柯萨奇病毒A组16型病毒样颗粒的制备及其免疫原性

Preparation and immunogenicity of virus-like particles of Coxsackievirus A16

目的应用Bac-to-Bac杆状病毒系统表达柯萨奇病毒A组16型(Coxsackievirus A16,CVA16)病毒样颗粒(virus-like particle,VLP),通过条件优化,提高VLP的表达量,纯化后检测其免疫原性。方法对CVA16结构蛋白基因P1及蛋白酶基因3CD进行密码子优化,插入启动子P10改造为CMV的重组杆状病毒表达载体p Fast Bac Dual-CMV中,获得重组穿梭质粒p Fast Bac Dual-CMV-P1-3CD,转化DH10 Bac TM Competent Cells,提取重组杆粒Bacmid-CMVP1-3CD,转染Sf9细胞,组装成重组杆状病毒Ac MNPV-CMV-P1-3CD,并进行扩增,采用间接免疫荧光法、Western blot法及透射电镜观察对表达产物进行初步鉴定。对重组CVA16 VLP的表达条件(病毒接种MOI值、感染时间及细胞培养方式)进行优化后,通过20%蔗糖垫层及氯化铯连续密度梯度离心法纯化CVA16VLP,并通过腹部皮下注射免疫ICR小鼠3次,采用间接ELISA法检测小鼠血清特异性Ig G抗体滴度,微量中和试验法检测血清中和抗体滴度。结果重组杆状病毒Ac MNPV-CMV-P1-3CD经PCR及测序鉴定证实组装成功;重组CVA16 VLP在Sf9细胞中成功表达,优化的表达条件为:重组杆状病毒Ac MNPV-CMV-P1-3CD按MOI=5接种,Sf9细胞采用悬浮培养方式悬浮培养96 h,收集细胞及培养上清;纯化的CVA16 VLP纯度可达90%以上,针对VP2的单克隆抗体能与VP0及VP2发生特异性反应,电镜下可见大量形态规则的类球形VLP,直径约为27 nm;纯化的CVA16 VLP免疫ICR小鼠后,诱导机体产生的特异性血清Ig G滴度可达1∶105,中和抗体滴度达1∶358。结论应用杆状病毒表达系统成功表达了CVA16的P1和3CD蛋白,并组装形成完整的VLP;通过质粒启动子的改造及表达条件的优化,有效提高了CVA16 VLP的表达量;纯化的CVA16 VLP可诱导小鼠产生特异性体液免疫应答,为CVA16亚单位疫苗的研究提供了参考。

Objective To express the virus-like particles（VLPs） of Coxsackievirus（CV） A16 by using Bac-to-Bac system,increase the expression level by optimization of expression condition,purify the expressed product and determine its immunogenicity. Methods The P10 promoter of vector p Fast Bac Dual was replaced by CMV promoter with a low starting efficiency. The codon optimized P1 and 3CD genes were cloned into the vector p Fast Bac Dual-CMV in which promoter P10 was modified to CMV. The obtained recombinant shuttle plasmid p Fast Bac dual-CMV-P1-3CD was transformed to DH10 BacTMCompetent Cells. Bacmid-CMV-P1-3CD was extracted and transfected to Sf9 cells for packaging,and the obtained recombinant baculovirus was propagated. The expressed product was preliminarily identified by indirect IFA,Western blot and electron microscopy. The expression condition of CVA16 VLP（MOI,infection time and cell culture method） was optimized,and CVA16 VLP was purified by 20% glucose cushion and cesium chloride continuous density gradient centrifugation,and injected s.c. into ICR mice for 3 times. Specific Ig G titer in murine sera was determined by indirect ELISA,while the neutralizing antibody titer by microneutralization test. Results PCR analysis and sequencing proved that recombinant baculovirus Ac MNPV-CMV-P1-3CD was assembled successfully as proved by PCR and sequencing,and recombinant CVA16 VLP was expressed successfully in Sf9 cells. The expression condition was optimized as follows：recombinant baculovirus Ac MNPV-CMV-P1-3CD was inoculated at a MOI of 5,while Sf9 cells were cultured in suspension for 96 h then the cells and culture supernatant were collected. The purified CVA16 VLP reached a purity of more than 90%. The monoclonal antibody against VP2 showed specific reactions with VP0 and VP2.A large quantity of spherical-like VLPs,at a mean diameter of about 27 nm,were observed under electron microscope.The specific serum Ig G titer induced in ICR mice by the purified CVA16 VLPs reached 1 ∶ 105,while the neutralizing antibody titer reached 1 ∶ 358. Conclusion The P1 and 3CD proteins of CVA16 were successfully expressed by Bac-toBac system and were assembled into intact VLPs. The modification of plasmid promoter and optimization of expression condition increased the expression level of CVA16 VLPs. Purified CVA16 VLPs induced specific humoral immune response in mice,which provided a reference for development of CVA16 subunit vaccine.