CN114539396B - 一种牛肠道病毒卵黄抗体及制剂 - Google Patents
一种牛肠道病毒卵黄抗体及制剂 Download PDFInfo
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- CN114539396B CN114539396B CN202210105610.2A CN202210105610A CN114539396B CN 114539396 B CN114539396 B CN 114539396B CN 202210105610 A CN202210105610 A CN 202210105610A CN 114539396 B CN114539396 B CN 114539396B
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Abstract
针对反刍动物的生理特点,为了解决卵黄抗体在瘤胃中消化、失活的问题,本发明创造性地提出了两种制剂方式,一种是肠道制剂,其能避免卵黄抗体经过瘤胃,研究表明,对于犊牛施用肠道制剂后,其能对于BEV感染导致的犊牛腹泻具有治疗作用,与传统的药物疗法效果接近,但是相比抗生素疗法更为环保、安全;另一种是过瘤胃制剂,其可以作为日粮精料的一部分添加,使用方便,将卵黄抗体制成过瘤胃制剂后,其能避免卵黄抗体在瘤胃中降解,并在肠道定点释放,对于BEV感染导致的腹泻具有治疗作用。
Description
技术领域:
本发明属于生物技术领域,具体涉及一种牛肠道病毒卵黄抗体及制剂。
背景技术:
牛肠道病毒(BEV)感染是由小RNA病毒科肠道病毒属的牛肠道病毒(Bovine enterovirus,BEV)引起的以消化道和呼吸道症状为主要特征的一种新发动物传染病。据报道,牛肠道病毒的感染谱较宽,各种品种和日龄的牛群均易感,具有较高的发病率,牛群感染该病毒后常表现为发热、咳嗽、呼吸困难、腹泻等症状,严重者甚至出现突然死亡。本病在国内为新发传染病,BEV感染在牛群中非常普遍,且除感染牛只外,也可通过口-口、粪-口等接触传播方式感染人、羊及其它脊柱动物。感染牛肠道病毒的病牛其排泄物和分泌物也携带有大量病原体,通过污染水源、圈舍、饲料等迅速感染整个牛群,加之该病原体具有可在环境中存活较长时间和其感染谱较广等特点,造成了牛肠道病毒感染的广泛传播;同时,牛肠道病毒常与其它病原体一起呈现混合感染或者继发感染的状态,以致对当今养牛业的健康发展起到了较为严重的阻碍作用,给养牛业造成巨大的经济损失。此外,部分牛群感染肠道病毒后,不表现临床症状,为隐性感染。
由于BEV感染为国内新发传染病,现在对此病尚无有效的免疫制剂进行预防,也无有效的药物用于临床治疗,山东省农业科学院奶牛研究中心等单位研究了比阿培南、头孢拉定、H-Lys-Trp-Lys-OH等在制备防治牛肠道病毒感染药物中的应用,然而,其仍处于研究的初级阶段,尚未进入临床治疗。
卵黄抗体IgY抗体在被动免疫保护中表现出良好的应用前景,如在猪传染性胃肠炎病毒、预防轮状病毒感染、犬细小病毒感染、鸭肝炎病毒、小鹅瘟病毒及传染性法氏囊病毒病等方面,但将卵黄抗体应用在反刍动物尚未有相关报道。反刍动物与单胃动物的消化道结构不同,反刍动物胃包括瘤胃、网胃、瓣胃、皱胃四个胃,其中瘤胃存在大量的微生物,如细菌、真菌、纤毛虫,具有很强的消化作用,反刍动物消化道的特殊结构导致其具有很强的消化和降解代谢能力,许多营养物质和药物在瘤胃就被微生物降解。如何有效地将药物活性成分通过瘤胃,达到肠道是目前反刍动物肠道给药的难点。
过瘤胃技术是利用物理或化学的方法把一些容易被瘤胃微生物破坏的营养物质保护起来使之不被瘤胃微生物分解,完好的通过瘤胃,到达皱胃和肠道中再释放出来,在小肠中发挥其作用,从而满足机体对营养物质的需求。壳聚糖是甲壳素的脱乙基产物,为阳离子型聚电解质天然多糖,来源广泛,具有生物官能性、相容性、安全性等特点,已被广泛应用于医药、食品、化工等行业。壳聚糖不溶于水和碱性溶液,在pH<6.5的酸性溶液中溶解,在过瘤胃保护产品的研制中备受关注。
鉴于国内目前分离的毒株较少,对于其研究不够深入,因而,分离毒株,并对其进行深入研究,研发能预防或治疗牛肠道病毒感染的药物对我国养牛业具有重大意义。
发明内容:
针对目前缺乏预防或治疗牛肠道病毒感染的药物的现状,本发明旨在提供一种牛肠道病毒卵黄抗体及制剂,以分离得到的牛肠道病毒1型EV-E-HN1817株为抗原,制备得到卵黄抗体,而后进一步将其制成肠道制剂和过瘤胃制剂,经小规模试验验证,对于BEV感染导致的犊牛和育肥牛腹泻具有一定的治疗作用。
为解决以上技术问题,本发明采用如下技术方案:
一种牛肠道病毒卵黄抗体,其特征在于,所述卵黄抗体是以牛肠道病毒1型EV-E-HN1817株制备得到的灭活疫苗免疫健康产蛋鸡制备得到,所述牛肠道病毒1型EV-E-HN1817株于2020年10月14日在中国典型培养物保藏中心CCTCC进行用于专利程序的培养物的保藏,其保存号为:CCTCC No.V202071。
一种牛肠道病毒卵黄抗体肠道制剂,其特征在于,所述肠道制剂由水相和油相混合后均质得到,所述水相中由牛肠道病毒卵黄抗体、β-环糊精水溶液和吐温-80组成;所述油相由卵磷脂与玉米油组成。
优选的,所述牛肠道病毒卵黄抗体肠道制剂的制备方法,包括如下步骤:将琼扩效价为1:64的牛肠道病毒卵黄抗体溶液、质量体积浓度为10%的β-环糊精水溶液、吐温-80按照80:15:5的体积比例混合作为水相,将卵磷脂与玉米油按照5:95的质量比例混合均匀作为油相,将水相和油相按照7:3的体积比混合,60-80MPa均质2-3次,每次均质3min,即得到乳化的牛肠道病毒卵黄抗体肠道制剂。
本发明还请求保护一种牛肠道病毒卵黄抗体过瘤胃制剂及其制备方法,所述牛肠道病毒卵黄抗体过瘤胃制剂采用如下方法制备得到:
步骤一,将琼扩效价为1:64的牛肠道病毒卵黄抗体溶液与β-环糊精按照质量比1:1的比例混合,搅拌均匀后进行真空冷冻干燥,低温粉碎后过200目筛,制备得到含水量低于8%的牛肠道病毒卵黄抗体粉;
步骤二,将牛肠道病毒卵黄抗体粉与热保护剂-蔗糖、微晶纤维素、滑石粉按照5:1:3:1的质量比例混合均匀,按照质量体积比4:3的比例加入浓度为85% V/V 的乙醇溶液混合均匀制备得到软材;
步骤三,采用制粒机将上述软材进行剪切、滚圆和烘干制备得到芯材;
步骤四,将壳聚糖溶解于浓度为2%的乙酸溶液制备得到质量体积浓度为5%的壳聚糖溶液;而后将上述芯材加入空气流化床包衣机,喷入配制的质量体积浓度为5%的壳聚糖溶液作为内层包衣,其中芯材与壳聚糖溶液的质量比例为1:0.8,干燥后,过50目筛,即得到内层包衣芯材;
步骤五,将棕榈油脂肪粉加热至100℃进行熔融,而后加入脂肪粉质量5%的滑石粉搅板均匀,冷却至80℃作为外层包衣溶液,而后将上述内层包衣芯材加入空气流化床包衣机,喷入配制的外层包衣溶液,内层包衣芯材与外层包衣溶液的质量比为1:1.2,干燥后,过20目筛,即得到牛肠道病毒卵黄抗体过瘤胃制剂。
基于以上技术方案,本发明具有如下优点和有益效果:
首先,本发明以分离得到的牛肠道病毒1型EV-E-HN1817株为抗原,免疫蛋鸡后制备得到卵黄抗体,并将所述的卵黄抗体进一步制成制剂用于BEV感染导致的牛腹泻,其丰富了牛肠道病毒感染的治疗手段,对于养牛业具有重要的意义。
其次,针对反刍动物的生理特点,为了解决卵黄抗体在瘤胃中消化、失活的问题,本发明创造性地提出了两种制剂方式,一种是肠道制剂,其能避免卵黄抗体经过瘤胃,研究表明,对于犊牛施用肠道制剂后,其能对于BEV感染导致的犊牛腹泻具有治疗作用,与传统的药物疗法效果接近,但是相比抗生素疗法更为环保、安全;另一种是过瘤胃制剂,其可以作为日粮精料的一部分添加,使用方便,将卵黄抗体制成过瘤胃制剂后,其能避免卵黄抗体在瘤胃中降解,并在肠道定点释放,对于BEV感染导致的腹泻具有治疗作用。
再次,本发明将卵黄抗体用微胶囊包膜技术制成过瘤胃的制剂,能够使得卵黄抗体在反刍动物的瘤胃中不易被降解,使卵黄抗体能顺利通过反刍动物的瘤胃和皱胃,并在小肠快速定点释放,解决了目前卵黄抗体在反刍动物体内易被降解,难以穿过瘤胃和皱胃的问题。内层包材制备中添加β-环糊精作为包埋剂,其能有效地吸附卵黄抗体,采用蔗糖做保热护剂,其与β-环糊精都可以提高卵黄抗体的耐热程度,从而避免了干燥和包衣过程中热处理对于卵黄抗体活性的影响;采用双层包衣技术,其中外层采用棕榈油脂肪粉,在棕榈油存在的情况下,卵黄抗体颗粒与瘤胃中的瘤胃液隔离,亲水性的瘤胃液无法进入内层与卵黄抗体接触,避免了卵黄抗体在瘤胃中的降解;进入小肠后,外层的包衣基本溶解,内层包衣壳聚糖的包衣厚度较薄,具有较好的释放性能,使得卵黄抗体在肠道内能够有效释放,发挥相应的功效。
附图说明:
图1:分离毒株的RT-PCR鉴定,通道1,分离毒株;通道2,阴性对照;M,marker。
具体实施方式:
实施例1:BEV病毒的分离与鉴定
1.1样品及样品的处理
南阳地区某养牛场发生疑似牛肠道病毒感染,经取样RT-PCR检测,其BEV感染率高达80%。在该牛场中采集病牛的粪便样品5份,采集的粪便样品分别用灭菌的PBS缓冲液(pH=7.2)按照1:4(W/V)稀释后,充分研磨匀浆,8000r/min离心15min,收集上清,加入2000U/mL的青霉素-链霉素混合液,4℃静置4h后,用0.22μm滤膜过滤两次,-80℃保存备用。
病毒的分离培养
取制备好的上清样品1mL,接种到长满单层的MDBK细胞,同时接种1mLPBS作为阴性对照,置于37℃培养箱中孵育吸附2h,而后弃去接种物,DMEM培养液洗涤两次后,加入含2%FBS的DMEM培养基,置于5%CO2、37℃培养箱中继续培养,盲传3代。
采用以上同样的方法,将第3代病毒液接种至长满单层的MDBK细胞,每隔6h观察1次细胞。接种48-72h后,将接毒细胞反复冻融3次,离心获得病毒液再次接种MDBK细胞,重复上述操作,将病毒传至5代。
病毒TCID50的测定
对F5代病毒液进行连续10倍稀释,具体方法是:
(1)从-80℃超低温冰箱取出病毒液,反复冻融三次,将病毒液做10-2~10-13连续稀释,
(2)拿出已经铺满培养瓶底部的96孔MDBK细胞培养板,弃去培养液后,将病毒液接种到MDBK细胞中,每孔100 μL,并设置重复和阴性对照,感作2 h 后加入2% DMEM 的维持液。
(3)放入37℃,5%CO2细胞培养箱底部孵育2h,然后将病毒液弃去,更换100μL 1%细胞维持液,继续置入37℃,5%CO2细胞培养箱培养。连续观察72h,计录每个稀释度出现CPE 孔数,按照Reed-Muench 方法计算病毒的TCID50。
经计算,F5代病毒的滴度为108.9TCID50/mL;继续传代后,F10的病毒滴度为1012.5TCID50/mL。
分离毒株的RT-PCR检测
采用文献报道的BEV 5’-UTR-F(5’-tttaaaacagcctgggggttgtac-3’)和BEV 5’-UTR-R(5’-cggagtaccgaaagtagtctgttc-3’)(张姗等,中国兽医科学,2018,48(07))对分离的病毒进行RT-PCR检测鉴定,经RT-PDR检测,其能扩增出于预期相符的667bp的特异性片段,如图1所示。
分离病毒的理化特性鉴定
对该病毒进行乙醚敏感性试验、氯仿敏感性试验、胰蛋白酶敏感性试验、耐热性试验、耐酸性试验等理化特性鉴定。
1.5.1乙醚敏感性试验:取2mL F5代病毒液与0.5mL乙醚混匀置4℃过夜,1200g离心30min,吸取下层病毒液并过滤除菌,于MDBK细胞中测定得到处理后的病毒液的滴度为106.7TCID50 /mL。
1.5.2氯仿敏感性试验:F5病毒液以3000g离心15min去除细胞碎片,取1.2mL上清与200μL氯仿混匀置4℃过夜,次日取上层病毒液并过滤除菌,于MDBK细胞中测定得到处理后的病毒液的滴度为105.9TCID50 /mL。
1.5.3胰蛋白酶敏感性试验:取1mL病毒液与等体积0.5%胰蛋白酶溶液混匀(胰蛋白酶终浓度为0.25%),置37℃作用1h,立即加入4mL胎牛血清以终止胰蛋白酶的作用,过滤除菌后于MDBK细胞中测定得到处理后的病毒液的滴度为106.2TCID50 /mL。
1.5.4耐酸耐碱性试验:分别取2mL病毒液,将其pH调至3.0、5.0、9.0和10.0,置于37℃水浴中作用1h,再将pH调至7.0,过滤除菌后于MDBK细胞中测定得到处理后的病毒液的滴度为105.2TCID50 /mL(pH=3.0)、105.7TCID50 /mL(pH=5.0)、103.1TCID50 /mL(pH=9.0)102.9TCID50 /mL(pH=10.0)。
1.5.5温度敏感性试验:取1mL病毒液置于50℃水浴中作用30min,1200g离心30min,吸取下层病毒液并过滤除菌,于MDBK细胞中测定得到处理后的病毒液的滴度为103.7TCID50 /mL(50℃)。
基于以上结果可知,本发明所分离得到的BEV毒株对于乙醚、氯仿等脂溶性溶剂及胰蛋白酶具有一定的抵抗能力,可以耐受pH=3.0和5.0的酸性环境,然而,对pH=9.0和10.0的碱性环境较为敏感,50℃处理30min会使得病毒活性明显下降,表明其对热较为敏感。
病毒的保藏
2020年10月14日在中国典型培养物保藏中心CCTCC进行用于专利程序的培养物的保藏,其保存号为:CCTCC No.V202071,分类命名为牛肠道病毒1型EV-E-HN1817;保藏地址为:湖北省武汉市武汉大学保藏中心。
实施例2. BEV灭活疫苗的制备:
2.1病毒原液制备
将生长状态良好的MDBK细胞单层弃去培养液,按2%的量吸附接种牛肠道病毒1型EV-E-HN1817株,37℃吸附2小时后加入维持液继续培养,每日观察记录细胞病变情况。当细胞80%以上病变时收获,冻融2次,取样进行半成品检验,测定病毒滴度并将病毒液稀释至109.0 TCID50 /mL,即得到疫苗病毒原液,-80℃保存。
灭活
将检验合格的疫苗病毒原液澄清过滤去细胞碎片,边搅拌边加入10%甲醛溶液,使其终浓度为0.2%,37℃灭活16h,期间不断搅拌,灭活后病毒液于2~8℃保存。
疫苗制备及分装
2.3.1油相制备
将90重量份注射用白油、5重量份硬脂酸铝混匀并加热至70℃,再加入5重量份span-80,至温度达到115℃时维持40min,冷却后备用。
2.3.2水相制备
将吐温-80与灭活的病毒液按1:19的体积比例混合,搅拌30-35min使吐温-80完全溶解混匀。
2.3.3乳化
将油相和水相以1:2体积比例乳化25-30min,取10ml疫苗加入离心管中,4000rpm,离心10min,管底析出的水相≤0.5ml。
2.3.4分装定量分装,压盖,2-8℃保存。
实施例3. BEV卵黄抗体的制备
3.1免疫程序
(1)基础免疫:采用皮下注射的方式,健康产蛋鸡接种实施例2制备的BEV灭活疫苗,每羽1.0ml;
(2)加强免疫:基础免疫14日后,皮下注射实施例2制备的BEV灭活疫苗,每只1.0ml;
(3)强化免疫:加强免疫14日后,皮下注射实施例2制备的BEV灭活疫苗,每只1.5ml。
(4)维持免疫:在强化免疫接种后根据抗体效价,每隔2~3个月再加强接种1次,每只1.5ml。
高免蛋收集
强化免疫接种结束后10日,每隔5 日抽样测定高免蛋蛋黄中的抗体效价,当中和效价不低于1:256时,收集高免蛋,置10~15℃,应不超过10日。
采用辛酸-黄原胶法对蛋黄抗体进行提取和纯化
(1)蛋壳消毒:高免蛋浸入0.15%新洁尔灭水溶液中消毒15分钟,然后用甲醛熏蒸高免蛋30分钟。
(2)蛋黄分离:打破蛋壳,除去蛋清、胚盘和系带,收集卵黄。
(3)除脂:量取卵黄体积,加入灭菌玻瓶内,按其2体积量加入终浓度为0.1%黄原胶水溶液(W/V),混匀,并调至pH=5.2,置2~8℃静置16小时。
(4)萃取:吸取并量取上清液体积,加入终浓度2%正辛酸(v/v),室温静置4小时,取上清液经过滤后,滤液调至pH=7.2。
(5)过滤、浓缩:将加辛酸处理好的卵黄上清液用孔径为1.0μm和 0.45μm的筒式滤芯过滤,再经截留分子量为100kDa的中空纤维超滤柱进行浓缩10倍。
(6)灭活:按终浓度为0.05%加入甲醛溶液,充分搅拌混匀,室温灭活 24小时。
(7)半成品配制:将浓缩灭活卵黄抗体用pH=7.2的磷酸盐缓冲液稀释为琼扩抗体效价不低于1:8,再用孔径为0.22μm滤膜过滤除菌。
半成品检验
(1)无菌检验:按现行《中国兽药典》附录进行检验,无菌生长。
(2)中和效价测定:卵黄抗体中和效价不低于1:128。
(3)支原体检验:按现行《中国兽药典》附录进行检验,无支原体生长。
(4)甲醛残留量测定:按现行《中国兽药典》附录进行检验,符合规定。
(5)辛酸残留量检验辛酸残留量不高于0.1%。
(6)分装定量分装,加盖密封,2~8℃保存。
实施例4. BEV-卵黄抗体肠道制剂的制备
将琼扩效价为1:64的BEV卵黄抗体半成品、质量体积浓度为10%的β-环糊精水溶液、吐温-80按照80:15:5的体积比例混合作为水相,将卵磷脂与玉米油按照5:95的质量比例混合均匀作为油相,将水相和油相按照7:3的体积比混合,60-80MPa均质2-3次,每次均质3min,即得到乳化的BEV-卵黄抗体肠道制剂。
实施例5.BEV-卵黄抗体肠道制剂的效果评价
在南阳地区某发生疑似牛肠道病毒感染牛场,经取样RT-PCR检测,其BEV感染率高达35%以上,选择10头3-5周龄、腹泻症状明显的犊牛随机分为5组,经检测,其粪便样品均为BEV阳性,分别进行编号管理,其中组1-3依次为低剂量试验组、中剂量试验组和高剂量试验组,对犊牛进行灌肠处理,每头犊牛每2天灌肠一次,每次灌肠5mL、10mL、15mL本发明的BEV-卵黄抗体肠道制剂(具体灌肠方法是:取对应剂量的肠道制剂,采用生理盐水定容至500mL进行灌肠);组4为药物治疗组,采用药物治疗(具体用药如下:0.9%生理盐水50 mL,2 mL地塞米松,2支青霉素钾,1次/d,同时,采用20%磺胺嘧啶钠5 mL×1支,进行注射,2次/d,需连续使用6天);组5为对照组,每头犊牛相应通过肠道灌入500mL生理盐水,若腹泻停止、每日大便次数少于5次则相应试验动物的灌肠试验终止,计为治愈,试验持续16天。试验期间每日采食的同样的日粮,统计各试验组大便次数,具体结果见下表1。
表1 各实验组每头犊牛平均每天大便次数(单位:次/天)
0天 | 1-2天 | 3-4天 | 5-8天 | 9-12天 | 12-16天 | |
高剂量组 | 12.5*1 | 8.50 | 4.75 | 3.75 | 3.25 | 3.00 |
中剂量组 | 11.0 | 8.50 | 5.70 | 4.25 | 4.00 | 3.75 |
低剂量组 | 11.5 | 9.25*2 | 5.75 | 5.50 | 4.50 | 4.25 |
药物治疗组 | 11.0 | 8.00 | 5.25 | 4.50 | 5.25 | 3.50 |
对照组 | 12.5 | 11.5 | 12.75 | 12.0 | 12.25 | 13.5 |
注:*1,两头牛两天内合计大便50次,50次/(2头*2天)=12.5次/(头*天),即每头犊牛平均每天大便次数为:12.5次/天;
*2,两头牛两天内合计大便37次,37次/(2头*2天)=9.25次/(头*天),即每头犊牛平均每天大便次数为:9.25次/天。
基于以上试验结果可知,针对BEV感染导致的犊牛腹泻,采用本发明所述的BEV-卵黄抗体肠道制剂能够取得较好的治疗效果,在灌肠治疗的第3-4天基本能够控制腹泻状况,其中高剂量组的治疗效果与常规药物治疗组的作用基本相近,中、低剂量组相对而言效果略差。而在试验第12-16天,各剂量组和药物治疗组均恢复正常,而对照组的腹泻状况没有改善。以上试验表明,本发明的BEV-卵黄抗体肠道制剂对于BEV感染导致的犊牛腹泻具有治疗作用。
实施例7.BEV-卵黄抗体过瘤胃制剂的制备
步骤一,将琼扩效价为1:64的BEV卵黄抗体半成品溶液与β-环糊精按照质量比1:1的比例混合,搅拌均匀后进行真空冷冻干燥,低温粉碎后过200目筛,制备得到含水量低于8%的BEV卵黄抗体粉;
步骤二,将BEV卵黄抗体粉与热保护剂-蔗糖、微晶纤维素、滑石粉按照5:1:3:1的质量比例混合均匀,按照质量体积比4:3的比例加入浓度为85%(V/V)的乙醇溶液混合均匀制备得到软材;
步骤三,采用制粒机将上述软材进行剪切、滚圆和烘干制备得到芯材
步骤四,将壳聚糖溶解于浓度为2%的乙酸溶液制备得到质量体积浓度为5%的壳聚糖溶液;而后将上述芯材加入空气流化床包衣机,喷入配制的质量体积浓度为5%的壳聚糖溶液作为内层包衣,其中芯材与壳聚糖溶液的质量比例为1:0.8,干燥后,过50目筛,即得到内层包衣芯材;
步骤五,将棕榈油脂肪粉加热至100℃进行熔融,而后加入脂肪粉质量5%的滑石粉搅板均匀,冷却至80℃作为外层包衣溶液,而后将上述内层包衣芯材加入空气流化床包衣机,喷入配制的外层包衣溶液,内层包衣芯材与外层包衣溶液的质量比为1:1.2,干燥后,过20目筛,即得到BEV-卵黄抗体过瘤胃制剂。
实施例8.BEV-卵黄抗体过瘤胃制剂的效果评价
在南阳地区某发生疑似牛肠道病毒感染牛场,经取样RT-PCR检测,其BEV感染率高达35%以上,选择12头育肥牛随机分为3组,经检测,其粪便样品均为BEV阳性,分别进行编号管理,其中组1为试验组,组2为卵黄抗体粉组,组3为对照组,试验过程持续15天,育肥牛每日采食的日粮精粗比为1:3,其中对照组正常饲喂,试验组饲喂的精料中添加了5‰的BEV-卵黄抗体过瘤胃制剂,卵黄抗体粉组的精料中添加了5‰的卵黄抗体粉(将琼扩效价为1:64的BEV卵黄抗体半成品溶液与β-环糊精按照质量比1:1的比例混合,搅拌均匀后进行真空冷冻干燥,低温粉碎后过200目筛,制备得到含水量低于8%的BEV卵黄抗体粉),每头牛在各饲养棚隔离饲喂。统计各试验组大便次数,具体结果见下表2。
表2 各实验组每头犊牛平均每天大便次数(单位:次/天)
0天 | 1-2天 | 3-4天 | 5-8天 | 9-12天 | 12-16天 | |
组1-试验组 | 9.50 | 6.25 | 5.75 | 4.50 | 4.25 | 3.50 |
组2-卵黄抗体粉组 | 9.25 | 8.00 | 7.75 | 7.25 | 6.50 | 6.25 |
组3-对照组 | 8.75 | 9.00 | 8.75 | 8.25 | 7.50 | 7.75 |
基于以上试验结果可知,本发明采用壳聚糖对卵黄抗体进行包衣制备得到BEV-卵黄抗体过瘤胃制剂,将其与精料混合后用于育肥牛的饲喂可以改善因BEV感染导致的腹泻状况,而未经包被的卵黄抗体粉对于腹泻状况无明显改善。
最后应说明的是:以上各实施例仅用以说明本发明的技术方案,而非对其限制,对前述各实施例所记载的技术方案进行修改,或者对其中部分或者全部技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
Claims (1)
1.一种牛肠道病毒卵黄抗体过瘤胃制剂,其特征在于,所述牛肠道病毒卵黄抗体过瘤胃制剂在反刍动物的瘤胃中不易被降解,使卵黄抗体能顺利通过反刍动物的瘤胃和皱胃,并在小肠快速定点释放,所述牛肠道病毒卵黄抗体过瘤胃制剂用于对于BEV感染导致的腹泻的治疗;所述牛肠道病毒卵黄抗体过瘤胃制剂采用如下方法制备得到:
步骤一,将琼扩效价为1:64的牛肠道病毒卵黄抗体溶液与β-环糊精按照质量比1:1的比例混合,搅拌均匀后进行真空冷冻干燥,低温粉碎后过200目筛,制备得到含水量低于8%的牛肠道病毒卵黄抗体粉;
步骤二,将牛肠道病毒卵黄抗体粉与热保护剂-蔗糖、微晶纤维素、滑石粉按照5:1:3:1的质量比例混合均匀,按照质量体积比4:3的比例加入浓度为85% V/V 的乙醇溶液混合均匀制备得到软材;
步骤三,采用制粒机将上述软材进行剪切、滚圆和烘干制备得到芯材;
步骤四,将壳聚糖溶解于浓度为2%的乙酸溶液制备得到质量体积浓度为5%的壳聚糖溶液;而后将上述芯材加入空气流化床包衣机,喷入配制的质量体积浓度为5%的壳聚糖溶液作为内层包衣,其中芯材与壳聚糖溶液的质量比例为1:0.8,干燥后,过50目筛,即得到内层包衣芯材;
步骤五,将棕榈油脂肪粉加热至100℃进行熔融,而后加入脂肪粉质量5%的滑石粉搅板均匀,冷却至80℃作为外层包衣溶液,而后将上述内层包衣芯材加入空气流化床包衣机,喷入配制的外层包衣溶液,内层包衣芯材与外层包衣溶液的质量比为1:1.2,干燥后,过20目筛,即得到牛肠道病毒卵黄抗体过瘤胃制剂;
所述牛肠道病毒卵黄抗体是以牛肠道病毒1型EV-E-HN1817株制备得到的灭活疫苗免疫健康产蛋鸡制备得到,所述牛肠道病毒1型EV-E-HN1817株于2020年10月14日在中国典型培养物保藏中心CCTCC进行用于专利程序的培养物的保藏,其保存号为:CCTCCNo.V202071;
所述牛肠道病毒卵黄抗体的制备方法包括如下步骤:
步骤一 免疫程序:
(1)基础免疫:采用皮下注射的方式,健康产蛋鸡接种BEV灭活疫苗,每羽1.0ml;
(2)加强免疫:基础免疫14日后,皮下注射BEV灭活疫苗,每只1.0ml;
(3)强化免疫:加强免疫14日后,皮下注射BEV灭活疫苗,每只1.5ml;
(4)维持免疫:在强化免疫接种后根据抗体效价,每隔2~3个月再加强接种1次,每只1.5ml;
步骤二 高免蛋收集:强化免疫接种结束后10日,每隔5 日抽样测定高免蛋蛋黄中的抗体效价,当中和效价不低于1:256时,收集高免蛋,置10~15℃,应不超过10日;
步骤三:采用辛酸-黄原胶法对蛋黄抗体进行提取和纯化:
(1)蛋壳消毒:高免蛋浸入0.15%新洁尔灭水溶液中消毒15分钟,然后用甲醛熏蒸高免蛋30分钟;
(2)蛋黄分离:打破蛋壳,除去蛋清、胚盘和系带,收集卵黄;
(3)除脂:量取卵黄体积,加入灭菌玻瓶内,按其2体积量加入终浓度为0.1%黄原胶水溶液(W/V),混匀,并调至pH=5.2,置2~8℃静置16小时;
(4)萃取:吸取并量取上清液体积,加入终浓度2%正辛酸(v/v),室温静置4小时,取上清液经过滤后,滤液调至pH=7.2;
(5)过滤、浓缩:将加辛酸处理好的卵黄上清液用孔径为1.0μm和 0.45μm的筒式滤芯过滤,再经截留分子量为100kDa的中空纤维超滤柱进行浓缩10倍;
(6)灭活:按终浓度为0.05%加入甲醛溶液,充分搅拌混匀,室温灭活 24小时;
(7)半成品配制:将浓缩灭活卵黄抗体用pH=7.2的磷酸盐缓冲液稀释为琼扩抗体效价不低于1:8,再用孔径为0.22μm滤膜过滤除菌。
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