间日疟疾传播阻断疫苗候选抗原Pvs25在家蚕杆状病毒表达系统中的表达  

Expression of Plasmodium vivax Malaria Transmission-blocking Vaccine Candidate Antigen Pvs25 in Silkworm Baculovirus Expression System

作　　者：盛稳稳 郭庆拓 陈剑清[1] 崔立旺[2] 张耀洲[1,3,4] 于威[1,5] 舒特俊[1] 盛清[1] 

机构地区：[1]浙江理工大学生物化学研究所,杭州310018 [2]宾夕法尼亚州立大学,美国宾夕法尼亚州16802 [3]天津市国际生物医药联合研究院,天津310450 [4]天津大学,天津310450 [5]浙江省家蚕生物反应器和生物医药重点实验室,杭州310018

出　　处：《蚕业科学》2014年第4期672-680,共9页ACTA SERICOLOGICA SINICA

基　　金：国家高技术研究发展计划"863"项目(No.2011AA100603);浙江省自然科学基金项目(No.207217)

摘　　要：间日疟疾传播阻断疫苗候选抗原Pvs25是间日疟原虫有性生殖期表达的表面蛋白,可以抑制疟原虫在媒介按蚊体内的有性生殖及孢子增殖,有效地阻断疟疾病原从按蚊向人体的传播。为了探索该疫苗高效、低成本生产的新途径,进行了Pvs25在家蚕杆状病毒表达系统中表面展示表达的初步试验。首先将经过非功能区去除和密码子优化的间日疟原虫Pvs25基因克隆至转移载体pFastBacHTb,构建重组质粒pET32a-Pvs25并转化E.coli Rosetta菌株,诱导后成功表达了Pvs25蛋白,利用Ni-NTA亲和层析的方法纯化重组Pvs25蛋白,免疫BALB/c雌鼠制备多克隆抗体,利用Protein A抗体纯化柱纯化后的抗体经间接ELISA法测定其效价为1∶12 800;利用Bac-to-Bac系统构建含Pvs25基因的重组杆状病毒vBmPvs25,接种BmN细胞和家蚕5龄幼虫,经SDS-PAGE与Western blotting分析表明Pvs25在BmN细胞和幼虫中均成功表达;构建vBmgp64-CMV-Pvs25表面展示重组病毒,检测到Pvs25在BmN细胞中表达并展示于细胞和杆状病毒囊膜上。研究结果为利用家蚕杆状病毒表达系统生产间日疟疾传播阻断候选疫苗奠定了一定的试验基础。Pvs25,a vivax malaria transmission-blocking vaccine candidate antigen,is a surface protein of Plasmodium vivax expressed during its sexual reproduction.It can inhibit sexual reproduction and spore reproduction of Plasmodium vivax parasite in media mosquitoes,thereby blocking the transmission of vivax malaria from media mosquitoes to human effectively.To explore new ways of producing this vaccine with high efficiency and low cost,a preliminary expression test of Pvs25 was carried out in baculovirus expression system of silkworm（Bombyx mori）.Firstly,Pvs25 was cloned into the transfer vector pFastBacHTb to construct recombinant plasmid pET32a-Pvs25.Then,it was transformed into E.coli Rosetta.Pvs25 protein wasexpressed successfully after induction.Recombinant protein Pvs25 was purified by Ni-NTA affinity chromatography and used to immune BALB /c female mice to produce polyclonal antibody.The titer of purified antibody was 1∶ 12 800 as determined by indirect ELISA.We used Bac-to-Bac system to construct recombinant virus vBmPvs25 and inoculated it into BmN cells and the 5th instar larvae of silkworm.SDS-PAGE and Western blotting analysis showed that Pvs25 was successfully expressed in both BmN cells and silkworm larvae.Furthermore,Pvs25 was displayed on BmN cells and viron membrane by recombinant virus vBmgp64-CMV-Pvs25.This study establishes a good basis for producing vivax malaria transmission-blocking candidate vaccine using baculovirus expression system of silkworm.

关 键 词：间日疟原虫 Pvs25蛋白 传播阻断疫苗 家蚕杆状病毒表达系统 表面展示 多克隆抗体 

分 类 号：S884.5[农业科学—特种经济动物饲养]