CN110354098B - 重组蛋白cfp-10纳米颗粒的制备及应用 - Google Patents
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Abstract
本发明一种重组蛋白CFP‑10纳米颗粒的制备方法及其在制备预防结核疫苗中的应用,其中制备步骤为:(1)聚乳酸‑羟基乙酸共聚物PLGA溶液的配置;(2)重组蛋白CFP‑10溶液的配置;(3)初乳的制备;(4)复乳的制备;(5)纳米微粒的形成。采用本发明所述方法制备的重组蛋白CFP‑10纳米颗粒,通过滴鼻方式对BCG免疫小鼠进行加强免疫,证实其具有提高黏膜免疫水平、促进BCG免疫的效果。
Description
技术领域
本发明属于新型疫苗用途领域,尤其涉及一种涉及重组蛋白CFP-10纳米颗粒的制备及在制备预防结核疫苗中的应用。
背景技术
牛结核是一种重要的人畜共患病,危害养殖业发展和公共卫生安全。牛结核的致病菌为牛分枝杆菌,它可以突破种间屏障在其他哺乳动物与人种间传播,宿主范围广,对许多哺乳动物群都有影响。目前的调查研究中显示我国牛结核病存在着易反复、全国散发的特点,并通过食物、接触等方式传播给人。卡介苗(BCG)是最广泛使用的预防结核病的疫苗,对于儿童的保护效果显著,但对成年人保护效力有限,与牛分枝杆菌相比,BCG存在一定的基因缺陷,比如BCG缺失RD1区域,其基因编码的蛋白与结核分枝杆菌的毒力密切相关。聚乳酸-羟基乙酸共聚物(PLGA),是FDA批准的具有无毒、易制备、可生物降解等优势,该材料已经长期用于生产生物降解的外科缝线以及包封需延长释放的药物。目前作为可以控制释放疫苗的递送系统材料,将抗原肽或蛋白包封在其中或者吸附在PLGA颗粒表面,其疫苗制剂具有持续长时间释放包被抗原的优势,使包封的抗原肽或蛋白免于水解。
培养滤液蛋白10(culture filtrate protein 10,CFP-10)由结核分枝杆菌的RD1区基因Rv3874编码,在体内和早期分泌抗原靶蛋白6(ESAT-6)形成1∶1异二聚体复合物而发挥生物学功能,激发强烈的特异性T细胞应答。由于CFP-10所在的RD1区只存在于结核分枝杆菌和牛分枝杆菌中,而其他分枝杆菌及卡介苗(BCG)中均缺失该区序列,因此,CFP-10已成为结核病诊断和疫苗研究的一大热点。纳米亚单位疫苗具有高效免疫作用、靶向性、可控释性等优势。用PLGA包被优势抗原制备成纳米亚单位疫苗具有潜在的应用价值。
发明内容
本发明的目的在于提供一种新的结核纳米疫苗,具体是其在制备预防结核疫苗中的应用。本发明通过BCG初免、重组蛋白CFP-10纳米疫苗加强免疫,然后用牛分枝杆菌建立小鼠感染模型证实其免疫活性。
为了实现上述目的,本发明首先提供一种重组蛋白CFP-10纳米颗粒的制备方法,其特征在于,具体包括如下操作步骤:
(1)聚乳酸-羟基乙酸共聚物PLGA溶液的配置;
(2)重组蛋白CFP-10溶液的配置;
(3)初乳的制备;
(4)复乳的制备;
(5)纳米微粒的形成。
其中,所述聚乳酸-羟基乙酸共聚物PLGA溶液的配置是将聚乳酸-羟基乙酸共聚物PLGA溶解于乙酸乙酯中形成有机相,PLGA终浓度为36mg/mL;
其中,所述重组蛋白CFP-10溶液的配置是将冻干的重组蛋白CFP-10粉末溶解于PBS溶液中作为内水相;
其中,所述初乳的制备是将步骤(2)中获得的所述内水相注入到步骤(1)形成的所述有机相中,超声形成初乳;
特别地,步骤(3)中超声处理的参数为:40%功率,超声2s,间隔2s,工作总时间6min;
其中,所述复乳的制备是将步骤(3)中形成的所述初乳注入到1%PVA溶液中,超声形成复乳;
特别地,步骤(4)中超声处理的参数为:60%功率,超声2s,间隔2s,工作总时间8min;
其中,所述纳米微粒的形成是将步骤(4)形成的所述复乳倒入0.5%PVA溶液中,700rpm,搅拌3~4h,挥发油相,向制得的溶液中加入5倍体积的双蒸水水进行洗涤,4℃、5000rpm,离心3min,洗涤2次,用适量无菌双蒸水重悬即得纳米微粒乳液;
其中,所述重组蛋白CFP-10纳米微粒平均粒径约247nm,电位为-28.8mV;
其中,所述重组蛋白CFP-10纳米微粒包封率为80.53%。
本发明还提供按照本发明所述方法制备的重组蛋白CFP-10纳米微粒在制备预防结核杆菌疫苗中的应用。
本发明的有益效果在于:采用本发明所述方法制备的重组蛋白CFP-10纳米微粒获得的疫苗与传统BCG免疫对照相比,使用重组蛋白CFP-10纳米微粒疫苗可以明显提高黏膜免疫IgA水平、减少组织载菌量,促进BCG免疫效果。
附图说明
图1:按照本发明所述方法制备的重组蛋白CFP-10纳米微粒扫描电镜图;
图2:重组蛋白CFP-10纳米微粒对BCG免疫小鼠产生IgA的影响,其中*P<0.05,***P<0.01;
图3:重组蛋白CFP-10纳米微粒对BCG免疫小鼠肺组织载菌量影响,其中*P<0.05,***P<0.01。
具体实施方式
下面结合附图,对实施例作详细说明。
实施例1:重组蛋白CFP-10纳米疫苗的制备方法
复乳法制备纳米微粒:根据牛分支杆菌菌株的CFP10基因序列,设计引物,扩增目的基因,并将目的基因连接到pET30a(+)载体构建pET30a(+)-CFP-10重组质粒,将重组质粒转化到E.coli BL21(DE3)感受态细胞进行诱导表达,并对表达的蛋白进行纯化冻干。将纯化冻干后的蛋白溶解于PBS溶液中形成内水相,将内水相注入到有机相中,超声形成初乳,将初乳注入到1%PVA溶液中,超声形成复乳,将复乳倒入0.5%PVA溶液中,搅拌3~4h,挥发油相。离心洗涤,冷冻干燥可获得重组蛋白CFP-10纳米微粒。具体操作步骤如下:
(1)聚乳酸-羟基乙酸共聚物PLGA溶液的配置;
将聚乳酸-羟基乙酸共聚物PLGA溶解于乙酸乙酯中形成有机相,PLGA终浓度为36mg/mL;
(2)重组蛋白CFP-10溶液的配置
将冻干的重组蛋白CFP-10粉末溶解于PBS溶液中作为内水相;
(3)初乳的制备
将内水相注入到有机相中,超声形成初乳,超声参数:40%功率,超声2s,间隔2s,工作总时间6min;
(4)复乳的制备
将初乳注入到1%PVA溶液中,超声形成复乳,超声参数:60%功率,超声2s,间隔2s,工作总时间8min;
(5)纳米微粒的形成
将复乳倒入0.5%PVA溶液中,700rpm,搅拌3~4h,挥发油相,向制得的溶液中加入5倍体积的双蒸水水进行洗涤,4℃、5000rpm,离心3min,洗涤2次。用适量无菌双蒸水重悬制成微粒乳液。
实施例2:纳米微粒物理表征和形态观察
将内水相和油相比例为1:9制备的样品进行表征,使用Malvern Zetasizer纳米粒度电位仪测定粒径和电位。制备的CFP10纳米微粒平均粒径约247nm,电位为-28.8mV。使用紫外分光光度计测量后,经计算纳米微粒包封率为80.53%。扫描电镜结果显示CFP10纳米微粒大小较均一,呈表面较光滑的球形(图1)。
实施例3:重组CFP-10蛋白纳米微粒增强BCG免疫效果评价
试验动物随机分为5组,每组9只小鼠,分为PBS(不免疫仅攻毒)、BCG对照组(BCG+PBS)、BCG+rCFP-10、BCG+PBS-NPs(BCG+空白纳米微粒)、BCG+rCFP-10-NPs(BCG+重组蛋白纳米微粒)。使用BCG进行首免(106CFU/只),4周后采用滴鼻方式进行纳米微粒第1次加强免疫(rCFP-10蛋白剂量为50μg/只),间隔2周,共3次。第3次免疫后的4周,每组随机选取3只小鼠进行相关免疫指标检测。其余通过滴鼻方式、使用NTSE-2菌株进行攻毒(1000CFU/只),4周后采样进行后续检测。
实施例4:重组CFP-10蛋白纳米微粒对BCG免疫小鼠产生IgA水平影响
攻毒前对小鼠支气管肺泡灌洗液中IgA含量测定来评估黏膜免疫水平。如图2所示,BCG首免后的加强免疫可以明显提高黏膜免疫水平,纳米微粒引起的黏膜免疫水平出现极显著高于BCG对照组(P<0.001),显著高于BCG+rCFP10组(P<0.05)。结果说明通过鼻腔免疫纳米微粒、特异性抗原都可以促进BCG首免的黏膜免疫水平。
实施例5:重组CFP-10蛋白纳米微粒对BCG免疫小鼠肺组织载菌量影响
攻毒四周后对小鼠肺脏载菌量检测,如图3,结果显示,与PBS组相比,各处理组肺脏的载菌量均不同程度减少,与BCG对照组相比,重组蛋白CFP-10组(P<0.05)和纳米微粒组(P<0.01)肺脏的载菌量显著减少,表明在BCG预免疫的基础上,重组蛋白CFP-10纳米微粒能显著减少感染小鼠肺脏中的载菌量。
此实施例仅为本发明较佳的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,可轻易想到的变化或替换,都应涵盖在本发明的保护范围之内。因此,本发明的保护范围应该以权利要求的保护范围为准。
Claims (3)
1.一种重组蛋白CFP-10纳米颗粒的制备方法,其特征在于,具体包括如下操作步骤:
(1)聚乳酸-羟基乙酸共聚物PLGA溶液的配置:将聚乳酸-羟基乙酸共聚物PLGA溶解于乙酸乙酯中形成有机相,PLGA终浓度为36mg/mL;
(2)重组蛋白CFP-10溶液的配置:将冻干的重组蛋白CFP-10粉末溶解于PBS溶液中作为内水相;
(3)初乳的制备:将步骤(2)中获得的所述内水相注入到步骤(1)形成的所述有机相中,超声形成初乳,其中超声处理的参数为:40%功率,超声2s,间隔2s,工作总时间6min;
(4)复乳的制备:将步骤(3)中形成的所述初乳注入到1%PVA溶液中,超声形成复乳,其中超声处理的参数为:60%功率,超声2s,间隔2s,工作总时间8min;
(5)纳米颗粒的形成:将步骤(4)形成的所述复乳倒入0.5%PVA溶液中,700rpm,搅拌3~4h,挥发油相,向制得的溶液中加入5倍体积的双蒸水进行洗涤,4℃、5000rpm,离心3min,洗涤2次,用适量无菌双蒸水重悬即得重组蛋白CFP-10纳米颗粒乳液。
2.根据权利要求1所述的制备方法,其特征在于,所述重组蛋白CFP-10纳米颗粒平均粒径约247nm,电位为-28.8mV,封率为80.53%。
3.根据权利要求1-2任一项所述制备方法获得的重组蛋白CFP-10纳米颗粒在制备预防结核杆菌疫苗中的应用。
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