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中蜂囊状幼虫病毒结构蛋白VP2的密码子优化表达及其免疫原性研究 被引量:5

Optimized Expression of the VP2 Code of the Structural Protein of CSBV and its Immunogenicity
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摘要 利用生物信息学软件对编码中蜂囊状幼虫病毒(Chinese sacbrood virus,CSBV)VP2结构蛋白的密码子进行优化来提高VP2蛋白表达量,并进行免疫原性研究确定VP2蛋白的免疫原性。通过对17株囊状幼虫病毒(SBV)和7株中蜂囊状幼虫病毒(CSBV)的VP2基因序列进行比对,选取代表毒株(GenBank:HM237361.1)CSBV-LN株VP2基因作为参考序列,并用在CSBV所有毒株中保守性相对较强的氨基酸替换CSBV-LN株VP2对应位置的氨基酸,通过在线软件(http://www.jcat.de/和http://genomes.urv.es/OPTIMIZER/)对其密码子进行优化,构建原核表达质粒pET-28a-optiVP2,并转化入BL21(DE3)中,进行诱导表达。表达产物经SDS-PAGE和Westren blot鉴定正确后,将表达蛋白纯化并免疫至白来杭母鸡,对免疫鸡的抗体水平和淋巴细胞增值指数进行检测,研究CSBV VP2蛋白的免疫原性。同时,收集免疫后鸡蛋制备卵黄抗体,经纯化再与CSBV相互作用后,接种于3日龄蜜蜂幼虫,观察接种后幼虫死亡率,研究卵黄抗体中和CSBV病毒能力。结果显示,优化后的optiVP2能够在大肠杆菌中高效表达,其表达产物具有良好免疫原性,表达产物接种白来杭母鸡可诱导产生抗体,制备的卵黄抗体具有中和CSBV的作用。本实验实现了CSBV结构基因VP2在原核系统的高效表达,鸡免疫实验表明其具有良好免疫原性,并且可诱导鸡产生保护作用中和抗体,为进一步开发防治CSBV的生物制剂提供了帮助。 We optimized and enabled prokaryotic expression of the VP2-gene code of the structural protein of the Chinese sacbrood virus(CSBV)with bioinformatics software,and studied its immunogenicity.Through comparison of the VP2 gene sequence of 17 SBV and 7CSBV,we selected the VP2 gene sequence of CSBV-LN of GenBank HM237361.1as the sequence for reference.Relatively conventional amino acids were replaced by the amino acid in the corresponding position of VP2 of CSBV-LN/.The code was optimized through software(www.jcat.de/and http://genomes.urv.es/OPTIMIZER/)to construct the prokaryotic-expressed plasmid pET-28a-optiVP2.The latter was transformed into BL21(DE3)to induce expression.The expression product was purified and used to immunize White Leghorn hens after assessment by SDS-PAGE and western blotting.Then,indices of reproduction and the lymph system were measured,and the immunogenicity of the CSBV VP2 protein studied.Egg-yolk antibodies were prepared and,after purification and interaction with CSBV,were inserted into 3-day-old larvae.Then,the mortality rate of larvae was observed,and the virus capacity in egg yolks studied.Results showed that optimized optiVP2 could be expressed efficiently.The product had considerable immunogenicity and,after vaccination,antibodies could be induced by White Leghorn hens.The prepared yolk antibodies could neutralize CSBV.Our study showed efficient expression of VP2,which is the structural gene of CSBV in the prokaryotic expression system.Our study showed that White Leghorn hens have high immunogenicity and can induce antibody production,which may aid further development of biologic agents to counteract CSBV infection.
出处 《病毒学报》 CAS CSCD 北大核心 2017年第2期231-237,共7页 Chinese Journal of Virology
基金 国家自然科学基金项目(项目号:31372435) 题目:中蜂囊状幼虫病毒培养体系建立及其受体与受体结合蛋白筛选 辽宁省高等学校优秀科技人才支持计划(项目号:LR2015038) 题目:中蜂囊状幼虫病毒优势抗原表位筛选及其与宿主互作蛋白
关键词 中蜂囊状幼虫病毒(CSBV) VP2基因 系统进化 密码子优化 原核表达 Chinese sacbrood virus(CSBV) VP2 structural protein Phylogenic evolution Codon optimization Prokaryotic expression
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