CN111714626A - 一种采用mdck细胞系生产禽流感疫苗的方法及其制品 - Google Patents
一种采用mdck细胞系生产禽流感疫苗的方法及其制品 Download PDFInfo
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Classifications
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0684—Cells of the urinary tract or kidneys
- C12N5/0686—Kidney cells
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- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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Abstract
本发明涉及一种采用MDCK细胞系生产禽流感疫苗的方法及其制品,所述方法包括将贴壁的MDCK细胞驯化为稳定增殖的无血清全悬浮培养型MDCK细胞系,然后接毒增殖,分离收集病毒液,灭活,添加佐剂配制成疫苗。本发明用细胞系代替鸡胚组织培养制造禽流感病毒以及禽流感疫苗,可解决鸡胚自身及受外源病毒污染的问题,通过原材料和培养条件的严格控制,保证生产出来的疫苗纯粹,确保了疫苗的安全性,可在安全、经济的条件下应用于大规模生产,获得高纯度、工业产率理想的有效禽流感疫苗。
Description
技术领域
本发明涉及疫苗制备技术领域,具体涉及一种采用MDCK细胞系生产禽流感疫苗的方法及其制品。
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,严重威胁养殖业和人类健康,并给国民经济带来巨大损失。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有效手段。
用于禽流感疫苗制备的传统鸡胚工艺存在诸多问题,生产周期长、操作繁琐、工作量大,收获病毒液后,还需对剩余鸡胚进行无害化处理,易于感染禽源外源病毒,产生生物安全隐患。如遇禽流感大规模爆发,鸡胚的供应还会存在很大问题。以细胞作为介质是目前疫苗制备的趋势,犬肾(Madin-Darby canine kidney cells,MDCK)细胞由Madin和Darby分离培育建立,培养容易、增殖快、流感病毒感染效率高,是国内外公认的最适合禽流感病毒培养的细胞系。其通常是以贴壁培养生长,细胞之间互相衔接或呈镶嵌状排列,并会在表面集合形成紧密的连接蛋白,从而形成单层贴壁细胞。但是,该培养方式存在一些缺陷,比如贴壁培养受基质表面积限制导致病毒产量下降;贴壁培养过程中换液等程序较为复杂繁琐,血清的使用易受微生物、病毒和支原体等的污染,无血清培养技术是阐明细胞生成、增殖、分化以及基因表达调控的基础问题的有效工具,但即使无血清贴壁培养也只适用于试验研究,不适用于大规模培养,不能满足病毒疫苗等实际生产需要。
因此,如何将MDCK细胞系作为种细胞高效培养禽流感病毒,进而获得禽流感疫苗,是一项值得技术人员深入研究和解决的技术问题。同时,疫苗质量取决于病毒抗原含量,细胞的高感染率是实现病毒增殖的前提。因此,寻找一种细胞培养方法能够生产出高滴度的病毒,满足免疫生产要求,在发生疫情时能够快速提供疫苗,显得迫在眉睫。
发明内容
本发明的目的在于克服现有技术存在的不足之处,提供一种应用生物反应器培养悬浮细胞生产禽流感病毒以及制备禽流感疫苗的方法。该方法用来生产的禽流感病毒不仅产毒高效稳定,病毒血细胞凝集效价高,而且可以大量降低生产成本,不会受制于原料供应,占用场地小,自动化程度高,易于快速扩大生产规模,显著提高疫苗产量和质量。
为达到上述目的,本发明采取了如下的技术方案:一种采用MDCK细胞系生产禽流感疫苗的方法,包括如下步骤:
(1)细胞培养:复苏培养贴壁的MDCK细胞,待细胞生长至致密单层后,消化传代;逐步减少FBS比例直至0%;摇床扩大培养,直至驯化为稳定增殖的无血清全悬浮培养型MDCK细胞系;接种于生物反应器,完成初级细胞培养;
(2)病毒接种:将鸡胚繁殖的禽流感病毒液接种至致密单层的贴壁MDCK细胞,获得悬浮MDCK细胞培养用种毒;取上述禽流感病毒种毒接种于悬浮MDCK细胞,进行培养;
(3)收集病毒液:分离收获病毒液;
(4)病毒灭活:采用化学试剂灭活病毒;
(5)纯化病毒液:采用离心或澄清膜处理病毒液,去除杂质碎片;进一步采用柱层析法纯化病毒液;
(6)疫苗配制:添加佐剂将病毒液配制成疫苗形式。
进一步的,所述细胞种毒的病原类型为禽流感H5N1亚型或H9N1亚型。
优选的,一种采用MDCK细胞系生产禽流感疫苗的方法,包括如下步骤:
(1)细胞培养:将DMEM、F12和RPMI1640按照一定比例进行混合均匀,以此为基础培养基并添加10%FBS,复苏培养贴壁的MDCK细胞,待细胞生长至致密单层后,0.25%EDTA-胰酶消化,传代5次;逐步减少含10%FBS的DMEM-F12-RPMI1640培养基比例、增加无血清的DMEM-F12-RPMI1640培养基比例,使血清含量逐渐从10%减至0%;
将生长良好的已适应无血清培养的MDCK细胞经0.25%EDTA-胰酶消化后,进行摇床培养,转速40r/min;待细胞生长速率稳定后传代,逐步扩大培养,并逐步提高摇床转速,直至驯化为稳定增殖的无血清全悬浮培养型MDCK细胞系;
将上述驯化完成的MDCK细胞系接种于5L生物反应器,接种密度为2×106cells/ml,培养条件为温度37℃,pH值7.2,溶氧40%,110r/min;扩增至1×107cells/ml时完成初级细胞培养;
(2)病毒接种:将鸡胚繁殖的禽流感H5N1亚型病毒液接种至致密单层的贴壁MDCK细胞,以病毒维持液进行培养;传代5次;待细胞病变达90%时收集培养上清液,以3500r/min离心15min,沉淀弃去,取上清液作为悬浮MDCK细胞培养用种毒;
取上述适量贴壁培养MDCK细胞增殖的H5N1亚型禽流感病毒,按照MOI为10-3-10-1接种于5.0×106cells/mL悬浮MDCK细胞,培养条件为34℃,pH7.3,溶氧40%,搅拌转速110r/min;
(3)收集病毒液:接毒后培养48-72h,当细胞活力降至40-60%时分离上清液收获病毒液;收集病毒液后,还可连续多次添加新鲜培养基继续培养,重复多次收获病毒液;
(4)病毒灭活:采用化学试剂如β丙内酯、烷化剂、苯酚、甲醛等灭活病毒,使病毒失去感染力,但保持病毒的抗原性和免疫原性。所述甲醛浓度为1/1500-1/6500,37℃下灭活16-48h,室温下灭活6-15天;多次收获的病毒液可合并后进行灭活,灭活前后采样进行无菌测定,灭活时还可加入防腐剂α-生育酚琥珀酸盐或汞化合物;
(5)纯化病毒液:采用离心或澄清膜处理病毒液,去除杂质碎片,如4℃,8000-10000r/min,离心10min,所述澄清膜选择0.4μm膜;进一步采用柱层析法纯化病毒液,具体使用凝胶过滤层析法有效去除病毒液中残留的小分子杂质;
(6)疫苗配制:添加佐剂将病毒液配制成疫苗形式,所述佐剂为矿物质类或双相乳剂。
进一步的,所述步骤(1)中生物反应器为能够自动控制温度、PH、溶氧、搅拌速度等参数,适用于悬浮培养的生物反应器。
进一步的,所述步骤(2)中病毒维持液的组成为,各组分的浓度以mg/L计: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;次黄嘌呤20、胸苷5、腺苷5、尿苷5、鸟苷5;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;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;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;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;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;碳酸氢钠1000;酚红20;PluronicF68150;地塞米松0.02;两性霉素B 500。
进一步的,所述步骤(3)中收获病毒液次数可为2-8次。
进一步的,所述步骤(4)中病毒灭活采用浓度为1/3000的甲醛,37℃下灭活24h;防腐剂为1/15000硫柳汞。
进一步的,所述步骤(5)中离心后的病毒液先进行超滤膜浓缩,再进行柱层析,其中超滤膜截留分子量为100kda,凝胶过滤的温度为4-16℃,洗脱速度为5-220ml/min;
进一步的,所述步骤(6)中矿物质类佐剂为Al(OH)3;所述双相乳剂为白油乳剂。
进一步的,所述步骤(6)中疫苗配制方法为,油相:取94%白油、6%Span-80、1-2%硬脂酸铝,混合搅拌均匀,灭菌备用;水相:取所述灭活后的病毒液加2-4%Span-80,摇匀备用;将上述制备得到的油相和水相按照3:1-2:1比例进行配制,缓慢搅拌混合,再通过胶体磨充分乳化,得粘稠的W/O乳剂疫苗,备用;再加2%吐温-80生理盐水,通过胶体磨充分乳化,即制成禽流感双相乳剂疫苗。
优选的,一种采用MDCK细胞系生产禽流感疫苗的方法,包括如下步骤:
(1)细胞培养:将DMEM、F12和RPMI1640按照3∶1∶1的比例进行混合均匀,以此为基础培养基并添加10%FBS,复苏培养贴壁的MDCK细胞,待细胞生长至致密单层后,0.25%EDTA-胰酶消化,传代5次;逐步减少含10%FBS的DMEM-F12-RPMI1640培养基比例、增加无血清的DMEM-F12-RPMI1640培养基比例,使血清含量逐渐从10%减至0%;
将生长良好的已适应无血清培养的MDCK细胞经0.25%EDTA-胰酶消化后,进行摇床培养,转速40r/min;待细胞生长速率稳定后传代,逐步扩大培养,并逐步提高摇床转速,直至驯化为稳定增殖的无血清全悬浮培养型MDCK细胞系;
将上述驯化完成的MDCK细胞系接种于5L生物反应器,接种密度为2×106cells/ml,培养条件为温度37℃,pH值7.2,溶氧40%,110r/min;扩增至1×107cells/ml时完成初级细胞培养;
(2)病毒接种:将鸡胚繁殖的禽流感H5N1亚型病毒液接种至致密单层的贴壁MDCK细胞,以病毒维持液进行培养;传代5次;待细胞病变达90%时收集培养上清液,以3500r/min离心15min,沉淀弃去,取上清液作为悬浮MDCK细胞培养用种毒;
取上述适量贴壁培养MDCK细胞增殖的H5N1亚型禽流感病毒,按照MOI为10-2接种于5.0×106cells/mL悬浮MDCK细胞,培养条件为34℃,pH7.3,溶氧40%,搅拌转速110r/min;
(3)收集病毒液:接毒后培养60h,当细胞活力降至50%时分离上清液收获病毒液;收集病毒液后,连续添加5次新鲜培养基继续培养,重复收获病毒液;
(4)病毒灭活:将5次收获的病毒液进行合并,添加浓度为1/3000的甲醛和1/15000的硫柳汞灭活病毒,37℃下灭活24h,使病毒失去感染力,但保持病毒的抗原性和免疫原性。灭活前后采样进行无菌测定,灭活后所得病毒液即为疫苗原液;
(5)纯化疫苗原液:采用4℃,8000-10000r/min,离心10min处理病毒液,去除杂质碎片;进一步先进行截留分子量为100kda的超滤膜浓缩,并用pH7.6-7.8的PBS缓冲液平衡,浓缩20倍,收集浓缩液,再进行凝胶过滤柱层析,过滤温度为4-16℃,洗脱速度为5-220ml/min,去除病毒液中残留的小分子杂质;
(6)疫苗配制:油相:取94%白油、6%Span-80、2%硬脂酸铝,混合搅拌均匀,灭菌备用;水相:取所述灭活后的病毒液加2%Span-80,摇匀备用;将上述制备得到的油相和水相按照2:1比例进行配制,缓慢搅拌混合,再通过胶体磨充分乳化,得粘稠的W/O乳剂疫苗,备用;再加2%吐温-80生理盐水,通过胶体磨充分乳化,即制成禽流感双相乳剂疫苗。
进一步的,所述疫苗还可包含细胞因子诱导剂白介素、干扰素、肿瘤坏死因子、淋巴毒素、集落刺激因子等,优选IL-1、IL-2、INF-γ、TNF-α、GM-CSF中的一种或多种。
进一步的,疫苗配制结束后进行质量检定,包括无菌实验、热源质实验、外观检查、毒性试验、效力试验、甲醛含量测定、硫柳汞含量测定、pH值测定等,检定合格后分装,即为成品。
本发明的另一个目的在于提供根据本发明所述的MDCK细胞系生产禽流感疫苗的方法制得的疫苗制品。所述疫苗产品可以为单价苗、二价苗或三价苗,有助于扩大疫苗的免疫适用范围,加强免疫效果,实用性较强。
与现有技术比较,本发明采用MDCK细胞系生产禽流感疫苗的方法具有以下显著的有益效果:本发明用细胞系代替鸡胚组织培养制造禽流感病毒以及禽流感疫苗,可解决鸡胚自身及受外源病毒污染的问题,通过原材料和培养条件的严格控制,保证生产出来的疫苗纯粹,确保了疫苗的安全性。而且,在制备过程中将MDCK细胞驯化培养和病毒增殖过程关联起来,细胞传代稳定,形态好、质量高,可用于高效生产禽流感病毒,在高效、安全、减少污染几率的情况下,完成灭活病毒、纯化疫苗原液、配制疫苗的全过程,便于标准化生产,提高了生产安全系数,适用于大规模高效生产禽流感疫苗。
具体实施方式
下面结合实施例对本发明的实施方案进行详细描述,但是本领域技术人员将会理解,下列实施例仅用于说明本发明,而不应视为限制本发明的范围。
实施例1
一种采用MDCK细胞系生产禽流感疫苗的方法,包括如下步骤:
(1)细胞培养:将DMEM、F12和RPMI1640按照3∶1∶1的比例进行混合均匀,以此为基础培养基并添加10%FBS,复苏培养贴壁的MDCK细胞,待细胞生长至致密单层后,0.25%EDTA-胰酶消化,传代5次;逐步减少含10%FBS的DMEM-F12-RPMI1640培养基比例、增加无血清的DMEM-F12-RPMI1640培养基比例,使血清含量逐渐从10%减至0%;
将生长良好的已适应无血清培养的MDCK细胞经0.25%EDTA-胰酶消化后,进行摇床培养,转速40r/min;待细胞生长速率稳定后传代,逐步扩大培养,并逐步提高摇床转速,直至驯化为稳定增殖的无血清全悬浮培养型MDCK细胞系;
将上述驯化完成的MDCK细胞系接种于5L生物反应器,接种密度为2×106cells/ml,培养条件为温度37℃,pH值7.2,溶氧40%,110r/min;扩增至1×107cells/ml时完成初级细胞培养;
(2)病毒接种:将鸡胚繁殖的禽流感H5N1亚型病毒液接种至致密单层的贴壁MDCK细胞,以病毒维持液进行培养;传代5次;待细胞病变达90%时收集培养上清液,以3500r/min离心15min,沉淀弃去,取上清液作为悬浮MDCK细胞培养用种毒;
取上述适量贴壁培养MDCK细胞增殖的H5N1亚型禽流感病毒,按照MOI为10-2接种于5.0×106cells/mL悬浮MDCK细胞,培养条件为34℃,pH7.3,溶氧40%,搅拌转速110r/min;
(3)收集病毒液:接毒后培养60h,当细胞活力降至50%时分离上清液收获病毒液;收集病毒液后,连续添加5次新鲜培养基继续培养,重复收获病毒液;
(4)病毒灭活:将5次收获的病毒液进行合并,添加浓度为1/3000的化学试剂甲醛和1/15000的硫柳汞灭活病毒,37℃下灭活24h,使病毒失去感染力,但保持病毒的抗原性和免疫原性。灭活前后采样进行无菌测定,灭活后进行灭活测定实验;灭活后所得病毒液即为疫苗原液;
(5)纯化疫苗原液:采用4℃,10000r/min,离心10min处理病毒液,去除杂质碎片;进一步先进行截留分子量为100kda的超滤膜浓缩,并用pH7.6的PBS缓冲液平衡,浓缩20倍,收集浓缩液,再进行凝胶过滤柱层析,过滤温度为8℃,洗脱速度为110ml/min,去除病毒液中残留的小分子杂质;
(6)疫苗配制:添加0.5mg/ml的Al(OH)3佐剂将病毒液配制成疫苗。
实施例2
一种采用MDCK细胞系生产禽流感疫苗的方法,包括如下步骤:
(1)细胞培养:将DMEM、F12和RPMI1640按照3∶1∶1的比例进行混合均匀,以此为基础培养基并添加10%FBS,复苏培养贴壁的MDCK细胞,待细胞生长至致密单层后,0.25%EDTA-胰酶消化,传代5次;逐步减少含10%FBS的DMEM-F12-RPMI1640培养基比例、增加无血清的DMEM-F12-RPMI1640培养基比例,使血清含量逐渐从10%减至0%;
将生长良好的已适应无血清培养的MDCK细胞经0.25%EDTA-胰酶消化后,进行摇床培养,转速40r/min;待细胞生长速率稳定后传代,逐步扩大培养,并逐步提高摇床转速,直至驯化为稳定增殖的无血清全悬浮培养型MDCK细胞系;
将上述驯化完成的MDCK细胞系接种于5L生物反应器,接种密度为2×106cells/ml,培养条件为温度37℃,pH值7.2,溶氧40%,110r/min;扩增至1×107cells/ml时完成初级细胞培养;
(2)病毒接种:将鸡胚繁殖的禽流感H5N1亚型病毒液接种至致密单层的贴壁MDCK细胞,以病毒维持液进行培养;传代5次;待细胞病变达90%时收集培养上清液,以3500r/min离心15min,沉淀弃去,取上清液作为悬浮MDCK细胞培养用种毒;
取上述适量贴壁培养MDCK细胞增殖的H5N1亚型禽流感病毒,按照MOI为10-2接种于5.0×106cells/mL悬浮MDCK细胞,培养条件为34℃,pH7.3,溶氧40%,搅拌转速110r/min;
(3)收集病毒液:接毒后培养60h,当细胞活力降至50%时分离上清液收获病毒液;收集病毒液后,连续添加5次新鲜培养基继续培养,重复收获病毒液;
(4)病毒灭活:将5次收获的病毒液进行合并,添加浓度为1/3000的甲醛和1/15000的硫柳汞灭活病毒,37℃下灭活24h,使病毒失去感染力,但保持病毒的抗原性和免疫原性。灭活前后采样进行无菌测定,灭活后所得病毒液即为疫苗原液;
(5)纯化疫苗原液:采用4℃,8000r/min,离心10min处理病毒液,去除杂质碎片;进一步先进行截留分子量为100kda的超滤膜浓缩,并用pH7.7的PBS缓冲液平衡,浓缩20倍,收集浓缩液,再进行凝胶过滤柱层析,过滤温度为4℃,洗脱速度为100ml/min,去除病毒液中残留的小分子杂质;
(6)疫苗配制:油相:取94%白油、6%Span-80、2%硬脂酸铝,混合搅拌均匀,灭菌备用;水相:取所述灭活后的病毒液加2%Span-80,摇匀备用;将上述制备得到的油相和水相按照2:1比例进行配制,缓慢搅拌混合,再通过胶体磨充分乳化,得粘稠的W/O乳剂疫苗,备用;再加2%吐温-80生理盐水,通过胶体磨充分乳化,即制成禽流感双相乳剂疫苗。
实施例3
本实施例说明实施例2制得的疫苗产品的检验测定及效果实验。
1、半成品检验:
无菌检验:按《中华人民共和国兽用生物制品质量标准》附录301页进行,无菌生长。
灭活检验:灭活后,无菌条件下从病毒灭活液中取样,经尿囊腔途径接种10日龄SPF鸡胚10枚,0.1ml/胚,37℃孵化,每天照蛋观察,120小时内应无死亡,若有死亡,尿囊液应无血凝价。
2、成品检验
性状:外观为白色乳状液,剂型呈油包水包油型。
稳定性:37℃保存21d或以3000r/min离心15分钟,未破乳,管底无水相析出。
无菌检验:按《中华人民共和国兽用生物制品质量标准》附录301页进行。
甲醛含量测定:按《中华人民共和国兽用生物制品质量标准》附录311页进行。
安全检验:用3-5周龄非免疫鸡各10只,皮下、肌肉注射疫苗0.6ml,观察10日,无因疫苗注射引起的全身反应和严重的局部反应。
表1采用MDCK细胞系生产得到的禽流感疫苗检定结果
疫苗批次 | 无菌检验 | 外源因子检查 | 甲醛含量 | 安全检验 | 抗原含量 |
201901 | 合格 | 合格 | <0.02% | 全部存活 | 1:64 |
201902 | 合格 | 合格 | <0.02% | 全部存活 | 1:64 |
201903 | 合格 | 合格 | <0.02% | 全部存活 | 1:64 |
201904 | 合格 | 合格 | <0.02% | 全部存活 | 1:64 |
3、免疫评价
采用贴壁MDCK细胞按普通培养工艺制备禽流感H5N1亚型病毒苗,采用传统鸡胚工艺制备禽流感H5N1亚型病毒苗,将上述贴壁细胞源、鸡胚源和本发明实施例2的悬浮细胞源禽流感病毒液分别制得的灭活疫苗进行分析比较。各组免疫10日龄SPF鸡各10只,每只皮下注射0.2mL;另取10只注射PBS做空白对照。免疫后21d和28d,于鸡翼下静脉采血,以3000r/min离心15min,取上清液分离血清,进行抗体HI效价检测,评价疫苗的免疫效果。由表2可知,细胞毒疫苗免疫效果相比于鸡胚毒免疫效果维持较高抗体水平,本发明悬浮细胞源禽流感病毒液制得的灭活疫苗免疫效果显著,符合兽医药典规定,能够适应规模化、工业化生产优质禽流感灭活疫苗的需求。
表2禽流感细胞毒灭活疫苗抗体检测
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 (10)
1.一种采用MDCK细胞系生产禽流感疫苗的方法,其特征在于,包括如下步骤:
(1)细胞培养:复苏培养贴壁的MDCK细胞,待细胞生长至致密单层后,消化传代;逐步减少FBS比例直至0%;摇床扩大培养,直至驯化为稳定增殖的无血清全悬浮培养型MDCK细胞系;接种于生物反应器,完成初级细胞培养;
(2)病毒接种:将鸡胚繁殖的禽流感病毒液接种至致密单层的贴壁MDCK细胞,获得悬浮MDCK细胞培养用种毒;取上述禽流感病毒种毒接种于悬浮MDCK细胞,进行培养;
(3)收集病毒液:分离收获病毒液;
(4)病毒灭活:采用化学试剂灭活病毒;
(5)纯化病毒液:采用离心或澄清膜处理病毒液,去除杂质碎片;
(6)疫苗配制:添加佐剂将病毒液配制成疫苗形式。
2.根据权利要求1所述一种采用MDCK细胞系生产禽流感疫苗的方法,其特征在于,所述步骤(5)还包括采用柱层析法纯化病毒液。
3.根据权利要求2所述一种采用MDCK细胞系生产禽流感疫苗的方法,其特征在于,具体包括如下步骤:
(1)细胞培养:将DMEM、F12和RPMI1640按照3∶1∶1的比例进行混合均匀,以此为基础培养基并添加10%FBS,复苏培养贴壁的MDCK细胞,待细胞生长至致密单层后,0.25%EDTA-胰酶消化,传代5次;逐步减少含10%FBS的DMEM-F12-RPMI1640培养基比例、增加无血清的DMEM-F12-RPMI1640培养基比例,使血清含量逐渐从10%减至0%;
将生长良好的已适应无血清培养的MDCK细胞经0.25%EDTA-胰酶消化后,进行摇床培养,转速40r/min;待细胞生长速率稳定后传代,逐步扩大培养,并逐步提高摇床转速,直至驯化为稳定增殖的无血清全悬浮培养型MDCK细胞系;
将上述驯化完成的MDCK细胞系接种于5L生物反应器,接种密度为2×106cells/ml,培养条件为温度37℃,pH值7.2,溶氧40%,110r/min;扩增至1×107cells/ml时完成初级细胞培养;
(2)病毒接种:将鸡胚繁殖的禽流感H5N1亚型病毒液接种至致密单层的贴壁MDCK 细胞,以病毒维持液进行培养;传代5次;待细胞病变达90%时收集培养上清液,以3500r/min离心15min,沉淀弃去,取上清液作为悬浮MDCK细胞培养用种毒;
取上述适量贴壁培养MDCK细胞增殖的H5N1亚型禽流感病毒,按照MOI为10-3-10-1接种于5.0×106cells/mL悬浮MDCK细胞,培养条件为34℃,pH7.3,溶氧40%,搅拌转速110r/min;
(3)收集病毒液:接毒后培养48-72h,当细胞活力降至40-60%时分离上清液收获病毒液;收集病毒液后,还可连续多次添加新鲜培养基继续培养,重复多次收获病毒液;
(4)病毒灭活:采用化学试剂如β丙内酯、烷化剂、苯酚、甲醛等灭活病毒,使病毒失去感染力,但保持病毒的抗原性和免疫原性。所述甲醛浓度为1/1500-1/6500,37℃下灭活16-48h,室温下灭活6-15天;多次收获的病毒液可合并后进行灭活,灭活前后采样进行无菌测定,灭活时还可加入防腐剂α-生育酚琥珀酸盐或汞化合物;
(5)纯化病毒液:采用离心或澄清膜处理病毒液,去除杂质碎片,如4℃,8000-10000r/min,离心10min,所述澄清膜选择0.4μm膜;进一步采用柱层析法纯化病毒液,具体使用凝胶过滤层析法有效去除病毒液中残留的小分子杂质;
(6)疫苗配制:添加佐剂将病毒液配制成疫苗形式,所述佐剂为矿物质类或双相乳剂。
4.根据权利要求3所述一种采用MDCK细胞系生产禽流感疫苗的方法,其特征在于,步骤(2)所述接毒量MOI为10-2,步骤(3)中所述接毒后培养时间为60h。
5.根据权利要求3所述一种采用MDCK细胞系生产禽流感疫苗的方法,其特征在于,所述步骤(3)中收获病毒液次数可为2-8次。
6.根据权利要求3所述一种采用MDCK细胞系生产禽流感疫苗的方法,其特征在于,所述步骤(4)中病毒灭活采用浓度为1/3000的甲醛,37℃下灭活24h;所述防腐剂为1/15000硫柳汞。
7.根据权利要求3所述一种采用MDCK细胞系生产禽流感疫苗的方法,其特征在于,所述步骤(5)中离心后的病毒液先进行超滤膜浓缩,再进行柱层析,其中所述超滤膜截留分子量为100kda,凝胶过滤的温度为4-16℃,洗脱速度为5-220ml/min。
8.根据权利要求3所述一种采用MDCK细胞系生产禽流感疫苗的方法,其特征在于,所述步骤(6)中矿物质类佐剂为Al(OH)3;所述双相乳剂为白油乳剂。
9.根据权利要求1-8任一项所述一种采用MDCK细胞系生产禽流感疫苗的方法,其特征在于,所述步骤(6)中疫苗配制方法为,取94%白油、6%Span-80、2%硬脂酸铝,混合搅拌均匀,灭菌备用;水相:取所述灭活后的病毒液加2%Span-80,摇匀备用;将上述制备得到的油相和水相按照2:1比例进行配制,缓慢搅拌混合,再通过胶体磨充分乳化,得粘稠的W/O乳剂疫苗,备用;再加2%吐温-80生理盐水,通过胶体磨充分乳化,即制成禽流感双相乳剂疫苗。
10.根据权利要求1-9任一项所述的方法制备得到的疫苗制品;所述疫苗可以为单价苗、二价苗或三价苗。
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CN115537379A (zh) * | 2022-12-01 | 2022-12-30 | 天信和(苏州)生物科技有限公司 | 一种用于培养mdck细胞及扩增病毒的组合培养基及其应用 |
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