染的阳性反应。由此证明PRP可辅助免疫球蛋白IgA的生成，以将于猪场中欲穿透猪只黏膜进入体内的PRRS病毒于黏膜中和，因而可减少PRRS病毒穿透黏膜，而刺激淋巴免疫系统产生免疫球蛋白IgG。
【表2】
| 编号 | 试验组 | 对照组 |
| 1 | 0.019 | 0.126 |
| 2 | 0.010 | 0.084 |
| 3 | 0.000 | 0.015 |
| 4 | 0.014 | 0.077 |
| 5 | 0.008 | 0.065 |
| 6 | 0.000 | 0.081 |
| 7 | 0.000 | 0.112 |
| 8 | 0.008 | 0.090 |
【表3】
8周龄小猪血液中免疫球蛋白IgG
注.抗体力价0.4以下为阴性。
序列表
<110> 虹广生物科技有限公司
<120> 经修饰的初乳蛋白质及其应用
<130> 2016
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<170> PatentIn version 3.5
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Claims (12)
1.一种经修饰的初乳蛋白质，其氨基酸序列如SEQ ID NO：1所示的氨基酸序列，为自如SEQ ID NO：2所示的野生型初乳蛋白质的氨基酸序列的第33位置的异亮氨酸被丙氨酸取代，第101位置的谷氨酸被半胱氨酸取代，以及第175位置的精氨酸被半胱氨酸取代而产生。
2.一种编码如权利要求1所述的经修饰的初乳蛋白质的氨基酸序列的DNA，其特征在于，具有SEQ ID NO：3所示的碱基序列。
3.一种口服剂型，其特征在于，包含如权利要求1所述的经修饰的初乳蛋白质。
4.一种动物饲料组成物，其特征在于，包含如权利要求1所述的经修饰的初乳蛋白质。
5.如权利要求4所述的动物饲料组成物，其特征在于，所述的经修饰的初乳蛋白质占所述的动物饲料组成物0.01wt％～0.02wt％。
6.一种医药组成物，其特征在于，包含：
医药载剂及疫苗佐剂；以及
如权利要求1所述的经修饰的初乳蛋白质。
7.一种如权利要求1所述的经修饰的初乳蛋白质用于制备增加一动物的免疫的饲料的应用。
8.如权利要求7所述的应用，其特征在于，通过增加所述的动物的免疫球蛋白IgA生成而增加所述的动物的免疫。
9.一种如权利要求1所述的经修饰的初乳蛋白质用于制备投递于动物的饲料的应用。
10.一种如权利要求1所述的经修饰的初乳蛋白质用于制备投递于动物的医药组成物的应用。
11.一种如权利要求1所述的经修饰的初乳蛋白质用于制备预防或治疗猪繁殖和呼吸障碍综合症的饲料的应用。
12.一种如权利要求1所述的经修饰的初乳蛋白质用于制备预防或治疗猪繁殖和呼吸障碍综合症的医药组成物的应用。
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| AU2017200707B2 (en) | 2018-04-19 |
| EP3219722A1 (en) | 2017-09-20 |
| US9834592B2 (en) | 2017-12-05 |
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