CN110327462A - 一种脂质体疫苗及其制备方法 - Google Patents
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Abstract
本发明涉及一种脂质体疫苗及其制备方法，属于病毒疫苗技术领域。主要技术方案为：一种脂质体疫苗的制备方法，提取病毒抗原表位信息，选择病毒包膜蛋白的跨膜序列或者选择细胞上膜受体的跨膜序列，并将跨膜序列通过基因工程技术以基因的形式分别设计在串联病毒表位蛋白的两端，利用基因工程方法表达具有跨膜序列的串联病毒表位蛋白，保证串联病毒表位蛋白表达后能够有效镶嵌在脂质体上，构建脂质体疫苗。本发明利用跨膜序列将疫苗的抗原表位有效固定在脂质体上，是通过模拟天然病毒包膜蛋白的组装形式构建疫苗，有利于提高疫苗的免疫活性。
Description
技术领域
本发明涉及病毒疫苗技术领域，特别涉及一种脂质体疫苗及其制备方法。
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、亚单位疫苗。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。亚单位疫苗，尤其是针对具有包膜结构病毒的基因工程疫苗主要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结构，免疫效果不如完整的病毒颗粒。
发明内容
为解决现有技术中存在的不足，本发明利用脂质体镶嵌病毒的抗原表位蛋白，使疫苗具有与天然病毒颗粒相似的包膜结构，提高疫苗的免疫活性。
本发明的发明构思是：将病毒的抗原表位串联表达，将在串联表达的病毒抗原表位蛋白两端增加可以镶嵌于脂质体佐剂上的跨膜序列，通过两端的跨膜序列把串联表达的病毒抗原表位区镶嵌在脂质体表面，形成与天然病毒颗粒相似的包膜结构，提高疫苗的免疫活性。
本发明的技术方案如下：一种脂质体疫苗的制备方法，提取病毒抗原表位信息，利用基因工程方法表达具有跨膜序列的串联病毒表位蛋白用于疫苗制备。
进一步的，选择病毒包膜蛋白的跨膜序列或者选择细胞上膜受体的跨膜序列，并将跨膜序列通过基因工程技术以基因的形式分别设计在串联病毒表位蛋白的两端，保证串联病毒表位蛋白表达后能够有效镶嵌在脂质体上，构建脂质体疫苗。其中跨膜结构选自于病毒的包膜蛋白跨膜序列，如乙肝病毒的跨膜序列：LYSILSPFLPLLPIFFCLWVYI、FIIFLFILLLCLIFLLVLL等，带状疱疹的跨膜序列：A AWTGGLAAVVLLCLVIFLIC等。
进一步的，制备方法具体为：
S1利用生物信息学技术与结构生物学技术确定具体致病性病毒的抗原表位；
S2利用生物信息学技术与结构生物学技术确定获取病毒包膜蛋白的跨膜区序列，以及相应的编码基因；
S3设计甘氨酸肽链将各种抗原表位连接在一起，利用化学合成法获取目的基因，并且在目的基因两端分别设计跨膜序列的编码基因；
S4利用基因工程方法完成相应表达载体的构建，将构建好的表达载体转入受体细胞表达具有跨膜序列的串联病毒表位蛋白；
S5选择分离膜蛋白用表面活性剂，利用生化分离技术完成具有跨膜序列的串联病毒表位蛋白的分离纯化；
S6以卵磷脂与胆固醇制备脂质体，或与人工合成类脂共同制备脂质体；
S7分别用表面活性剂溶液溶解脂质体与串联病毒表位蛋白溶液，充分混合均匀；
S8通过逐步去除表面活性剂的方法使具有跨膜序列的串联病毒表位蛋白镶嵌在脂质体上；具体方法是利用含有适宜表面活性剂的溶剂(如：LDAO、Brij-35、Triton X-100、CHAPS、DDM、SC等)进行含有跨膜区的病毒包膜蛋白的分离与纯化，利用表面活性剂保证病毒包膜蛋白在溶液中的溶解性；把制备好的脂质体同样用表面活性剂溶解，可以保证与病毒包膜蛋白溶液混合过程中不会造成病毒包膜蛋白的沉淀或析出；表面活性剂能够保证病毒包膜蛋白与脂质体(类脂)充分混合，向混合溶液中加入可以吸附表面活性剂的疏水性树脂(也称beads，美国伯乐公司产品，型号：Bio-Beads SM-2)，疏水性树脂吸附表面活性剂同时使类脂与病毒包膜蛋白组装形成脂质体，通过离心去除疏水性树脂及其吸附的表面活性剂，最终获得镶嵌有病毒包膜蛋白的脂质体。也可以使用透析的方法缓慢去除表面活性剂，能够达到相同的效果。
进一步的，所述的脂质体为天然磷脂脂质体、天然磷脂、人工类脂组合装备的脂质体，脂质体可以含有胆固醇，也可以不含。
进一步的，使用甘氨酸肽链进行病毒抗原表位的连接，甘氨酸的数目为1-30个。
本发明同时请求保护按照所述方案制备得到的脂质体疫苗。
本发明的有益效果如下：本发明利用跨膜序列将疫苗的抗原表位有效固定在脂质体上，是通过模拟天然病毒包膜蛋白的组装形式构建疫苗，有利于提高疫苗的免疫活性。
具体实施方式
下面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
乙肝病毒表面抗原表位主要位于124-147序列中(CMTTAQGTSMYPSCCCTKPSDGNC)，将该序列与乙肝病毒的包膜蛋白跨膜序列(LYSILSPFLPLLPIFFCLWVYI)通过连接链(GGGGGAGGGGG)拼接在一起，利用化学合成法制备拼接多肽的全长基因，基因两端分别设计了限制性内切酶BamHI和XhoI位点，利用以上两个限制酶酶切位点将合成的拼接多肽编码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，利用表面活性剂LDAO促进溶解并完成纯化过程。
利用天然磷脂与胆固醇(质量比为5:1)制备脂质体，用表面活性剂LDAO溶解后，与乙肝病毒完整包膜蛋白全蛋白的表面活性剂LDAO溶液混合，混合均匀后逐级去除表面活性剂LDAO，制备镶嵌有乙肝病毒完整包膜蛋白全蛋白的脂质体，即：乙肝疫苗。乙肝病毒表面抗原表位主要位于124-147序列的编码基因：
TGCACGACTCCTGCTCAAGGAACCTCTATGTTTCCCTCATGTTGCTGTACCAAACCTACGGACGGAAACTGC
跨膜区的编码基因：
CTGTACAACATCTTGAGTCCCTTTATGCCGCTGTTACCAATTTTCTTtTGTCTTTGGGTATACATT
含有10个甘氨酸的连接链编码基因：
GGAGGTGGAGGCGGTGCTGGAGGCGGCGGTGGA。
实施例2
将带状疱疹病毒包膜上(糖蛋白E)中主要的抗原表位序列：RKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQED与AAWTGGLAAVVLLCLVIFLIC(带状疱疹病毒包膜上糖蛋白E的跨膜序列)拼接在一起，在两个序列中间引入(GGGGGAGGGGGSGGGGGAGGGGGAGGGGGSGGGGG)含有30个甘氨酸的连接链；最后利用化学合成法制备拼接全长序列的编码基因，与大肠杆菌表达载体pET30a重组后转化大肠杆菌DE3菌株并表达，利用表面活性剂Brij-35促进溶解并完成纯化过程。
利用天然磷脂与胆固醇(质量比为8:1)制备脂质体，用表面活性剂Brij-35溶解后，与乙肝病毒完整包膜蛋白全蛋白的表面活性剂Brij-35溶液混合，混合均匀后逐级去除表面活性剂Brij-35，制备镶嵌有带状疱疹病毒完整包膜蛋白全蛋白(糖蛋白E)的脂质体，即：带状疱疹疫苗。
实施例3
乙肝病毒表面抗原表位主要位于124-147序列中(CMTTAQGTSMYPSCCCTKPSDGNC)，将该序列与乙肝病毒的疱膜蛋白跨膜序列(FIIFLFILLLCLIFLLVLL)通过连接链(GGGGG)拼接在一起，利用化学合成法制备拼接多肽的全长基因，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，利用表面活性剂LDAO促进溶解并完成纯化过程。
利用天然磷脂与人工类脂(质量比为5:1)制备脂质体，用表面活性剂LDAO溶解后，与乙肝病毒完整包膜蛋白全蛋白的表面活性剂LDAO溶液混合，混合均匀后逐级去除表面活性剂LDAO，制备镶嵌有乙肝病毒完整包膜蛋白全蛋白的脂质体。将疫苗脂质体进行过滤除菌，制得具有完整包膜蛋白的乙肝病毒疫苗。
上述实施例只是用于对本发明的举例和说明，而非意在将本发明限制于所描述的实施例范围内。此外本领域技术人员可以理解的是，本发明不局限于上述实施例，根据本发明的教导还可以做出更多种的变型和修改，这些变型和修改均落在本发明所要求保护的范围内。
Claims (6)
1.一种脂质体疫苗的制备方法，其特征在于，提取病毒抗原表位信息，利用基因工程方法表达具有跨膜序列的串联病毒表位蛋白用于疫苗制备。
2.根据权利要求1所述的一种脂质体疫苗的制备方法，其特征在于，通过在串联病毒表位蛋白两端设计可以镶嵌于脂质体的跨膜序列，使具有跨膜序列的串联病毒表位蛋白镶嵌在脂质体上，构建脂质体疫苗。
3.根据权利要求1所述的一种脂质体疫苗的制备方法，其特征在于，包括以下步骤：
S1确定致病性病毒的抗原表位；
S2确定获取病毒包膜蛋白的跨膜区序列，以及相应的编码基因；
S3设计甘氨酸肽链，将抗原表位连接在一起，形成目的基因，在目的基因两端加入可组装到脂质体膜的跨膜序列的编码基因；
S4构建表达载体，将表达载体转入受体细胞表达具有跨膜序列的串联病毒表位蛋白；
S5分离纯化串联病毒表位蛋白；
S6制备脂质体；
S7分别用表面活性剂溶液溶解脂质体与串联病毒表位蛋白溶液，充分混合均匀；
S8使用逐步去除表面活性剂的方法将串联病毒表位蛋白镶嵌在脂质体上。
4.根据权利要求3所述的一种脂质体疫苗的制备方法，其特征在于，所述的脂质体为天然磷脂脂质体、天然磷脂或人工类脂组合装备的脂质体。
5.根据权利要求3所述的一种脂质体疫苗的制备方法，其特征在于，使用甘氨酸肽链进行病毒抗原表位的连接，甘氨酸的数目为1-30个。
6.一种根据权利要求1-3所述方法制备的脂质体疫苗。
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