CN111588841A - 一种可气溶胶化的seb类毒素疫苗干粉吸入剂 - Google Patents
一种可气溶胶化的seb类毒素疫苗干粉吸入剂 Download PDFInfo
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Abstract
本发明公开了一种可气溶胶化的SEB类毒素疫苗干粉吸入剂。本发明保护一种干粉制剂:灭活的SEB蛋白0.049‑0.0.051g:CpG 0.049‑0.0.051g:甘露醇0.98‑1.02g:肌醇0.98‑1.02g:亮氨酸0.49‑0.51g:泊洛沙姆0.049‑0.051ml。本发明还保护一种干粉制剂的制备方法:将SEB类毒素溶液进行喷雾冷冻干燥,得到干粉制剂;SEB类毒素溶液中,含灭活的SEB蛋白0.049‑0.0.051g/100mL、CpG 0.049‑0.0.051g/100mL、甘露醇0.98‑1.02g/100mL、肌醇0.98‑1.02g/100mL、亮氨酸0.49‑0.51g/100mL、泊洛沙姆0.05%。本发明对于金黄色葡萄球菌肠毒素B的防控具有重大的应用价值。
Description
技术领域
本发明涉及一种可气溶胶化的SEB类毒素疫苗干粉吸入剂。
背景技术
金黄色葡萄球菌属于杆菌纲杆菌目葡萄球菌科,是一种常见的致病菌,在自然界广泛存在于空气、水、人和动物排泄物、地面及物体表面,是一种重要的人畜共患病。典型的金黄色葡萄球菌为球形,直径0.8μm左右,光镜下排列成葡萄串状;无芽孢、鞭毛,大多数无荚膜;可在普通培养基形成湿润、光滑、隆起的圆形菌落,菌落颜色依菌株而异,初呈灰白色,继而为金黄色、白色或柠檬色。革兰氏染色阳性,衰老或死亡后可转为阴性。对不良的环境抵抗能力较强,在干燥空气中可以存活数月,但不繁殖;耐热,70℃下1h,80℃下30min不会被杀死;耐低温,在冷冻食品中不易死亡;耐高渗,可以在15%Nacl和40%胆汁中生长。
金黄色葡萄球菌肠毒素是金黄色葡萄球菌分泌的一组具有超抗原活性的细菌毒素,低分子量可溶,有强大的激活淋巴细胞的能力,可使淋巴细胞产生强烈的细胞毒性作用和高水平的细胞因子(如肿瘤坏死因子TNF-α等),对肿瘤细胞具有强大的杀伤作用。目前发现的肠毒素分型有A、B、C1、C2、C3、D和E等。其中金黄色葡萄球菌肠毒素B(Staphylococcalenterotoxin B,SEB)能在很低浓度条件下激活大量T细胞,并刺激释放细胞因子,能引起人和动物呕吐、腹泻、腹痛等中毒症状,甚至造成机体不可逆的病变导致死亡。1.7μg的SEB就可杀死一个成年人,是被列入联合国《禁止生物武器公约》核查清单的11种生物毒素之一。
SEB感染一个重要的临床症状是毒性休克综合征(TSS),这与SEB的超抗原特性有关,是SEB刺激免疫细胞分泌过量的细胞因子的结果。目前对SEB中毒的治疗仅限于支持疗法,尚缺乏更有效的手段。而SEB疫苗的思路是对SEB蛋白进行基因突变或修饰,使之失去或降低超抗原的毒性作用,但能刺激机体产生具有保护作用的特异抗体,用于防治SEB的毒性效应。
发明内容
本发明的目的是提供一种可气溶胶化的SEB类毒素疫苗干粉吸入剂。
本发明保护一种干粉制剂(干粉制剂甲),原料组成为:灭活的SEB蛋白、CpG、甘露醇、肌醇、亮氨酸和泊洛沙姆。
本发明还保护一种干粉制剂(干粉制剂乙),原料组成以及配比如下:
灭活的SEB蛋白0.049-0.0.051g:CpG 0.049-0.0.051g:甘露醇0.98-1.02g:肌醇0.98-1.02g:亮氨酸0.49-0.51g:泊洛沙姆0.049-0.051ml。
本发明还保护一种干粉制剂(干粉制剂丙),原料组成以及配比如下:
灭活的SEB蛋白0.05g:CpG 0.05g:甘露醇1g:肌醇1g:亮氨酸0.5g:泊洛沙姆0.05ml。
本发明还保护一种干粉制剂的制备方法(方法甲),包括如下步骤:
将SEB类毒素溶液进行喷雾冷冻干燥,得到干粉制剂;
SEB类毒素溶液中,含有灭活的SEB蛋白、CpG、甘露醇、肌醇、亮氨酸和泊洛沙姆。
本发明还保护一种干粉制剂的制备方法(方法乙),包括如下步骤:
将SEB类毒素溶液进行喷雾冷冻干燥,得到干粉制剂;
SEB类毒素溶液中,含有灭活的SEB蛋白0.049-0.0.051g/100mL、CpG0.049-0.0.051g/100mL、甘露醇0.98-1.02g/100mL、肌醇0.98-1.02g/100mL、亮氨酸0.49-0.51g/100mL、泊洛沙姆0.049-0.051%(体积百分含量)。
本发明还保护一种干粉制剂的制备方法(方法丙),包括如下步骤:
将SEB类毒素溶液进行喷雾冷冻干燥,得到干粉制剂;
SEB类毒素溶液中,含有灭活的SEB蛋白0.05g/100mL、CpG 0.05g/100mL、甘露醇1g/100mL、肌醇1g/100mL、亮氨酸0.5g/100mL、泊洛沙姆0.05%(体积百分含量)。
以上任一所述SEB类毒素溶液中,余量为水。
以上任一所述SEB类毒素溶液的pH值为7.2。
以上任一所述SEB类毒素溶液用1M氢氧化钠溶液调pH值至7.2。
以上任一所述灭活的SEB蛋白可为将SEB蛋白进行甲醛进行灭活得到的。甲醛和SEB蛋白的摩尔比可为(4300-21500):1。甲醛和SEB蛋白的摩尔比为4300:1。
以上任一所述喷雾冷冻干燥的方法具体可为:冰浴预冷2小时后的SEB类毒素溶液通过二流体喷头的进口直接喷雾至冷介质(例如液氮)中,将冰晶进行真空冷冻干燥。
以上任一所述喷雾冷冻干燥的方法具体可为:将SEB类毒素溶液冰浴预冷2小时,然后取20ml转入注射器中,注射器出口连接二流体喷头的进口,直接喷雾至液氮中(不锈钢盆中装有3/4体积的液氮,置于磁力搅拌器上进行搅拌;如有需要,喷雾中途添加液氮;喷头离液氮液面≈10cm,设置气泵气压0.15兆帕),喷出来的雾化液滴,在液氮作用下迅速冷冻成冰晶;将冰晶连同少量剩余液氮转移至不锈刚杯中,开口处封上一层纱布,置于真空冷冻干燥机中干燥36h以上。
本发明还保护以上任一所述方法制备得到的干粉制剂。
本发明还保护以上任一所述干粉制剂在制备金黄色葡萄球菌肠毒素B疫苗中的应用。
本发明还保护一种金黄色葡萄球菌肠毒素B疫苗,其活性成分为以上任一所述干粉制剂。
以上任一所述疫苗可为治疗疫苗或预防疫苗。
以上任一所述金黄色葡萄球菌肠毒素B疫苗为用于治疗和/或预防金黄色葡萄球菌肠毒素B中毒的疫苗。
以上任一所述干粉制剂为可气溶胶化的干粉吸入剂。
SEB蛋白即金黄色葡萄球菌肠毒素B。
以上任一所述SEB蛋白即序列表的序列1所示的蛋白质。
以上任一所述亮氨酸为L-亮氨酸。
本发明中制备得到的干粉制剂,总粒子空气动力学质量中值直径为5.41μm,粒子呈圆球状,形态疏松多孔,粒径分布较均匀。干粉制剂有利于疫苗沉积到肺深部,可以增加抗原与粘膜组织接触反应的时间,有助于提高免疫保护效果。
将本发明提供的干粉制剂通过手持式干粉气溶胶肺递送装置递送至小鼠肺部进行三次免疫,血清和肺匀浆液中抗体(IgG、IgG1、Ig2a、IgM和IgA)的滴度随时间推移逐渐升高,表明三次免疫起到了免疫增强效果,可以刺激机体产生体液免疫、细胞免疫及粘膜免疫。三次免疫后进行SEB攻毒,SEB液体组和SEB干粉组小鼠生存率可达100%,感染后无明显临床症状。相反,未经过SEB类毒素疫苗干粉免疫的小鼠则生存率为0%,表明SEB类毒素疫苗干粉对小鼠起到很好的免疫保护效果。
本发明为金黄色葡萄球菌肠毒素B干粉疫苗的研发可行性提供了支持。本发明对于金黄色葡萄球菌肠毒素B的防控具有重大的应用价值。
附图说明
图1为SEB干粉吸入剂的粒子形态。
图2为生存率的结果。
图3为血清中IgG滴度的结果。
图4为血清中IgG1滴度的结果。
图5为血清中Ig2a滴度的结果。
图6为血清中IgM滴度的结果。
图7为血清中IgA滴度的结果。
图8为肺匀浆上清中IgG滴度的结果。
图9为肺匀浆上清中IgG1滴度的结果。
图10为肺匀浆上清中Ig2a滴度的结果。
图11为肺匀浆上清中IgM滴度的结果。
图12为肺匀浆上清中IgA滴度的结果。
图13为血清中IFN-γ的浓度的结果。
图14为血清中TNF-α的浓度的结果。
图15为肺匀浆上清中IFN-γ的浓度的结果。
图16为肺匀浆上清中TNF-α的浓度的结果。
具体实施方式
以下的实施例便于更好地理解本发明,但并不限定本发明。下述实施例中的实验方法,如无特殊说明,均为常规方法。下述实施例中所用的试验材料,如无特殊说明,均为自常规生化试剂商店购买得到的。以下实施例中的定量试验,均设置三次重复实验,结果取平均值。
金黄色葡萄球菌肠毒素B又称为SEB蛋白,其氨基酸序列如序列表的序列1所述。CpG,全称为Class B CpG oligonucleotide(B类CpG寡核苷酸),具有免疫激活功能,是针对人或小鼠TLR9的免疫刺激剂,是一种典型的黏膜免疫佐剂。实施例中所用的CpG,为sigma公司产品目录号为tlrl-2006的产品,该产品的网址链接为:https://www.invivogen.com/ odn2006。甘露醇:Sigma公司,货号为M1902。肌醇:Sigma公司,货号为I7508。亮氨酸(L-亮氨酸):Sigma公司,货号为L8912。泊洛沙姆(Pluronic F-68浓度为10g/100mL的F-68溶液):Sigma公司,货号为P5556。LPS(脂多糖):来源于大肠杆菌055:B5,Sigma,货号L2880。
小鼠细胞因子检测试剂盒:eBioscience,EPX170-26090-901。HRP标记的山羊抗鼠IgG、HRP标记的山羊抗鼠IgG1、HRP标记的山羊抗鼠Ig2a、HRP标记的山羊抗鼠IgA、HRP标记的山羊抗鼠IgM抗体,均为abcam产品。
手持式液体气溶胶肺递送装置:北京慧荣和科技有限公司的肺部液体定量雾化器。手持式干粉气溶胶肺递送装置:北京慧荣和科技有限公司的肺部干粉定量雾化器。扫描电子显微镜:S-3400N,日本日立公司。小型气溶胶沉降评测舱:北京慧荣和科技有限公司。激光粒度分析仪TSI3321:APS-3321,美国TSI公司。
实施例1、可气溶胶化的SEB类毒素疫苗干粉吸入剂的制备
一、灭活的SEB类毒素蛋白(干粉)的制备
1、制备SEB类毒素蛋白(干粉)。
SEB类毒素蛋白即序列表的序列1所示的蛋白质。
2、制备灭活的SEB类毒素蛋白(干粉)。
将甲醛和步骤1制备的SEB类毒素蛋白混匀,37℃静置反应30天,然后转移到透析袋(截留分子量为15KD)中,将透析袋置于pH7.2的PBS缓冲液中透析48小时(每12小时更换新的PBS缓冲液),然后将透析袋置于水中透析24小时(每4小时更换新的水),然后收集透析袋中的液体,进行冷冻干燥。
本步骤中甲醛和SEB类毒素蛋白的摩尔比为4300:1。
实际操作中,甲醛和SEB类毒素蛋白的摩尔比可为(4300-21500):1。
二、可气溶胶化的干粉吸入剂的制备
1、SEB类毒素溶液的制备
用步骤一的2制备的灭活的SEB类毒素蛋白、CpG、甘露醇、肌醇、亮氨酸、泊洛沙姆和水制备SEB类毒素溶液。SEB类毒素溶液制备时用1M氢氧化钠溶液调节pH值至7.2,制备完成后进行过滤除菌。SEB类毒素溶液中,灭活的SEB类毒素蛋白的浓度为0.05g/100mL、CpG的浓度为0.05g/100mL、甘露醇的浓度为1g/100mL、肌醇的浓度为1g/100mL、亮氨酸的浓度为0.5g/100mL、泊洛沙姆的浓度为0.05%(体积百分含量)。每100ml SEB类毒素溶液中含有的干物质的量约等于2.65g。
2、CPG溶液的制备
用CpG、甘露醇、肌醇、亮氨酸、泊洛沙姆和水制备CPG溶液。CPG溶液制备时用1M氢氧化钠溶液调节pH值至7.2,制备完成后进行过滤除菌。CPG溶液中,CpG的浓度为0.05g/100mL、甘露醇的浓度为1g/100mL、肌醇的浓度为1g/100mL、亮氨酸的浓度为0.5g/100mL、泊洛沙姆的浓度为0.05%(体积百分含量)。每100ml CPG溶液中含有的干物质的量约等于2.60g。
3、可气溶胶化的干粉吸入剂的制备
取20mL样本液(SEB类毒素溶液或CPG溶液),冰浴2小时进行预冷,然后转入20mL注射器(务必一次性使用)中,连接上进口的二流体喷头,直接喷雾至液氮中(不锈钢盆中装有3/4体积的液氮,置于磁力搅拌器上进行搅拌;如有需要,喷雾中途添加液氮),喷头离液氮液面≈10cm,设置气泵气压0.15兆帕(1.5Bar)。喷出来的雾化液滴,在液氮作用下迅速冷冻成冰晶。冰晶连同少量剩余液氮,转至不锈刚杯中,开口处封上一层纱布,置于冷冻干燥机中干燥至少36h。收集干粉样品至5mL的带橡胶塞的西林瓶中,-20℃密封储存。喷雾用时5分40秒。
采用SEB类毒素溶液作为样本液,得到的干粉命名为SEB干粉吸入剂。20mL样本液得到了182mg干粉(三次重复试验的平均值)。得率=SEB干粉吸入剂的产量÷20ml样本液中含有的干物质的量×100%。20ml样本液中含有的干物质的量=2.65g*0.2=530mg。因此,用SEB类毒素溶液作为样本液制备SEB干粉吸入剂的得率为34.3%。
采用CPG溶液作为样本液,得到的干粉命名为CPG干粉吸入剂。
三、制剂学特性分析
粒子形态和粒度分布:通过扫描电镜,取多个视野,观察粒子形态,并用软件估算粒子绝对质量中值直径。
SEB干粉吸入剂的粒子形态见图1(左图比例尺=100微米,右图比例尺=10微米)。粒子疏松多孔,干粉粒子绝对质量中值直径为7.29μm。
四、空气动力学特性分析
1、开启小型气溶胶沉降评测舱(舱内底部直径0.76m、高0.73m),调至循环风模式。
2、开启质量浓度检测仪TSI8530,实时监测舱内气溶胶粒子质量浓度,采样流量3L/min,直至舱内QRJ质量浓度降低至0.005mg/m3。
3、关闭舱内循环风,开启舱内底部放置的小功率风扇。
4、打开评测舱采样口,使用手持式干粉气溶胶肺递送装置将供试制剂向舱内发生干粉气溶胶,发生量5mg/次,只发生1次。
5、干粉气溶胶发生后风扇混匀30S,关闭风扇。
6、使用激光粒度分析仪TSI3321,并在采样口前段连接慧荣和生产的稀释器,采样流量5L/min,采样时间5min。测试指标为单次采样的总粒子数、总粒子空气动力学质量中值直径、总粒子空气动力学质量中值直径。
供试制剂为SEB干粉吸入剂。
单次采样总粒子数为342243,总粒子空气动力学数量中值直径为0.744μm,总粒子空气动力学质量中值直径为5.41μm。
实施例2、干粉制剂的效果检测
一、免疫
BALB/c小鼠(雌性,6-8周,北京维通利华实验动物有限公司)分为四组,每组30只。
试验过程:试验第1天进行第一次免疫,试验第8天进行第二次免疫,试验第22天进行第三次免疫;试验第36天进行攻毒,攻毒4小时后腹腔注射LPS(LPS发挥致敏作用,单只剂量75μg)。
第一组(SEB干粉组):每次每只小鼠免疫0.53mg实施例1的步骤二的3制备的SEB干粉吸入剂;
第二组(SEB液体组):每次每只小鼠免疫50μl悬液(其中含有0.53mg实施例1的步骤二的3制备的SEB干粉吸入剂,溶剂为pH7.2的PBS缓冲液);
第三组(CPG干粉组):每次每只小鼠免疫0.52mg实施例1的步骤二的3制备的CPG干粉吸入剂;
第四组(CPG液体组):每次每只小鼠免疫50μl悬液(其中含有0.52mg实施例1的步骤二的3制备的CPG干粉吸入剂,溶剂为pH7.2的PBS缓冲液);
干粉吸入剂采用手持式干粉气溶胶肺递送装置给药(插入气管递送至肺部),递送至小鼠肺部。悬液采用手持式液体气溶胶肺递送装置给药(插入气管递送至肺部),递送至小鼠肺部。
攻毒:采用手持式液体气溶胶肺递送装置给予攻毒液(插入气管递送至肺部),递送至小鼠肺部;攻毒液是将实施例1的步骤一的1制备的SEB类毒素蛋白(干粉)溶于pH7.2的PBS缓冲液得到的;每只小鼠的攻毒剂量为4μg SEB类毒素蛋白。
二、临床症状观察
CPG液体组和CPG干粉组小鼠在攻毒后均有耸毛现象、外部刺激后反应迟钝等症状,随后小鼠症状逐渐加重,可见强迫性腹部呼吸,最后小鼠死亡。
SEB液体组和SEB干粉组小鼠有轻微耸毛现象,后来耸毛症状消失。
三、生存曲线绘制
每组取10只小鼠,在攻毒后0d、1d、2d、2.5d、3d、3.5d、4d、5d、6d、7d、8d、9d、10d、11d、12d、13d、14d时间点,记录小鼠死亡只数,绘制生存曲线。
结果见图2。SEB液体组和SEB干粉组小鼠全部存活,CPG液体组和CPG干粉组小鼠在攻毒后1天内全部死亡。
四、特异性抗体检测
时间点1(第一次免疫前2天)、时间点2(第二次免疫前2天)、时间点3(第三次免疫前2天)、时间点4(攻毒前2天),时间点5(攻毒2周后),每组随机取4只存活小鼠。摘眼球取小鼠全血,收集血清。解剖,取肺,每个肺加入0.8毫升pH7.2的PBS缓冲液,进行充分匀浆,然后3000g离心10min,收集上清,即为肺匀浆上清。
采用酶联免疫吸附法检测血清/肺匀浆上清中5种特异性抗体(IgG、IgG1、IgG2a、IgM、IgA)的滴度。包被原为实施例1的步骤一的1制备的SEB类毒素蛋白(干粉),包被浓度为1μg/ml。一抗为血清的梯度稀释液或肺匀浆上清的梯度稀释液(用无菌生理盐水进行梯度稀释)。二抗为HRP标记的山羊抗鼠IgG、HRP标记的山羊抗鼠IgG1、HRP标记的山羊抗鼠Ig2a、HRP标记的山羊抗鼠IgA或HRP标记的山羊抗鼠IgM抗体。显色液为TMB显色液。
血清中IgG滴度的结果见图3。血清中IgG1滴度的结果见图4。血清中Ig2a滴度的结果见图5。血清中IgM滴度的结果见图6。血清中IgA滴度的结果见图7。SEB液体组和SEB干粉组血清中IgG抗体、IgG1抗体、Ig2a抗体滴度随时间推移呈逐渐升高趋势,IgM抗体和IgA抗体滴度相对较低。在平行时间点,SEB液体组和SEB干粉组血清中5种抗体均无显著差异。CPG液体组和CPG干粉组在前4个时间点均未检测到相应抗体,在时间点5动物均死亡无检测结果。
肺匀浆上清中IgG滴度的结果见图8。肺匀浆上清中IgG1滴度的结果见图9。肺匀浆上清中Ig2a滴度的结果见图10。肺匀浆上清中IgM滴度的结果见图11。肺匀浆上清中IgA滴度的结果见图12。SEB液体组和SEB干粉组肺匀浆上清中IgG抗体、IgG1抗体、Ig2a抗体滴度随时间推移呈逐渐升高趋势,IgM抗体和IgA抗体滴度相对较低。CPG液体组和CPG干粉组在前4个时间点均未检测到相应抗体,在时间点5动物均死亡无检测结果。
图3至图12中,△表示与同组别一免前2天时间点有显著差异,☆表示SEB液体组与相同时间点SEB干粉组有显著差异或CPG液体组与相同时间点CPG干粉组有显著差异。
五、细胞因子检测
时间点1(第一次免疫前2天)、时间点2(第二次免疫前2天)、时间点3(第三次免疫前2天)、时间点4(攻毒前2天),时间点5(攻毒2周后)每组随机取4只存活小鼠。摘眼球取小鼠全血,收集血清。解剖,取肺,每个肺加入0.8毫升pH7.2的PBS缓冲液,进行充分匀浆,然后3000g离心10min,收集上清,即为肺匀浆上清。
采用Th1/Th2/Th9/Th17/Th22/Treg Cytokine 17-Plex Mouse Panel(Invivogen,货号EPX170-26090-901)检测血清/肺匀浆上清中2种细胞因子(IFN-γ、TNF-α)的浓度。
血清中IFN-γ的浓度的结果见图13。血清中TNF-α的浓度的结果见图14。肺匀浆上清中IFN-γ的浓度的结果见图15。肺匀浆上清中TNF-α的浓度的结果见图16。
图13至图16中,△表示与同组别一免前2天时间点有显著差异,☆表示SEB液体组与相同时间点SEB干粉组有显著差异或CPG液体组与相同时间点CPG干粉组有显著差异。
SEQUENCE LISTING
<110> 中国人民解放军军事科学院军事医学研究院
<120> 一种可气溶胶化的SEB类毒素疫苗干粉吸入剂
<130> GNCYX190498
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 266
<212> PRT
<213> Artificial sequence
<400> 1
Met Tyr Lys Arg Leu Phe Ile Ser His Val Ile Leu Ile Phe Ala Leu
1 5 10 15
Ile Leu Val Ile Ser Thr Pro Asn Val Leu Ala Glu Ser Gln Pro Asp
20 25 30
Pro Lys Pro Asp Glu Leu His Lys Ser Ser Lys Phe Thr Gly Leu Met
35 40 45
Glu Asn Met Lys Val Leu Tyr Asp Asp Asn His Val Ser Ala Ile Asn
50 55 60
Val Lys Ser Ile Asp Gln Phe Leu Tyr Phe Asp Leu Ile Tyr Ser Ile
65 70 75 80
Lys Asp Thr Lys Leu Gly Asn Tyr Asp Asn Val Arg Val Glu Phe Lys
85 90 95
Asn Lys Asp Leu Ala Asp Lys Tyr Lys Asp Lys Tyr Val Asp Val Phe
100 105 110
Gly Ala Asn Tyr Tyr Tyr Gln Cys Tyr Phe Ser Lys Lys Thr Asn Asp
115 120 125
Ile Asn Ser His Gln Thr Asp Lys Arg Lys Thr Cys Met Tyr Gly Gly
130 135 140
Val Thr Glu His Asn Gly Asn Gln Leu Asp Lys Tyr Arg Ser Ile Thr
145 150 155 160
Val Arg Val Phe Glu Asp Gly Lys Asn Leu Leu Ser Phe Asp Val Gln
165 170 175
Thr Asn Lys Lys Lys Val Thr Ala Gln Glu Leu Asp Tyr Leu Thr Arg
180 185 190
His Tyr Leu Val Lys Asn Lys Lys Leu Tyr Glu Phe Asn Asn Ser Pro
195 200 205
Tyr Glu Thr Gly Tyr Ile Lys Phe Ile Glu Asn Glu Asn Ser Phe Trp
210 215 220
Tyr Asp Met Met Pro Ala Pro Gly Asp Lys Phe Asp Gln Ser Lys Tyr
225 230 235 240
Leu Met Met Tyr Asn Asp Asn Lys Met Val Asp Ser Lys Asp Val Lys
245 250 255
Ile Glu Val Tyr Leu Thr Thr Lys Lys Lys
260 265
Claims (9)
1.一种干粉制剂,原料组成为:灭活的SEB蛋白、CpG、甘露醇、肌醇、亮氨酸和泊洛沙姆。
2.一种干粉制剂,原料组成以及配比如下:
灭活的SEB蛋白0.049-0.0.051g:CpG 0.049-0.0.051g:甘露醇0.98-1.02g:肌醇0.98-1.02g:亮氨酸0.49-0.51g:泊洛沙姆0.049-0.051ml。
3.一种干粉制剂,原料组成以及配比如下:
灭活的SEB蛋白0.05g:CpG 0.05g:甘露醇1g:肌醇1g:亮氨酸0.5g:泊洛沙姆0.05ml。
4.一种干粉制剂的制备方法,包括如下步骤:
将SEB类毒素溶液进行喷雾冷冻干燥,得到干粉制剂;
SEB类毒素溶液中,含有灭活的SEB蛋白、CpG、甘露醇、肌醇、亮氨酸和泊洛沙姆。
5.一种干粉制剂的制备方法,包括如下步骤:
将SEB类毒素溶液进行喷雾冷冻干燥,得到干粉制剂;
SEB类毒素溶液中,含有灭活的SEB蛋白0.049-0.0.051g/100mL、CpG 0.049-0.0.051g/100mL、甘露醇0.98-1.02g/100mL、肌醇0.98-1.02g/100mL、亮氨酸0.49-0.51g/100mL、泊洛沙姆0.049-0.051%。
6.一种干粉制剂的制备方法,包括如下步骤:
将SEB类毒素溶液进行喷雾冷冻干燥,得到干粉制剂;
SEB类毒素溶液中,含有灭活的SEB蛋白0.05g/100mL、CpG 0.05g/100mL、甘露醇1g/100mL、肌醇1g/100mL、亮氨酸0.5g/100mL、泊洛沙姆0.05%。
7.权利要求4或5或6所述方法制备得到的干粉制剂。
8.权利要求1或2或3或7所述干粉制剂在制备金黄色葡萄球菌肠毒素B疫苗中的应用。
9.一种鼠疫金黄色葡萄球菌肠毒素B疫苗,其活性成分为权利要求1或2或3或7所述干粉制剂。
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