柯萨奇病毒A组16型病毒样颗粒的制备及其免疫原性

发布时间：2018-05-28 08:11

本文选题：手足口病 + 柯萨奇病毒A组型　；参考：《中国生物制品学杂志》2014年11期

【摘要】：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。
[Abstract]:Objective to express coxsackievirus A group 16 (Coxsackievirus A16) virus like particles like particle VLP by Bac-to-Bac baculovirus system, and to improve the expression of VLP by optimizing the conditions, and to detect its immunogenicity after purification. Methods the CVA16 structural protein gene P1 and protease gene 3CD were codon optimized and inserted into the recombinant baculovirus expression vector p Fast Bac Dual-CMV, which was transformed into CMV by promoter P10. The recombinant shuttle plasmid p Fast Bac Dual-CMV-P1-3 CD1 was obtained. The recombinant DH10 Bac TM Competent Cells, was transformed into Bacmid-CMVP1-3 CD. then transfected into Sf9 cells, the recombinant baculovirus Ac MNPV-CMV-P1-3 CD was assembled and amplified. The expression product was preliminarily identified by indirect immunofluorescence assay and transmission electron microscopy (TEM). After optimizing the expression conditions of recombinant CVA16 VLP (virus inoculation MOI value, infection time and cell culture mode), CVA16 VLP was purified by 20% sucrose cushion and cesium chloride continuous density gradient centrifugation, and ICR mice were immunized three times by abdominal subcutaneous injection. Indirect ELISA method was used to detect the titer of specific IgG antibody in serum of mice and the neutralization test was used to detect the titer of neutralized antibody in serum. Results the recombinant baculovirus ac MNPV-CMV-P1-3CD was successfully assembled by PCR and sequencing, and the recombinant CVA16 VLP was successfully expressed in Sf9 cells. The optimal expression conditions were as follows: the recombinant baculovirus ac MNPV-CMV-P1-3CD was inoculated with MOI=5 and cultured with Sf9 cells for 96 h, the cells were collected and supernatant was collected, and the purity of purified CVA16 VLP was more than 90%. Monoclonal antibodies against VP2 can react specifically with VP0 and VP2. A large number of regular spherical VLPs with a diameter of about 27 nm can be seen under electron microscope. The purified CVA16 VLP can be immunized with ICR mice. The titer of specific serum IgG and neutralizing antibody were 1: 105 and 1: 358 respectively. Conclusion the P1 and 3CD proteins of CVA16 were successfully expressed by baculovirus expression system, and the whole CVA16 VLP was assembled, and the expression level of CVA16 VLP was improved by the modification of plasmid promoter and the optimization of expression conditions. The purified CVA16 VLP can induce specific humoral immune response in mice and provide a reference for the study of CVA16 subunit vaccine.
【作者单位】：首都医科大学附属北京儿童医院北京市儿科研究所儿科学国家重点学科教育部儿科重大疾病研究重点实验室;中国疾病预防控制中心病毒病预防控制所卫生部医学病毒和病毒病重点实验室;
【基金】：(艾滋病和病毒性肝炎等重大传染病防治科技重大专项)病毒性传染病病原谱流行规律及变异研究(2013ZX10004202,2012ZX10004201-003)
【分类号】：R373.23;R392-33

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