CN110179755A - 一种脂质体型亚单位疫苗的制备方法 - Google Patents
一种脂质体型亚单位疫苗的制备方法 Download PDFInfo
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Abstract
本发明提供一种脂质体型亚单位疫苗的制备方法,使病毒包膜蛋白或抗原表位多肽通过形成希夫碱的方式有效组装在脂质体表面,使疫苗具有与天然病毒颗粒相似的包膜结构,提高疫苗活性。
Description
技术领域
本发明涉及病毒疫苗领域,特别涉及一种脂质体型亚单位疫苗的制备方法。
背景技术
疫苗是预防和控制传染病最经济、最有效的手段,从最早的接种用牛痘,到现在多种多样的疫苗,为保护人类健康起到重要作用。
当前使用的疫苗主要有三种类型:减毒活疫苗、灭活疫苗、基因工程疫苗。减毒活疫苗是利用低毒性为代价诱导机体形成抗体,免疫效果不理想并有安全隐患。灭活疫苗只是在使用阶段使病毒灭活,要生产过程中仍是使用有感染性的病毒,生产环节存在风险。基因工程疫苗通过异源表达病毒的包膜蛋白诱导人体形成抗体,过程中不使用活性病毒,过程安全;但基因工程疫苗多是病毒包膜蛋白的胞外域部分,没有完整的病毒包膜蛋白,缺少了跨膜区与胞内域,不能保持天然病毒包膜蛋白的排布形式,免疫效果不如完整的病毒包膜蛋白。
发明内容
本发明为解决现有技术中存在不足,利用脂质体镶嵌病毒的包膜蛋白,使疫苗具有与天然病毒包膜的相似的包膜结构,提高疫苗的免疫活性。
本发明的发明构思是:利用脂质体模拟天然病毒的包膜结构,在脂质体表面组装病毒包膜蛋白或病毒抗原表位,用没有病毒基因组DNA的病毒颗粒类似物替代减毒病毒或病毒包膜蛋白进行机体免疫诱导。
为解决上述问题,本发明的技术方案如下:一种脂质体型亚单位疫苗的制备方法,包括以下步骤:
S1利用基因工程方法获取病毒包膜蛋白,或者利用多肽固相合成技术合成病毒抗原表位多肽;在病毒包膜蛋白或病毒抗原表位多肽制备时,要注意在病毒包膜蛋白或病毒抗原表位多肽末端直接进行赖氨酸修饰或通过甘氨酸连接链与赖氨酸连接,可以单端修饰或在氨酸端与羧基端同时修饰赖氨酸。
S2取带有芳环上醛基的人工类脂,与阳离子类脂或阴离子类脂制备脂质体,脂质体因为使用了荷电人工类脂可以带正电荷或负电荷;如天然磷脂、人工荷电类脂、胆固醇;
S3利用缓冲液使病毒包膜蛋白或病毒抗原表位多肽带电荷,并且与脂质体带电荷相反,将病毒包膜蛋白或病毒抗原表位多肽溶液与脂质体混合后,由于电荷相互作用,病毒包膜蛋白或病毒抗原表位多肽在脂质体表面富集,利用脂质体表面特异微环境催化病毒包膜蛋白或病毒抗原表位多肽的修饰赖氨酸的氨基与类脂的芳环上醛基形成希夫碱结构,将病毒包膜蛋白或病毒抗原表位多肽锚定在脂质体表面。
S4利用氨基酸将脂质体表面剩余的醛基封闭,结合病毒包膜蛋白或抗原表位多肽的脂质体,使其具有与病毒包膜类似的结构,提高疫苗活性。
优选地,所述的病毒包膜蛋白利用基因工程法合成。
优选地,所述的病毒抗原表位多肽由基因工程法合成或化学法合成。
优选地,脂质体可以使用天然磷脂、人工合成类脂、带有芳环上醛基的人工类脂、胆固醇等类脂组装。天然磷脂包括磷脂酰胆碱、磷脂酰丝氨酸、磷脂酰乙醇胺。
优选地,根据具体等电点调整病毒包膜蛋白或病毒抗原表位多肽荷电特性,使用缓冲液为磷酸盐缓冲液,也可以是其他类型缓冲液。
优选地,脂质体表面剩余的醛基可以利用氨基酸封闭,也可用稍过量的病毒包膜蛋白或病毒抗原表位多肽完成。
本发明中各技术特征具有以下作用:病毒包膜蛋白或病毒抗原表位多肽以共价键连接在脂质体载体表面并模拟天然病毒表面结构,可以实现多种病毒包膜蛋白或病毒抗原表位多肽在脂质体表面的组合锚定,能够模拟天然病毒包膜结构,提高疫苗的免疫活性。
病毒包膜蛋白或病毒抗原表位多肽是利用基因工程技术表达,不含病毒基因组DNA,避免造成潜在病毒感染危险;也可以使用化学法固相合成病毒抗原表位多肽。
病毒包膜蛋白或病毒抗原表位多肽通过共价键锚定在脂质体表面,锚定过程是利用脂质体表面特异微环境催化氨基与人工类脂芳环上的醛基形成希夫碱结构完成,锚定过程仅仅是依靠脂质体本身完成不需要引入额外的催化剂,人工类脂的芳环上的醛基是指苯甲醛、水杨醛、吡哆醛,也可以是其他芳香醛基。
与现有技术相比,本发明的制备方法是以病毒包膜蛋白或病毒抗原表位多肽锚定到脂质体表面制备疫苗,可形成与天然病毒类似的包膜结构,提高疫苗免疫活性。
附图说明
图1为实施例1获得产品的SEM照片;
图2为实施例2获得产品的SEM照片;
图3为实施例3获得产品的SEM照片。
具体实施方式
下面结合附图、通过具体实施例对本发明作进一步详述。以下实施例只是描述性的,不是限定性的,不能以此限定本发明的保护范围。本发明所应用的化学试剂以及仪器如未经特别说明,均可从商业渠道购买。
实施例1
利用固相合成法制备带状疱疹病毒的抗原表位多肽,以带有目标多肽羧基端氨基酸的王树脂进行多肽的固相合成,合成仪为美国CS BIO公司的CS136多肽合成仪;以带有Fmoc保护基团的氨基酸(CS BIO公司产品)进行多肽合成,用溶解于DMF溶剂中的20%哌啶溶液脱保护基,利用DIC等缩合剂进行多肽链延伸,直至合成抗原表位多肽,然后继续在抗原表位多肽的氨基端引入赖氨酸,最后利用TFA脱保护基同时将合成后的带有赖氨酸修饰的抗原表位多肽从树脂上切割下来,用冷乙醚清洗获得赖氨酸修饰的带状疱疹病毒的抗原表位多肽。
带有芳环上醛基的人工类脂按以下方法合成,仅以盐酸吡哆醛为的原料的人工类脂合成为例:(1)取50ml三口瓶,加入1g(4.9mmol)盐酸吡哆醛,再加入11ml精制甲醇搅拌回流30min,待其完全溶解后停止加热,此时溶液已由无色变为浅黄色。冷却至室温,加入NaHCO30.42g(4.9mmol),继续回流搅拌反应2h。停止加热,冷却至室温,抽滤除去NaHCO3固体,并用冷甲醇洗涤此固体两次,使产物充分滤出,减压蒸馏除去溶剂,得淡黄色固体。产物用乙醚洗涤两次,抽滤除去乙醚,真空干燥4h,得淡黄色固体产物812.4mg(91.4%)。TLC检测只有单一点,Rf=0.50(氯仿:甲醇=8:1,v/v,紫外灯观察)。(2)取容量约30ml的细颈安瓿瓶(ampule tube),加入451mg(2.5mmol)上步产物、7ml精制甲醇完全溶解,然后加入2.9g(8.3mmol)碘十六烷、8ml精制苯,振荡呈均一相。经过三次液氮冷冻-抽真空-解冻循环充分抽出安瓿瓶内空气,安瓿瓶细颈处用火烧熔密封,100℃下避光搅拌反应160h。160h后打开安瓿瓶TLC检测有五个点(氯仿:甲醇=8:1,v/v为展开剂,紫外灯下观察)减压蒸去溶剂。固体产物用乙醚洗涤两次,抽滤除去乙醚,TLC检测固体物质变为四个点(展开剂同上,与产物最接近点已被除去)。粗产物硅胶柱层析(氯仿:甲醇=8:1,v/v为淋洗剂)分离,收集Rf=0.5处物质。真空干燥5h,得白色固体产物210mg(16.3%)。Rf=0.55(氯仿:甲醇=8:1,v/v,紫外灯观察)。(3)取50ml圆底烧瓶,加入上步产物100mg(0.55mmol),用10ml重蒸甲醇完全溶解,另取一50ml圆底烧瓶,加入400mg(2.96mmol)无水CuCl2,10ml重蒸甲醇完全溶解。两次溶液混合,可观察到金黄色沉淀产生,常温下搅拌反应0.5h。结束反应,过滤除去沉淀,滤液减压蒸馏除去溶剂,加入氯仿及饱和食盐水振荡折入分液漏斗中,充分振荡,静止分层,有机相用饱和食盐水洗涤两次,相分离滤纸分离,减压蒸去溶剂,真空干燥5h,得白色固体产物78mg(94.7%)。Rf=0.55(氯仿:甲醇=8:1,v/v,紫外灯观察)。(4)取上步产物20mg溶于15ml异丙醇中,避光搅拌10min使其充分溶解,然后缓慢滴加15ml超纯水,速度控制在10min左右滴完。1N盐酸调节溶液pH为1.0,常温下避光搅拌反应2h,结束反应,减压蒸馏将溶液浓缩至约6ml,即保证完全除去异丙醇,此时反应液已变混浊,10ml精制二氯甲烷分两次从水溶液中萃取出产物,饱和食盐水洗涤三次,相分离滤纸分离出有机相,TLC分离(10×10GF254制备用硅胶板,展开剂氯仿:甲醇=10:1,v/v,反复多次展开,其间用紫外灯观察直至板上亮黄色部分与其他组分分开为止),氯仿甲醇混合液洗脱收集得亮黄色产物11mg(57.1%)。Rf=0.35(氯仿:甲醇=8:1,v/v,紫外灯观察)
将天然磷脂、胆固醇、荷电人工类脂、带有芳环上醛基的人工类脂按(2:1:1:1)混合,以乙醇注入法等方法制备获得脂质体。具体过程这:(1)使用电子天平准确称取0.015g脂质混合物于广口烧瓶中,加入1ml无水乙醇,于恒温加热磁力搅拌器热水浴中磁力搅拌至完全溶解;(2)用微量注射器以适当的速率将其注入到5ml缓冲液中,并不断地磁力搅拌,使其充分水化(pH8.2PBS缓冲液);(3)使用旋转蒸发仪旋转蒸发去除乙醇;(4)对得到的脂质体混悬液用超声波破碎仪于冰浴中超声10min,工作2s,停3s,功率40%,即得到脂质体溶液。以pH8.2磷酸盐缓冲液使疱疹病毒的抗原表位多肽负电荷,利用负电荷使疱疹病毒的抗原表位多肽富集在阳离子脂质体表面;在脂质体表面微环境条件下,疱疹病毒的抗原表位多肽末端修饰的赖氨酸的氨基与脂质体表面芳环上的醛基形成希夫碱,使疱疹病毒的抗原表位多肽锚定在脂质体表面,形成疱疹病毒的脂质体型亚单位疫苗。
实施例2
利用基因技术合成带状疱疹病毒的包膜糖蛋白E的包膜外结构域,并且在基因构建时为糖蛋白E的包膜外结构域的羟基端通过含30个甘氨酸的连接链(GGGGGAGGGGGSGGGGGAGGGGGAGGGGGSGGGGG)与末端赖氨酸连接,利用基因技术合成还有长链甘氨酸的多肽是常规技术,一般技术人员都可以利用基因工程技术与分离纯化技术获得赖氨酸修饰的带状疱疹病毒的糖蛋白E的包膜外结构域。
带有水杨醛残基的人工类脂合成方法如下:5-溴水杨醛(Cas:1761-61-1)利用实施例1的方法在碱性条件(NaHCO3)在甲醇体系中回流完成醛基保护,然后加入双疏水链取代胺化合物继续回流,反应后分离纯化;最后用酸脱保护即得带有水杨醛残基的人工类脂。
双疏水链取代胺是由带有疏水链的胺与烷基溴在碱性条件下合成,利用重结晶法纯化。具体方法如下:取250ml三口瓶,其中加入66g(0.27mol)正十六烷基胺和39.2g(0.13mol)十六烷基溴,用100ml无水乙醇溶解,再加入56.3g(0.41mol)K2CO3,回流搅拌反应165h。结束反应,抽滤并用热无水乙醇洗涤两次,使产物充分滤出,滤液室温过夜重结晶,晶体进行真空抽滤。重复重结晶四次,经TLC(氯仿:甲醇=9:1为展开剂)检测比对,知十六烷基胺被除去。再用正己烷进行重结晶,晶体抽滤,重复重结晶六次。滤饼真空干燥6h,得白色固体产物。TLC检测只有单一点,Rf=0.45(氯仿:甲醇=8:1,v/v,碘瓶显色)。产物17.2g(28.5%)。
将天然磷脂、胆固醇、荷电人工类脂、带有水杨醛残基的人工类脂按(3:1:1:2)混合,以薄膜分散法等方法制备获得脂质体。称取类脂溶于氯仿,氮气流吹扫下使氯仿挥发制成薄膜,30℃真空干燥彻底去除残留氯仿,加入缓冲液(50mmol/L),漩涡振荡5min,再用杯式超声仪超声处理5min(60W、脉冲间隔1s),得到相应的混合囊泡悬浮液。以pH8.2磷酸盐缓冲液使赖氨酸修饰的带状疱疹病毒的糖蛋白E的包膜外结构域带负电荷,并利用负电荷富集在阳离子脂质体表面;在脂质体表面微环境条件下,糖蛋白E的包膜外结构域末端修饰的赖氨酸的氨基与脂质体表面水杨醛残基形成希夫碱,使带状疱疹病毒的糖蛋白E的包膜外结构域锚定在脂质体表面,形成带状疱疹病毒的脂质体型亚单位疫苗。
实施例3
在乙肝病毒表面抗原基因的两端分别加上赖氨酸的编码序列(赖氨酸的密码子为AAA和AAG),利用化学合成法制备编码赖氨酸修饰的乙肝病毒表面抗原全长目的基因,按常规基因工程操作方法将目的基因与大肠杆菌表达载体pET28a重组后转化大肠杆菌DE3菌株并表达,利用生化分离技术获得氨基端与羧基端均直接进行赖氨酸修饰的乙肝病毒表面抗原蛋白。
按实施例1的方法制备乙肝病毒的脂质体型亚单位疫苗。将天然磷脂、胆固醇、荷电人工类脂、带有苯甲醛残基的人工类脂按(3:2:1:1)混合,以逆相蒸发法等方法制备获得脂质体。以pH5.8磷酸盐缓冲液使赖氨酸修饰的乙肝病毒表面抗原带正电荷,并利用正电荷富集在阴离子脂质体表面;在脂质体表面微环境条件下,乙肝病毒表面抗原的末端修饰赖氨酸的氨基分别与脂质体表面苯甲醛残基形成希夫碱,使乙肝病毒表面抗原锚定在脂质体表面,形成乙肝病毒的脂质体型亚单位疫苗。
以上所述,仅为本发明创造较佳的具体实施方式,但本发明创造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创造披露的技术范围内,根据本发明创造的技术方案及其发明构思加以等同替换或改变,都应涵盖在本发明创造的保护范围之内。
Claims (8)
1.一种脂质体型亚单位疫苗的制备方法,其特征在于,包括以下步骤:
S1利用基因工程方法或多肽固相技术制备病毒包膜蛋白或抗原表位多肽,并且在病毒包膜蛋白或抗原表位的氨基端和羧基端分别加上赖氨酸,赖氨酸与病毒包膜蛋白及抗原表位之间可以加甘氨酸间隔序列;
S2利用带有芳香环上醛基的类脂与阳离子类脂或阴离子类脂等荷电类脂共同组装脂质体;
S3利用静电吸附作用使病毒包膜蛋白或抗原表位多肽在脂质体表面富集,并通过芳香环上醛基与氨基在脂质体表面特异环境下形成希夫碱的连接反应使类脂的芳香环上醛基与病毒包膜蛋白或抗原表位多肽的赖氨酸氨基有效连接在一起;
S4利用氨基酸将脂质体表面剩余的醛基封闭,或者使用稍稍过量的病毒包膜蛋白或病毒抗原表位多肽保证脂质体表面的芳环上醛基全部反应。
2.根据权利要求1所述的脂质体型亚单位疫苗的制备方法,其特征在于,病毒包膜蛋白或病毒抗原表位多肽是以形成共价键的连接方式锚定在脂质体载体表面并模拟天然病毒表面结构,不是简单的电荷吸附。
3.根据权利要求1所述的脂质体型亚单位疫苗的制备方法,其特征在于,病毒包膜蛋白或病毒抗原表位多肽是利用基因工程技术表达获取,也可以利用多肽固相合成技术获得。
4.根据权利要求1所述的脂质体型亚单位疫苗的制备方法,其特征在于,锚定过程是利用脂质体表面特异微环境催化多肽中氨基与脂质体表面的芳环上醛基形成希夫碱的共价键结构完成。
5.根据权利要求4所述的脂质体型亚单位疫苗的制备方法,其特征在于,所述脂质体包括天然磷脂、人工合成阳离子类脂或阴离子类脂、含芳香环上醛基的类脂、胆固醇等共同组装,通过电荷吸附作用使带相反电荷的多肽吸附到脂质体表面。
6.根据权利要求1所述的脂质体型亚单位疫苗的制备方法,其特征在于,病毒包膜蛋白或病毒抗原表位多肽在氨基端和羧基端分别增加赖氨酸修饰,或只在一端进行赖氨酸修饰。
7.根据权利要求6所述的脂质体型亚单位疫苗的制备方法,其特征在于,赖氨酸修饰可以通过含有0-30个甘氨酸的连接链与病毒包膜蛋白或病毒抗原表位多肽连接。
8.根据权利要求1所述的脂质体型亚单位疫苗的制备方法,其特征在于,利用病毒包膜蛋白或病毒抗原表位多肽具有等电点的特性,通过不同酸碱度的缓冲溶液调节病毒包膜蛋白或病毒抗原表位多肽的电荷完成与带相反电荷脂质体的吸附富集,所用缓冲液是磷酸盐缓冲液或其他缓冲液。
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CN111494723B (zh) * | 2020-04-22 | 2021-10-12 | 苏州大学附属第一医院 | 一种微环境响应性免疫调控促神经再生微纳米纤维的制备方法 |
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