CN111718906B - 适于禽流感病毒高效扩增的维持培养基 - Google Patents
适于禽流感病毒高效扩增的维持培养基 Download PDFInfo
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- CN111718906B CN111718906B CN202010591160.3A CN202010591160A CN111718906B CN 111718906 B CN111718906 B CN 111718906B CN 202010591160 A CN202010591160 A CN 202010591160A CN 111718906 B CN111718906 B CN 111718906B
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Abstract
本发明涉及生物制品领域,特别涉及适于禽流感病毒高效扩增的维持培养基,包括基础代谢营养物类、微量元素类、维生素类、盐类、病毒增殖促进剂、添加物等。制备方法简单,成本低,便于操作。制得的培养基具有支持MDCK细胞高效生产禽流感病毒的能力,也可为其他细胞系的病毒疫苗用培养基开发提供借鉴作用。
Description
技术领域
本发明涉及生物制品领域,具体涉及适于禽流感病毒高效扩增的维持培养基。
背景技术
禽流感(Bird Flu或Avian Influenza)是由甲型流感病毒的亚型(也称禽流感病毒)引起的一种急性传染病,能够感染人类,且人感染禽流感后的死亡率在30%以上,严重威胁养殖业和人类健康,并给社会带来巨大的经济损失。目前为止禽流感仍未找到理想的治疗药物,疫苗接种仍是当今防止疫病发生和流行的最主要预防措施。
禽流感疫苗的传统生产工艺是鸡胚培养法,这种以鸡胚作为生产基质的方式已使用多年,工艺成熟,但生产周期长、操作繁琐、工作量大、易污染,如遇禽流感大规模爆发,鸡胚的供应存在很大问题。以细胞作为介质是目前疫苗制备的趋势,与传统的鸡胚生产工艺相比,细胞培养生产工艺在禽流感爆发时操作更灵活,可快速大规模培养以应对SPF(无特定病原体)鸡蛋供应不足的问题。目前社会对于禽流感疫苗的需求量很大,但现有细胞培养生产工艺产能相对低下,因此,提高禽流感病毒在细胞中的扩增效率的研究工作尤为迫切。在基于细胞培养的禽流感疫苗生产工艺中,培养基是细胞生长和维持活性的基础,也是禽流感疫苗生产的关键因素。无血清单细胞悬浮培养因其易于放大、工艺稳定、过程可控等优势,正成为禽流感疫苗生产的主流技术。在目前的研究或生产中,在病毒生产阶段所用维持培养基大多与用于细胞生长的生长培养基是同一种,但由于病毒生产的特性不同于细胞,所需的生物合成原料与途径也不同于细胞,这种营养组分的不平衡限制了禽流感病毒的高效扩增。因此,针对特定的病毒生产阶段筛选适于禽流感病毒生产的维持培养基十分重要。
有大量文献报道MDCK细胞(Madin-Darby canine kidney cells)被广泛用于病毒的扩增和纯化,因其增殖快,且流感病毒对其感染效率高、不易变异,因此被公认为最适于甲、乙型流感病毒疫苗生产的细胞系之一。本发明即以MDCK细胞和禽流感病毒为研究对象,基于MDCK细胞悬浮培养扩增禽流感病毒的生产工艺,提供了一种支持禽流感病毒在MDCK悬浮细胞中高效扩增的培养基配方,该培养基使病毒产量获得极大提高,具有支持MDCK细胞高效生产禽流感病毒的能力,也可为其他细胞系的病毒疫苗用培养基开发提供借鉴作用。
发明内容
针对现有技术存在的不足,本发明的目的在于提供一种适于禽流感病毒高效扩增的维持培养基,该培养基能支持MDCK细胞高效生产禽流感病毒的能力,促进病毒的扩增效率。
为实现本发明的上述目的,采用如下技术方案:一种适于禽流感病毒高效扩增的维持培养基,其特征在于,各组分的浓度以mg/L计为:
基础代谢营养物类:所述基础代谢营养物类为丝氨酸20-250、精氨酸20-250、亮氨酸20-55、异亮氨酸20-250、丙氨酸1-2、酪氨酸20-55、天冬酰胺2-25、天冬氨酸2-25、盐酸半胱氨酸5-55、谷氨酸2-50、谷氨酰胺2-50、组氨酸20-250、赖氨酸20-250、苯丙氨酸10-55、苏氨酸5-55、色氨酸20-250、缬氨酸20-250;
核苷酸类:所述核苷酸类为次黄嘌呤2-20、胸苷2-5、腺苷2-5、尿苷2-5、鸟苷2-5;
微量元素类:所述微量元素类为硒0.05-0.1、铬0.05-0.1、钴0.02-0.05、镍0.02-0.05、锌0.05-0.1、铜0.05-0.1、锰0.05-0.1、钡0.05-0.1、镓0.02-0.05、锂0.05-0.1、锡0.05-0.1、碘0.05-0.1、钒0.05-0.1、锗0.02-0.05、钼0.02-0.05、硅0.05-0.1、铁0.05-0.1、铷0.02-0.05、锆0.02-0.05、镉0.05-0.1、铝0.05-0.1;
维生素类:所述维生素类为生物素0.02-0.05、D-泛酸钙0.05-0.1、叶酸0.02-0.05、烟酰胺0.02-0.05、盐酸吡哆醇0.02-0.05、核黄素0.05-0.1、盐酸硫胺素0.02-0.05、维生素B12 0.05-0.1、维生素B2 0.05-0.1、肌醇0.05-0.1;
盐类:所述盐类为氯化钠200-500、氯化钾200-500、硫酸铜2-55、硝酸铁2-55、硫酸亚铁2-55、氯化镁2-55、无水磷酸氢二钠20-250、磷酸二氢钠20-250、亚硒酸钠20-250、硫酸锌2-55;
病毒增殖促进剂:所述病毒增殖促进剂为胆固醇1-5、卵磷脂0.02-0.05、氯化钙1-5、亚油酸0.05-0.01、亚麻酸0.05-0.01、月桂酸0.05-0.1、棕榈酸0.05-0.1、维生素E 0.05-0.1、前列腺素20-55、吐温-80 1-5;
添加物:所述添加物为葡萄糖1000-8000、转铁蛋白2-55、胰岛素2-55、牛血清白蛋白500-1000、大豆水解物500-1000。
进一步的,所述培养基还包括:酸碱度缓冲剂碳酸氢钠10-1000mg/L;
进一步的,所述培养基还包括:酸碱度指示剂酚红1-20mg/L;
进一步的,所述培养基还包括:剪切力保护剂嵌段式聚醚F68 20-150mg/L;
进一步的,所述培养基还包括:激素地塞米松0.001-0.02mg/L;
进一步的,所述培养基还包括:抗生素两性霉素B 200-500mg/L。
发明人认为,本发明提供的适于禽流感病毒高效扩增的维持培养基中,基础营养代谢物包括碳源和氮源,为细胞正常生长、代谢、维持生命提供了物质基础;适当提高丝氨酸、精氨酸和异亮氨酸、组氨酸、色氨酸等的浓度,满足禽流感病毒的代谢需求,有效促进禽流感病毒在细胞内扩增。核苷酸类化合物是细胞物质能量代谢所必须的组成分子,同时也是DNA和RNA等遗传物质合成所需的物质基础;维生素类是细胞代谢必不可少的化合物,众所周知细胞的各种代谢生长活动都需要维生素的参与,许多维生素可作为辅酶或者辅酶的组成分子;盐类能调节细胞膜通透性,维持正常酸碱平衡和渗透压,维持细胞良好的形态和功能,有些无机化合物还构成酶的辅基、维生素、激素、核酸和蛋白质的组成成分,参与多种重要的生理功能;酸碱度缓冲剂能调控整体系统的酸碱度,使其达到平衡,避免过酸性或过碱性的环境造成细胞核损伤;酸碱指示剂能实时指示培养液的酸碱度变化,方便及时更换新鲜培养液、进行细胞传代等操作;添加物则包含血清替代物和为细胞生长提供重要的氨基酸、短肽等营养物的生命大分子物质,其中添加物中的牛血清白蛋白、转铁蛋白代替血清功能,提高培养基蛋白含量,保证细胞分化、增殖需要,胰岛素能促进葡萄糖代谢,保证MDCK细胞的生长活性;剪切力保护剂可削弱生物反应器中因搅拌、通气等机械外力对细胞的损伤作用;抗生素进一步保证细胞培养环境的安全性,避免细胞污染;病毒增殖促进剂能调控细胞生理状态、维持病毒活性、促进病毒高效扩增。
上述适于禽流感病毒高效扩增的维持培养基的制备方法,包括以下步骤:
1)制备混合液:将原料干粉用溶剂溶解并混合;
2)调节pH:调节混合液的pH;
3)定容:定容得到适于禽流感病毒高效扩增的维持培养基。
进一步的,所述原料干粉溶解之前需研磨过筛。
进一步的,所述溶剂选自蒸馏水、去离子水、低级醇中的一种或几种。
进一步的,所述调节pH的溶剂选自氢氧化钠、氨水、氢氧化钾中的一种或几种。
进一步的,所述定容溶剂选自蒸馏水、去离子水中的一种或几种。
作为本发明的进一步改进,上述适于禽流感病毒高效扩增的维持培养基的制备方法,优选包括以下步骤:
1)制备混合液:将原料干粉混合后磨粉过50-200目筛,然后将所得细粉于去离子水中搅拌溶解,得到混合液;
2)调节pH:加入碱液调节混合液的pH至6.0-7.0;
3)定容:去离子水定容得到适于禽流感病毒高效扩增的维持培养基。
进一步的,所述定容后还包括过滤除菌步骤。
作为本发明的进一步改进,上述适于禽流感病毒高效扩增的维持培养基的制备方法,优选包括以下步骤:
1)制备混合液:将原料干粉混合后磨粉过150目筛,然后将所得细粉于10-30℃无热源去离子水中搅拌溶解,得到混合液;
2)调节pH:加入氨水调节混合液的pH至6.7;
3)定容:去离子水定容得到适于禽流感病毒高效扩增的维持培养基;
4)过滤出菌:采用0.22μm滤膜过滤除菌。
本发明的有益效果在于:
1、本发明所述适于禽流感病毒高效扩增的维持培养基不含动物血清;成分明确,可以保证培养基批次间的一致性;
2、本发明添加的功能性生物材料如牛血清白蛋白、转铁蛋白、胰岛素等生物大分子物质,提高培养基的蛋白含量,支持MDCK细胞高密度全悬浮培养,细胞成活率达到96%以上,为禽流感病毒的高效扩增提供基础条件;
3、本发明培养基性质明确,杂质含量降低,简化后期MDCK细胞的分离纯化工艺,有利于进行细胞生长代谢、病毒扩增方面的研究,同时降低生产成本,显著提高生产过程效率;
4、本发明的制备方法简单,成本低效率高,安全性有保证,有利于大规模生产。
附图说明
图1为MDCK细胞在本发明适于禽流感病毒高效扩增的维持培养基中的形态图;
图2为MDCK细胞培养时活细胞生长随时间的变化图。
具体实施方式
以下介绍本发明适于禽流感病毒高效扩增的维持培养基的具体实施方式,应该指出,本发明的实施不限于以下的实施方式。
实施例1
本实施例公开了一种适于禽流感病毒高效扩增的维持培养基,其特征在于,各组分的浓度以mg/L计为:
基础代谢营养物类:所述基础代谢营养物类为丝氨酸20、精氨酸20、亮氨酸20、异亮氨酸20、丙氨酸1、酪氨酸20、天冬酰胺2、天冬氨酸2、盐酸半胱氨酸5、谷氨酸2、谷氨酰胺2、组氨酸20、赖氨酸20、苯丙氨酸10、苏氨酸5、色氨酸20、缬氨酸20;
核苷酸类:所述核苷酸类为次黄嘌呤2、胸苷2、腺苷2、尿苷2、鸟苷2;
微量元素类:所述微量元素类为硒0.05、铬0.05、钴0.02、镍0.02、锌0.05、铜0.05、锰0.05、钡0.05、镓0.02、锂0.05、锡0.05、碘0.05、钒0.05、锗0.02、钼0.02、硅0.05、铁0.05、铷0.02、锆0.02、镉0.05、铝0.05;
维生素类:所述维生素类为生物素0.02、D-泛酸钙0.05、叶酸0.02、烟酰胺0.02、盐酸吡哆醇0.02、核黄素0.05、盐酸硫胺素0.02、维生素B12 0.05、维生素B2 0.05、肌醇0.05;
盐类:所述盐类为氯化钠200、氯化钾200、硫酸铜2、硝酸铁2、硫酸亚铁2、氯化镁2、无水磷酸氢二钠20、磷酸二氢钠20、亚硒酸钠20、硫酸锌2;
病毒增殖促进剂:所述病毒增殖促进剂为胆固醇1、卵磷脂0.02、氯化钙1、亚油酸0.05、亚麻酸0.05、月桂酸0.05、棕榈酸0.05、维生素E 0.05、前列腺素20、吐温-80 1;
添加物:所述添加物为葡萄糖1000、转铁蛋白2、胰岛素2、牛血清白蛋白500、大豆水解物500;
酸碱度缓冲剂:所述酸碱度缓冲剂为碳酸氢钠10;
酸碱度指示剂:所述酸碱度指示剂为酚红1;
剪切力保护剂:所述剪切力保护剂为嵌段式聚醚F68 20;
激素:所述激素为地塞米松0.001;
抗生素:所述抗生素为两性霉素B 200。
根据以下步骤制备适于禽流感病毒高效扩增的维持培养基:
1)制备混合液:将原料干粉混合后磨粉过150目筛,然后将所得细粉于10℃无热源去离子水中搅拌溶解,得到混合液;
2)调节pH:加入氨水调节混合液的pH至6.7;
3)定容:去离子水定容得到适于禽流感病毒高效扩增的维持培养基;
4)过滤出菌:采用0.22μm滤膜过滤除菌。
实施例2
本实施例所述适于禽流感病毒高效扩增的维持培养基中,各组分的浓度以mg/L计为:
基础代谢营养物类:所述基础代谢营养物类为丝氨酸150、精氨酸150、亮氨酸35、异亮氨酸150、丙氨酸1、酪氨酸20、天冬酰胺25、天冬氨酸25、盐酸半胱氨酸55、谷氨酸50、谷氨酰胺50、组氨酸150、赖氨酸20、苯丙氨酸55、苏氨酸55、色氨酸150、缬氨酸20;
核苷酸类:所述核苷酸类为次黄嘌呤20、胸苷5、腺苷2、尿苷2、鸟苷2;
微量元素类:所述微量元素类为硒0.08、铬0.08、钴0.08、镍0.02、锌0.08、铜0.08、锰0.08、钡0.08、镓0.02、锂0.08、锡0.08、碘0.08、钒0.08、锗0.05、钼0.05、硅0.08、铁0.08、铷0.05、锆0.02、镉0.08、铝0.08;
维生素类:所述维生素类为生物素0.02、D-泛酸钙0.08、叶酸0.02、烟酰胺0.02、盐酸吡哆醇0.02、核黄素0.08、盐酸硫胺素0.05、维生素B12 0.08、维生素B2 0.08、肌醇0.08;
盐类:所述盐类为氯化钠500、氯化钾500、硫酸铜20、硝酸铁20、硫酸亚铁20、氯化镁20、无水磷酸氢二钠20、磷酸二氢钠20、亚硒酸钠250、硫酸锌20;
病毒增殖促进剂:所述病毒增殖促进剂为胆固醇1、卵磷脂0.05、氯化钙5、亚油酸0.08、亚麻酸0.08、月桂酸0.08、棕榈酸0.08、维生素E 0.08、前列腺素35、吐温-80 1;
添加物:所述添加物为葡萄糖4000、转铁蛋白55、胰岛素55、牛血清白蛋白800、大豆水解物800。
酸碱度缓冲剂:所述酸碱度缓冲剂为碳酸氢钠500;
酸碱度指示剂:所述酸碱度指示剂为酚红15;
剪切力保护剂:所述剪切力保护剂为嵌段式聚醚F68 100;
激素:所述激素为地塞米松0.005;
抗生素:所述抗生素为两性霉素B 200。
根据以下步骤制备适于禽流感病毒高效扩增的维持培养基:
1)制备混合液:将原料干粉混合后磨粉过150目筛,然后将所得细粉于10-30℃无热源去离子水中搅拌溶解,得到混合液;
2)调节pH:加入氨水调节混合液的pH至6.7;
3)定容:去离子水定容得到适于禽流感病毒高效扩增的维持培养基;
4)过滤出菌:采用0.22μm滤膜过滤除菌。
实施例3
本实施例所述适于禽流感病毒高效扩增的维持培养基中,各组分的浓度以mg/L计为:
基础代谢营养物类:所述基础代谢营养物类为丝氨酸250、精氨酸250、亮氨酸55、异亮氨酸250、丙氨酸2、酪氨酸55、天冬酰胺25、天冬氨酸25、盐酸半胱氨酸55、谷氨酸50、谷氨酰胺50、组氨酸250、赖氨酸250、苯丙氨酸55、苏氨酸55、色氨酸250、缬氨酸250;
核苷酸类:所述核苷酸类为次黄嘌呤20、胸苷5、腺苷5、尿苷5、鸟苷5;
微量元素类:所述微量元素类为硒0.1、铬0.1、钴0.05、镍0.05、锌0.1、铜0.1、锰0.1、钡0.1、镓0.05、锂0.1、锡0.1、碘0.1、钒0.1、锗0.05、钼0.05、硅0.1、铁0.1、铷0.05、锆0.05、镉0.1、铝0.1;
维生素类:所述维生素类为生物素0.05、D-泛酸钙0.1、叶酸0.05、烟酰胺0.05、盐酸吡哆醇0.05、核黄素0.1、盐酸硫胺素0.05、维生素B12 0.1、维生素B2 0.1、肌醇0.1;
盐类:所述盐类为氯化钠500、氯化钾500、硫酸铜55、硝酸铁55、硫酸亚铁55、氯化镁55、无水磷酸氢二钠250、磷酸二氢钠250、亚硒酸钠250、硫酸锌55;
病毒增殖促进剂:所述病毒增殖促进剂为胆固醇5、卵磷脂0.05、氯化钙5、亚油酸0.01、亚麻酸0.01、月桂酸0.1、棕榈酸0.1、维生素E 0.1、前列腺素55、吐温-80 5;
添加物:所述添加物为葡萄糖8000、转铁蛋白55、胰岛素55、牛血清白蛋白1000、大豆水解物1000;
酸碱度缓冲剂:所述酸碱度缓冲剂为碳酸氢钠1000;
酸碱度指示剂:所述酸碱度指示剂为酚红20;
剪切力保护剂:所述剪切力保护剂为嵌段式聚醚F68 150;
激素:所述激素为地塞米松0.02;
抗生素:所述抗生素为两性霉素B 500。
根据以下步骤制备适于禽流感病毒高效扩增的维持培养基:
1)制备混合液:将原料干粉混合后磨粉过150目筛,然后将所得细粉于30℃无热源去离子水中搅拌溶解,得到混合液;
2)调节pH:加入氨水调节混合液的pH至6.7;
3)定容:去离子水定容得到适于禽流感病毒高效扩增的维持培养基;
4)过滤出菌:采用0.22μm滤膜过滤除菌。
试验例
1、仪器 倒置显微镜(Motic)、生物安全柜(Heal Force)、CO2培养箱(HealForce)、4℃离心机(eppendrof)。
2、培养基 实验组采用本发明所述适于禽流感病毒高效扩增的维持培养基,对照组采用XenoTM-S001S MDCK培养基。
3、细胞株与病毒株 犬肾细胞(Madin-Darby canine kidney,MDCK),甲型流感病毒株H9N2。
4、试验方法 所述MDCK细胞培养的条件是本领域中常规的使用条件,生物反应器的参数设置参考生物反应器厂家的使用说明书设置。优选的培养条件:接种密度为5×105cells/ml,37℃,溶氧D0为50%,按70%,20%,5%,5%的比例通入压缩空气,氧气,二氧化碳和氮气,通气量为1.0,转速为110 rpm。记录MDCK细胞在本发明适于禽流感病毒高效扩增的维持培养基中的形态图,见图1。每24h取样进行活细胞计数,并计算细胞生长速率,结果如图2所示。
当MDCK细胞生长到10-12×106cells/mL时,接种甲型流感病毒株H9N2病毒液,对照组采用XenoTM-S001S MDCK培养基,实验组使用本发明实施例1-3所述适于禽流感病毒高效扩增的维持培养基维持病毒增殖。对使用本发明所述适于禽流感病毒高效扩增的维持培养基与对照组XenoTM-S001S MDCK培养基的病毒滴度对比如表1:
表1禽流感病毒滴度对比表 (Log(1/TCID50)/100μl)
时间(h) | 实施例1 | 实施例2 | 实施例3 | 对照组 |
24 | 5.2 | 5.4 | 5.8 | 4.5 |
48 | 6.4 | 6.6 | 6.8 | 5.2 |
72 | 7.5 | 7.8 | 8.2 | 6.0 |
96 | 8.6 | 8.6 | 8.9 | 6.9 |
120 | 7.3 | 7.6 | 8.5 | 5.2 |
5、试验结果 采用本发明提供的适于禽流感病毒高效扩增的维持培养基,在培养过程中MDCK细胞呈单个均匀分散状,悬浮生长,无结团现象,细胞形态饱满完整,轮廓清晰,细胞大小均一,活细胞比例增长幅度较大。相比于对照组培养基,本发明提供的适于禽流感病毒高效扩增的维持培养基促进病毒产量提高,这是由于本发明保证了合理的氨基酸浓度,达到了有效促进流感病毒在细胞内扩增的目的。
以上所述实施例仅表达了本发明的实施方式,其描述较为具体和详细,但并不能因此而理解为对本发明专利范围的限制,但凡采用等同替换或等效变换的形式所获得的技术方案,均应落在本发明的保护范围之内。
Claims (7)
1.一种适于禽流感病毒高效扩增的维持培养基,其特征在于,各组分的浓度以mg/L计为:
基础代谢营养物类:所述基础代谢营养物类为丝氨酸20-250、精氨酸20-250、亮氨酸20-55、异亮氨酸20-250、丙氨酸1-2、酪氨酸20-55、天冬酰胺2-25、天冬氨酸2-25、盐酸半胱氨酸5-55、谷氨酸2-50、谷氨酰胺2-50、组氨酸20-250、赖氨酸20-250、苯丙氨酸10-55、苏氨酸5-55、色氨酸20-250、缬氨酸20-250;
核苷酸类:所述核苷酸类为次黄嘌呤2-20、胸苷2-5、腺苷2-5、尿苷2-5、鸟苷2-5;
微量元素类:所述微量元素类为硒0.05-0.1、铬0.05-0.1、钴0.02-0.05、镍0.02-0.05、锌0.05-0.1、铜0.05-0.1、锰0.05-0.1、钡0.05-0.1、镓0.02-0.05、锂0.05-0.1、锡0.05-0.1、碘0.05-0.1、钒0.05-0.1、锗0.02-0.05、钼0.02-0.05、硅0.05-0.1、铁0.05-0.1、铷0.02-0.05、锆0.02-0.05、镉0.05-0.1、铝0.05-0.1;
维生素类:所述维生素类为生物素0.02-0.05、D-泛酸钙0.05-0.1、叶酸0.02-0.05、烟酰胺0.02-0.05、盐酸吡哆醇0.02-0.05、核黄素0.05-0.1、盐酸硫胺素0.02-0.05、维生素B12 0.05-0.1、维生素B2 0.05-0.1、肌醇0.05-0.1;
盐类:所述盐类为氯化钠200-500、氯化钾200-500、硫酸铜2-55、硝酸铁2-55、硫酸亚铁2-55、氯化镁2-55、无水磷酸氢二钠20-250、磷酸二氢钠20-250、亚硒酸钠20-250、硫酸锌2-55;
病毒增殖促进剂:所述病毒增殖促进剂为胆固醇1-5、卵磷脂0.02-0.05、氯化钙1-5、亚油酸0.05-0.01、亚麻酸0.05-0.01、月桂酸0.05-0.1、棕榈酸0.05-0.1、维生素E 0.05-0.1、前列腺素20-55、吐温-80 1-5;
添加物:所述添加物为葡萄糖1000-8000、转铁蛋白2-55、胰岛素2-55、牛血清白蛋白500-1000、大豆水解物500-1000;
酸碱度缓冲剂:所述酸碱度缓冲剂为碳酸氢钠10-1000;
酸碱度指示剂:所述酸碱度指示剂为酚红1-20;剪切力保护剂嵌段式聚醚F68 20-150mg/L;激素地塞米松0.001-0.02mg/L;抗生素两性霉素B 200-500mg/L。
2.如权利要求1所述适于禽流感病毒高效扩增的维持培养基,其特征在于:各组分的浓度以mg/L计为:
基础代谢营养物类:所述基础代谢营养物类为丝氨酸250、精氨酸250、亮氨酸55、异亮氨酸250、丙氨酸2、酪氨酸55、天冬酰胺25、天冬氨酸25、盐酸半胱氨酸55、谷氨酸50、谷氨酰胺50、组氨酸250、赖氨酸250、苯丙氨酸55、苏氨酸55、色氨酸250、缬氨酸250;
核苷酸类:所述核苷酸类为次黄嘌呤20、胸苷5、腺苷5、尿苷5、鸟苷5;
微量元素类:所述微量元素类为硒0.1、铬0.1、钴0.05、镍0.05、锌0.1、铜0.1、锰0.1、钡0.1、镓0.05、锂0.1、锡0.1、碘0.1、钒0.1、锗0.05、钼0.05、硅0.1、铁0.1、铷0.05、锆0.05、镉0.1、铝0.1;
维生素类:所述维生素类为生物素0.05、D-泛酸钙0.1、叶酸0.05、烟酰胺0.05、盐酸吡哆醇0.05、核黄素0.1、盐酸硫胺素0.05、维生素B12 0.1、维生素B2 0.1、肌醇0.1;
盐类:所述盐类为氯化钠500、氯化钾500、硫酸铜55、硝酸铁55、硫酸亚铁55、氯化镁55、无水磷酸氢二钠250、磷酸二氢钠250、亚硒酸钠250、硫酸锌55;
病毒增殖促进剂:所述病毒增殖促进剂为胆固醇5、卵磷脂0.05、氯化钙5、亚油酸0.01、亚麻酸0.01、月桂酸0.1、棕榈酸0.1、维生素E 0.1、前列腺素55、吐温-80 5;
添加物:所述添加物为葡萄糖8000、转铁蛋白55、胰岛素55、牛血清白蛋白1000、大豆水解物1000;
酸碱度缓冲剂:所述酸碱度缓冲剂为碳酸氢钠1000;
酸碱度指示剂:所述酸碱度指示剂为酚红20;
剪切力保护剂:所述剪切力保护剂为嵌段式聚醚F68 150;
激素:所述激素为地塞米松0.02;
抗生素:所述抗生素为两性霉素B 500。
3.如权利要求1-2任一所述适于禽流感病毒高效扩增的维持培养基的制备方法,其特征在于,包括以下步骤:
1)制备混合液:将原料干粉用溶剂溶解并混合;
2)调节pH:调节混合液的pH;
3)定容:定容得到适于禽流感病毒高效扩增的维持培养基;
4)过滤除菌。
4.如权利要求3所述的制备方法,其特征在于:所述原料干粉溶解之前需研磨过筛,所述溶剂选自蒸馏水、去离子水、低级醇中的一种或几种;所述调节pH的溶剂选自氢氧化钠、氨水、氢氧化钾中的一种或几种;所述定容溶剂选自蒸馏水、去离子水中的一种或几种。
5.如权利要求4所述的制备方法,其特征在于:所述过筛目数为50-200目,所述调节混合液的pH至6.0-7.0。
6.如权利要求5所述的制备方法,其特征在于:所述过筛目数为150目,所述调节混合液的pH至6.7。
7.如权利要求3所述的制备方法,其特征在于:所述过滤除菌为采用0.22μm滤膜过滤除菌。
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