基于序列和啟動子改進的CA16 VLP表達量提升及其免疫原性檢測
發(fā)布時間：2019-06-13 06:31
【摘要】：手足口病(hand,foot and mouth disease)作為一種嬰幼兒易感的病毒性傳染性疾病,其主要病原體是CA16(Coxsackievirus A16)和EV71(Enterovirus 71)。手足口病的流行并不具有明顯的地域性,因此在全球范圍內均有流行。該病多數(shù)病例伴隨較為溫和的臨床癥狀,但是不乏嚴重神經系統(tǒng)疾病及死亡病例的出現(xiàn)。近年來亞太地區(qū),尤其是中國爆發(fā)的大流行儼然使得手足口病成為兒童公共健康的最大威脅。缺乏特異性的治療方法和有效的預防控制手段使得研發(fā)有效的疫苗預防該病的流行已是共識。中國大陸于2016年3月正式批準了EV71全病毒滅活疫苗上市,該疫苗對EV71引起的重癥疾病具有良好的保護力,可有效預防EV71引發(fā)的手足口病。但此疫苗對于CA16感染缺乏交叉保護力,加之近年來手足口病流行呈現(xiàn)出CA16、EV71交叉流行的趨勢,使得研發(fā)CA16疫苗刻不容緩。病毒樣顆粒(Virus Like Particle,VLP)由于不含有病毒核酸,保留病毒完整的蛋白外殼,具有較好的免疫原性及安全性,已經逐漸成為疫苗研發(fā)的新候選形式。相比EV71 VLP疫苗的發(fā)展,CA16 VLP疫苗受限于較低的表達量而大幅滯后。因此,本論文選取昆蟲桿狀病毒表達系統(tǒng)并結合基因序列和啟動子的改進擬解決該問題。因此,在昆蟲桿狀病毒表達系統(tǒng)基礎上,對CA16 P1序列進行密碼子優(yōu)化(P1ori和P1opt),3CD序列進行替換(3CD和BJ3CD),并用ECMV啟動子替換pFastBacTM Dual載體中P10(P10和Enhancer CMV,ECMV)。構建不同優(yōu)化設計的重組質粒并摸索優(yōu)化表達條件篩選得到一個高表達量的質粒,從而突破CA16VLP表達量低這一瓶頸,同時對純化的CA16 VLP的抗原性及免疫原性進行評估。最終,本論文基于Bac-to-Bac系統(tǒng)共設計構建了Y(P1ori-3CD-PFD),BY(P1ori-BJ3CD-PFD),BU(P1opt-BJ3CD-PFD),BEY(P1ori-ECMV-BJ3CD-PFD),BEU(P1opt-ECMV-BJ3CD-PFD)5種重組質粒,并摸索優(yōu)化了表達溫度,收獲時間及感染復數(shù)三個基本參數(shù),最終確定蛋白表達優(yōu)化參數(shù)為T=21℃,MOI=0.1,TOH=120 h。在這個優(yōu)化參數(shù)下進行CA16 VLP的表達,重組質粒BU實現(xiàn)了表達量約5.7倍的提升,從原始表達量2.60 mg/L提高到14.7 mg/L。更為重要的是純化的CA16 VLP與CA16滅活病毒顆粒具有相似的結構并在小鼠體內誘導產生較高的中和抗體和IgG抗體。高表達量的重組質粒設計和優(yōu)化的表達參數(shù)會加快CA16 VLP量產的步伐。同時,ECMV啟動子的調節(jié)作用減緩了昆蟲細胞裂解及內容物的釋放,從而促進了VLP蛋白分泌表達。綜上所述,本論文的工作對提高CA16 VLP表達量做了一次有意義的嘗試,并為解決CA16 VLP疫苗的生產瓶頸提供了途徑。
[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，
本文編號：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，
本文編號：2498312
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