基于序列和启动子改进的CA16 VLP表达量提升及其免疫原性检测
发布时间：2019-06-13 06:31
【摘要】：手足口病(hand,foot and mouth disease)作为一种婴幼儿易感的病毒性传染性疾病,其主要病原体是CA16(Coxsackievirus A16)和EV71(Enterovirus 71)。手足口病的流行并不具有明显的地域性,因此在全球范围内均有流行。该病多数病例伴随较为温和的临床症状,但是不乏严重神经系统疾病及死亡病例的出现。近年来亚太地区,尤其是中国爆发的大流行俨然使得手足口病成为儿童公共健康的最大威胁。缺乏特异性的治疗方法和有效的预防控制手段使得研发有效的疫苗预防该病的流行已是共识。中国大陆于2016年3月正式批准了EV71全病毒灭活疫苗上市,该疫苗对EV71引起的重症疾病具有良好的保护力,可有效预防EV71引发的手足口病。但此疫苗对于CA16感染缺乏交叉保护力,加之近年来手足口病流行呈现出CA16、EV71交叉流行的趋势,使得研发CA16疫苗刻不容缓。病毒样颗粒(Virus Like Particle,VLP)由于不含有病毒核酸,保留病毒完整的蛋白外壳,具有较好的免疫原性及安全性,已经逐渐成为疫苗研发的新候选形式。相比EV71 VLP疫苗的发展,CA16 VLP疫苗受限于较低的表达量而大幅滞后。因此,本论文选取昆虫杆状病毒表达系统并结合基因序列和启动子的改进拟解决该问题。因此,在昆虫杆状病毒表达系统基础上,对CA16 P1序列进行密码子优化(P1ori和P1opt),3CD序列进行替换(3CD和BJ3CD),并用ECMV启动子替换pFastBacTM Dual载体中P10(P10和Enhancer CMV,ECMV)。构建不同优化设计的重组质粒并摸索优化表达条件筛选得到一个高表达量的质粒,从而突破CA16VLP表达量低这一瓶颈,同时对纯化的CA16 VLP的抗原性及免疫原性进行评估。最终,本论文基于Bac-to-Bac系统共设计构建了Y(P1ori-3CD-PFD),BY(P1ori-BJ3CD-PFD),BU(P1opt-BJ3CD-PFD),BEY(P1ori-ECMV-BJ3CD-PFD),BEU(P1opt-ECMV-BJ3CD-PFD)5种重组质粒,并摸索优化了表达温度,收获时间及感染复数三个基本参数,最终确定蛋白表达优化参数为T=21℃,MOI=0.1,TOH=120 h。在这个优化参数下进行CA16 VLP的表达,重组质粒BU实现了表达量约5.7倍的提升,从原始表达量2.60 mg/L提高到14.7 mg/L。更为重要的是纯化的CA16 VLP与CA16灭活病毒颗粒具有相似的结构并在小鼠体内诱导产生较高的中和抗体和IgG抗体。高表达量的重组质粒设计和优化的表达参数会加快CA16 VLP量产的步伐。同时,ECMV启动子的调节作用减缓了昆虫细胞裂解及内容物的释放,从而促进了VLP蛋白分泌表达。综上所述,本论文的工作对提高CA16 VLP表达量做了一次有意义的尝试,并为解决CA16 VLP疫苗的生产瓶颈提供了途径。
[Abstract]:Hand, foot and mouth disease (hand,foot and mouth disease), as a viral infectious disease susceptible to infants and young children, is mainly caused by CA16 (Coxsackievirus A16) and EV71 (Enterovirus 71). The epidemic of HFMD is not obvious regional, so it is popular all over the world. Most cases of the disease are accompanied by mild clinical symptoms, but there is no lack of serious nervous system diseases and deaths. In recent years, outbreaks in the Asia-Pacific region, especially in China, have made HFMD the biggest threat to children's public health. The lack of specific treatment and effective prevention and control means make it a consensus to develop an effective vaccine to prevent the epidemic of the disease. In March 2016, mainland China officially approved the listing of EV71 inactivated vaccine, which has a good protective effect on severe diseases caused by EV71 and can effectively prevent hand, foot and mouth disease caused by EV71. However, this vaccine lacks cross protection against CA16 infection, and the epidemic of hand, foot and mouth disease in recent years shows the trend of CA16,EV71 cross epidemic, which makes the research and development of CA16 vaccine urgent. Virus-like particle (Virus Like Particle,VLP), which does not contain viral nucleic acid and retains the complete protein shell of the virus, has good immunogenicity and safety, and has gradually become a new candidate form for vaccine research and development. Compared with the development of EV71 VLP vaccine, CA16 VLP vaccine lags behind because of its low expression. Therefore, in this paper, the insect baculovirus expression system was selected and combined with the improvement of gene sequence and promoter to solve this problem. Therefore, on the basis of insect baculovirus expression system, CA16 P1 sequence was optimized (P1ori and P1opt), 3CD sequence was replaced (3CD and BJ3CD), and ECMV promoter was used to replace P10 (P10 and Enhancer CMV,ECMV) in pFastBacTM Dual vector. The recombinant plasmid with different optimization design was constructed and a high expression plasmid was obtained by optimizing the expression conditions, so as to break through the bottleneck of low expression of CA16VLP. At the same time, the antigenicity and immunogenicity of purified CA16VLP were evaluated. Finally, five kinds of Y (P1ori-3CD-PFD), BY (P1ori-BJ3CD-PFD), BU (P1opt-BJ3CD-PFD), BEY (P1ori-ECMV-BJ3CD-PFD), BEU (P1opt-ECMV-BJ3CD-PFD) recombinant plasmids were designed and constructed based on Bac-to-Bac system, and three basic parameters, expression temperature, harvest time and infection complex number, were optimized. The optimal parameters of protein expression were determined to be T 鈮，
本文编号：2498312
[Abstract]:Hand, foot and mouth disease (hand,foot and mouth disease), as a viral infectious disease susceptible to infants and young children, is mainly caused by CA16 (Coxsackievirus A16) and EV71 (Enterovirus 71). The epidemic of HFMD is not obvious regional, so it is popular all over the world. Most cases of the disease are accompanied by mild clinical symptoms, but there is no lack of serious nervous system diseases and deaths. In recent years, outbreaks in the Asia-Pacific region, especially in China, have made HFMD the biggest threat to children's public health. The lack of specific treatment and effective prevention and control means make it a consensus to develop an effective vaccine to prevent the epidemic of the disease. In March 2016, mainland China officially approved the listing of EV71 inactivated vaccine, which has a good protective effect on severe diseases caused by EV71 and can effectively prevent hand, foot and mouth disease caused by EV71. However, this vaccine lacks cross protection against CA16 infection, and the epidemic of hand, foot and mouth disease in recent years shows the trend of CA16,EV71 cross epidemic, which makes the research and development of CA16 vaccine urgent. Virus-like particle (Virus Like Particle,VLP), which does not contain viral nucleic acid and retains the complete protein shell of the virus, has good immunogenicity and safety, and has gradually become a new candidate form for vaccine research and development. Compared with the development of EV71 VLP vaccine, CA16 VLP vaccine lags behind because of its low expression. Therefore, in this paper, the insect baculovirus expression system was selected and combined with the improvement of gene sequence and promoter to solve this problem. Therefore, on the basis of insect baculovirus expression system, CA16 P1 sequence was optimized (P1ori and P1opt), 3CD sequence was replaced (3CD and BJ3CD), and ECMV promoter was used to replace P10 (P10 and Enhancer CMV,ECMV) in pFastBacTM Dual vector. The recombinant plasmid with different optimization design was constructed and a high expression plasmid was obtained by optimizing the expression conditions, so as to break through the bottleneck of low expression of CA16VLP. At the same time, the antigenicity and immunogenicity of purified CA16VLP were evaluated. Finally, five kinds of Y (P1ori-3CD-PFD), BY (P1ori-BJ3CD-PFD), BU (P1opt-BJ3CD-PFD), BEY (P1ori-ECMV-BJ3CD-PFD), BEU (P1opt-ECMV-BJ3CD-PFD) recombinant plasmids were designed and constructed based on Bac-to-Bac system, and three basic parameters, expression temperature, harvest time and infection complex number, were optimized. The optimal parameters of protein expression were determined to be T 鈮，
本文编号：2498312
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