CN115058354A - 一种化学成分明确的猪肺炎支原体培养基及其制备方法 - Google Patents
一种化学成分明确的猪肺炎支原体培养基及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种化学成分明确的猪肺炎支原体培养基及其制备方法,用于为制备猪支原体肺炎灭活疫苗提供支原体,属于兽医微生物学技术领域,本发明的培养基含氨基酸、胆固醇、亚油酸、亚麻酸、葡萄糖酸锌、牛磺酸、牛血清白蛋白、肌醇、丙酮酸钠、谷氨酰胺、吡啶甲酸铬、维生素B12、叶酸、D泛酸钙、果糖、葡萄糖、磷脂和谷氨酰胺等物质,具有成分明确、活菌滴度高、血清用量低、培养周期短、批间差小、效果稳定的特点;本发明提供的制备方法,成分配置完成后溶解定容,调节pH值后除菌接种即可,具有简单高效的特点。
Description
技术领域
本发明属于兽医微生物学技术领域,用于为制备猪支原体肺炎灭活疫苗提供支原体,具体涉及一种化学成分明确的猪肺炎支原体培养基及其制备方法。
背景技术
猪支原体性肺炎是由猪肺炎支原体引发的一种慢性肺炎,又称猪地方流行性肺炎,临床上以咳嗽、气喘和肺部典型“虾肉”样病变为特征,发病率高,死亡率低。本病虽为老病,但近年来由于经常和PRRSV、圆环病毒等其它病原混合感染,造成猪死亡率增高,导致重大的经济损失而突显出了其重要性。长期以来,本病一直被认为是对养猪业造成重大经济损失最常发生、流行最广最难净化的重要疫病之一。由国内外临床经验可知,疫苗免疫是预防本病最重要的手段。
猪肺炎支原体为本病病原,可为猪支原体肺炎病原学鉴定提供可靠的病原材料,但猪肺炎支原体的营养要求非常苛刻,在一般的培养基中很难生长,是动物支原体中较难培养的支原体之一。目前在猪肺炎支原体疫苗生产中,其使用的猪肺炎支原体培养基有很多种,从最早的煮沸猪肺组织的灭活细胞培养基、猪肺埋块无细胞培养基,到后来的KM2培养基、A26培养基,均属于成份不明确配方培养基,这些培养基通常含有胰蛋白胨、脑心浸粉、水解乳蛋白、酵母浸出粉、PPLO粉、BHI粉等,成分复杂批间差大,培养效果不稳定。
现已公布的专利所涉及的猪肺炎支原体培养基有20个,20个专利中都含有成分不明确的脑心浸出液、PPLO肉汤、乳清蛋白水解物、酵母提取物等,其中专利号为CN111635876A的猪肺炎支原体培养基血清用量为3%-10%,活菌滴度是1*109-1*1010。
像专利号为CN102154167B的猪肺炎支原体培养基中就含有成分不明确的脑心浸出液、PPLO肉汤、乳清蛋白水解物等,制备方法复杂,活菌滴度是1*109-1*1010。以及专利号为CN103555641B的猪肺炎支原体培养基同样含有成分不明确的酵母粉,制备方法也非常复杂,配制时间也相对较长,活菌滴度是1*108-1*1010
目前猪肺炎支原体培养基主要应用于疫苗抗原的生产和相关科研及检测。已知的猪肺炎支原体培养基专利中使用大量动物源组分,包括胨、水解物、浸粉等,使用血清量也比较大。同时传统的培养基及培养工艺所培养的猪肺炎支原体存在生长速度较慢、培养时间长、活菌滴度低以及制备方法复杂等问题。
发明内容
有鉴于此,本发明提供一种化学成分明确的猪肺炎支原体培养基,具有成分明确、活菌滴度高、培养周期短、批间差小、效果稳定的特点。
本发明另一目的是提供该猪肺炎支原体的培养基的制备方法,具有简单高效的特点。
为了实现以上技术目的,本发明采用的技术方案:
一种化学成分明确的猪肺炎支原体培养基,包括以下成分:
通过以上技术方案,本发明的培养基成分明确,不含任何动物源组分,不含水解物及蛋白胨等,保证了培养基性能稳定,批间一致性;
猪肺炎支原体培养基通常含有的营养物质一般包括水、氮源、碳源、无机盐、生长因子等。传统猪肺炎支原体培养基通常加入蛋白胨、浸出液(如:胰蛋白胨、脑心浸出液、牛肉浸粉、酵母浸粉、乳清蛋白水解物等)来提供支原体所需要的氮源、维生素及无机盐类。但往往由于生产过程中厂家工艺不一、原料批间差异,导致蛋白胨、浸出液内某些有效成分损失或破坏,培养支原体也容易出现批间差异、效果不稳定。本发明培养基结合猪肺炎支原体所需营养物质通过添加L-盐酸精氨酸、L-丝氨酸、L-天冬氨酸、L-胱氨酸二盐酸盐、L-甲硫氨酸、L-亮氨酸、L-异亮氨酸、L-谷氨酸、L-苯丙氨酸、L-丙氨酸、L-缬氨酸、L-天冬酰胺、L-盐酸赖氨酸、L-色氨酸、L-脯氨酸、谷氨酰胺、L-苏氨酸、L-盐酸组氨酸氨基酸、盐酸硫胺、牛血清白蛋白、生物素、叶酸、泛酸钙、烟酰胺、肌醇、维生素A、维生素E、抗坏血酸、盐酸硫胺、氯化钙、氯化钾等成分为支原体的生长提供了足够的氮源、蛋白质、维生素、无机盐,来替代组分不明确的蛋白胨、浸出液、水解物等;
在支原体的细胞膜中,含有较多的脂类物质,其含量占膜成分的1/3,主要包括胆固醇、磷脂和脂多糖。胆固醇与膜结合可稳定膜的结构,使柔韧性减少。磷脂是支原体细胞膜的主要结构物质,是一种非常重要的复合脂,含有高度疏水(脂肪酸)和相对亲水(甘油)部分,因而能以不同的化学形式存在。支原体的膜普遍含有磷脂酰甘油和双脂甘油,其中的脂甘油含量只占细胞膜脂类总量的3%,但对保护ATP酶有重要意义。
亚油酸、亚麻酸为哺乳动物细胞所必须的脂肪酸能够有加快支原体生长繁殖,从而减少培养时间;
血清主要作用是提供激素和各种生长因子。葡萄糖酸锌和牛磺酸的添加可以大大的提高活菌滴度;肌醇是一种水溶性维生素,维生素B族中的一种,可促进新陈代谢;丙酮酸钠是重要的生理、能量代谢的中间体;谷氨酰胺则能够促进支原体的生长;牛血清白蛋白的添加可以促进分裂,促进支原体增长的作用,经过试验在该培养基中加入这些物质能够替代血清在培养基中的作用,从而降低一定血清含量也不影响培养效果;
铬作为GTF的活性成分协同胰岛素发挥作用,对糖、脂肪、蛋白质和核酸的代谢起着重要作用;
吡啶甲酸铬是一种矿物质,作为脂溶性的非电解质稳定性强,可顺利通过细胞膜直接作用于组织细胞,是葡萄糖耐量因子的组成成分,具有胰岛素作用,促进细胞对葡萄糖利用,促进葡萄糖的氧化磷酸化,促进糖原和脂肪酸的合成;
维生素B12和叶酸均为水溶性维生素,叶酸是合成四氢叶酸的重要原料,四氢叶酸在核酸的生物合成和蛋白质的生物合成过程中起重要作用,维生素B12是唯一含有金属元素的维生素,可促进蛋白质的生物合成,同时B12保护叶酸在细胞内的转移和贮存;
D泛酸钙在细胞内转变成酰基载体蛋白和辅酶,参与糖类、脂类、蛋白质代谢中的催化反应;
添加细胞相对葡萄糖不易利用的果糖,可减少代谢过程中乳酸的生成,从而稳定pH值;
优化的葡萄糖和谷氨酰胺配比,可以有效控制细胞代谢过程中乳酸和氨的生成量;
本发明运用微生物学、无机、有机分析化学的技术原理,对不同的氮源、碳源、蛋白质、无机盐和维生素等诸多营养组分的选择及其组合进行了大量实验和筛选,基于合理的组分选择以及合适配比,突破了猪肺炎支原体难培养的瓶颈。
本发明还提供一种适用于培养猪肺炎支原体的制备方法,包括如下步骤:
1)将上述的一种适用于培养猪肺炎支原体的培养基中的成分倒入容器中,使用纯化水完全溶解后定容,得到培养液;
2)采用灭菌的氢氧化钠溶液将步骤1)得到的培养液的pH值调节至7.5-7.7;
3)将步骤2)得到的培养液进行除菌操作;
4)在无菌的条件下往步骤3)得到的培养液中添加灭活的猪血清,其中,猪血清添加的体积比为5%。
进一步的,除菌操作为将步骤2)得到的培养液通过0.22μm微孔滤膜或滤芯过滤除菌。
进一步的,步骤3)中培养液进行除菌操作后,控制温度为2-8℃避光保存相对现有技术,本发明的有益效果是:
(1)本发明的培养基具有成分明确、批间差小和效果稳定的特点,本发明不含任何动物源组分,不含水解物及蛋白胨等,本发明没有使用常规培养基添加选项中的水解物和蛋白类营养,水解物为混合组分,组分不明确,批间差异大,蛋白类组分热稳定性差,极易失活,本发明组分配方巧妙的替代了这些物质;本发明有效避免了现有技术中采用牛心汤、消化肉汤、脑心浸粉、水解乳蛋白、胰蛋白胨、酵母浸出粉、PPLO粉、BHI粉等导致的营养成分浓度不一、生物污染等许多偏差问题;同时本发明也简化了下游产品的纯化步骤,减轻了下游纯化的压力,为大规模标准化生产提供了科学依据。
(2)本发明的血清用量低,本发明通过使用葡萄糖酸锌、牛磺酸、肌醇、丙酮酸钠、谷氨酰胺、牛血清白蛋白等来替代血清在本发明的培养基中的作用,起到了降低一定血清含量也不影响培养效果的作用,本发明可以使得血清的添加量降到5%,降低了成本,大大减轻了活疫苗中猪血清对猪体的过敏应激反应,具有更好的经济效益和实用性。
(3)本发明的培养基具有使猪肺炎支原体的活菌滴度高、培养周期短的特点,支原体的质量有保证,半成品生长滴度高而且稳定,用本发明进行培养42h左右,培养基pH从7.6降至6.8左右,培养的菌液CCU滴度为109-1010,其效果远优于现有技术的多种培养基。
(4)本发明中所采用的各组分材料均无特殊要求,均可以从市场上购买到标准化产品。
(5)本发明提供的猪肺炎支原体制备方法,制作步骤简单,成本低,培养效果优良且高效。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅用以解释本发明,并不用于限定本发明,即所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例
实施例1
一种适用于培养猪肺炎支原体培养基的制备,称取以下成分:
1)将上述成分依次倒入洁净容器中,添加800ml纯化水,搅拌至完全溶解,定容至1000ml,得到培养液;
2)采用灭菌的氢氧化钠溶液将步骤1)得到的培养液的pH值调节至7.6;
3)将步骤2)得到的培养液通过0.22μm微孔滤膜过滤除菌,控制温度为2-8℃避光保存,待用。
实施例2
一种适用于培养猪肺炎支原体培养基的制备,称取以下成分:
1)将上述成分依次倒入洁净容器中,添加800ml纯化水,搅拌至完全溶解,定容至1000ml,得到培养液;
2)采用灭菌的氢氧化钠溶液将步骤1)得到的培养液的pH值调节至7.6;
3)将步骤2)得到的培养液通过0.22μm微孔滤芯过滤除菌,控制温度为2-8℃避光保存,待用。
实施例3
一种适用于培养猪肺炎支原体培养基的制备,称取以下成分:
1)将上述成分依次倒入洁净容器中,添加800ml纯化水,搅拌至完全溶解,定容至1000ml,得到培养液;
2)采用灭菌的氢氧化钠溶液将步骤1)得到的培养液的pH值调节至7.6;
3)将步骤2)得到的培养液通过0.22μm微孔滤膜过滤除菌,控制温度为2-8℃避光保存,待用;
实施例4
本发明的培养基中猪血清添加量比较试验;
采用2组实施例1制备的培养基,分别在无菌的条件下往培养液中添加灭活的猪血清,其中,猪血清添加的体积分别占培养液的体积的5%和10%测定活菌滴度CCU(颜色改变单位),培养48h后试验数据如下:
培养基 | 血清含量 | CCU结果 | 平均结果 |
实施例1的第一组 | 5% | 9/10 | 1×10<sup>9</sup> |
实施例1的第二组 | 10% | 10/10 | 1×10<sup>10</sup> |
从实验数据上看:5%和10%的灭活的猪血清的活菌滴度CCU结果相当,本发明的培养基具有性能稳定,批间一致性好的特点;
此外,本发明可以使得血清的添加量降到5%,因此降低了成本,同时支原体的质量也有保证,半成品生长滴度高而且稳定,用本发明方法制备的半成品菌液效价高达1010CCU/ml,可用于疫苗等相关领域的生产。
实施例5
本发明的培养基与常用培养基培养猪肺炎支原体的比较;
1、猪肺炎支原体菌株和培养条件
猪肺炎支原体购自中国兽医药品监察所。分别将该菌株接种于本发明的实施例1、实施例2和实施例3的培养基,以及KM2猪肺炎支原体肉汤培养基(简称KM2培养基)和常用的支原体肉汤培养基,种子继代复壮后,分别按10%(V/V)的比例接种,37℃培养,当培养基的颜色变黄、PH由7.6降至6.8时,无菌取出培养物。
2、活菌滴度CCU(颜色改变单位)测定
每个培养菌液取13支无菌试管,每支试管装0.9ml不同配方的培养基,向第1管中加入0.1ml待测菌液,旋涡混合机上混匀后,更换吸头,吸取0.1ml到第2支试管,如此依次进行10倍系列稀释,直至第12管,第13管设为对照,仅添加培养基,不添加猪肺炎支原体菌液,每个培养菌液做3个重复。将所有试管放在37℃恒温培养箱中培养,每日观察试管菌液变色情况并作记录。观察时间为2周,最后发生颜色改变的试管稀释度即为该菌液的CCU。
3、猪肺炎支原体生长情况及CCU测定结果
猪肺炎支原体在不同配方培养基中的生长情况及培养时间见下表:
其中,“+”表示有颜色变化;“-”表示无颜色变化。
猪肺炎支原体在本发明实施例1、实施例2、实施例3支原体培养基中培养2天后均观察到颜色变化,CCU滴度为109-1010;在KM2培养基中培养3-4天变色,CCU滴度仅为108-109;在支原体肉汤培养基中培养3天变色,CCU滴度仅为108-109。可见,本发明的培养基非常适合猪肺炎支原体的生长,10%的接种比例传代,一般培养42h左右培养基pH从7.6降至6.8左右,培养的菌液CCU滴度为109-1010。
显而易见的,上面所述的实施例仅仅是本发明实施例中的一部分,而不是全部。基于本发明记载的实施例,本领域技术人员在不付出创造性劳动的情况下得到的其它所有实施例,或在本发明的启示下做出的结构变化,凡是与本发明具有相同或相近的技术方案,均落入本发明的保护范围之内。
Claims (4)
2.一种化学成分明确的猪肺炎支原体培养基制备方法,其特征在于,包括如下步骤:
1)将权利要求1所述的一种适用于培养猪肺炎支原体的培养基中的成分倒入容器中,使用纯化水完全溶解后定容,得到培养液;
2)采用灭菌的氢氧化钠溶液将步骤1)得到的培养液的pH值调节至7.5-7.7;
3)将步骤2)得到的培养液进行除菌操作;
4)在无菌的条件下往步骤3)得到的培养液中添加灭活的猪血清,其中,猪血清添加的体积比为5%。
3.根据权利要求2所述的一种化学成分明确的猪肺炎支原体培养基制备方法,其特征在于,所述除菌操作为将步骤2)得到的培养液通过0.22μm微孔滤膜或滤芯过滤除菌。
4.根据权利要求2所述的一种化学成分明确的猪肺炎支原体培养基制备方法,其特征在于,所述步骤3)中培养液进行除菌操作后,控制温度为2-8℃避光保存。
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