2019, Vol. 35中国科学院微生物研究所、中国微生物学会主办
文章信息
- 荣芮, 李婷婷, 张玉云, 顾颖, 夏宁邵, 李少伟
- Rong Rui, Li Tingting, Zhang Yuyun, Gu Ying, Xia Ningshao, Li Shaowei
- 昆虫杆状病毒表达载体系统在疫苗研究中的应用进展
- Progress in vaccine development based on baculovirus expression vector system
- 生物工程学报, 2019, 35(4): 577-588
- Chinese Journal of Biotechnology, 2019, 35(4): 577-588
- 10.13345/j.cjb.180301
-
文章历史
- Received: July 18, 2018
- Accepted: October 15, 2018
引用本文
|
2. 厦门大学 公共卫生学院 分子疫苗学与分子诊断学国家重点实验室，福建 厦门 361102
2. State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Public Health, Xiamen University, Xiamen 361102, Fujian, China
昆虫杆状病毒表达载体系统(Baculovirus expression vector system，BEVS)自20世纪80年代发现后，现已被广泛用于外源蛋白表达、药物筛选、疫苗开发、基因治疗等领域。BEVS因其安全性高、操作简便、可用于多亚基蛋白同时表达、适用于大规模培养等优势，具有极大的发展前景。文中将从BEVS的系统发展及其在疫苗开发中的应用等方面进行阐述。
1 杆状病毒杆状病毒(Baculovirus)是一类双链、环状闭合、基因组大小范围为80−180 kb的DNA病毒[1]。杆状病毒的形态一般为棍棒状，长度为200−400 nm，宽度为40-110 nm[2]。目前已分离得到了600多种杆状病毒，完成了近60多种杆状病毒的全基因组测序工作。根据国际病毒分类命名委员会第九次报告，杆状病毒共属一个科(Baculoviridae)，共4个属：甲型(Alphabaculovirus)、乙型(Betabaculovirus)、丙型(Gammabaculovirus)和丁型杆状病毒属(Deltabaculovirus)，它们分别以核型多角体病毒(Nuclear polyhedrosis virus，NPV)或颗粒体病毒(Granulovirus，GV)形式感染相应宿主[3]。此外，杆状病毒也可依据其含有包埋型病毒(Occlusion- derived virions，ODVs)的多少，分为3类：核型多角体病毒(含有较多个)、颗粒型病毒(含有一个)与非包埋型病毒(不含有)[4]。
其中，从苜蓿环纹夜蛾中分离出的苜蓿蠖核型多角体病毒(Autographa californica nuclear polyhedrosis virus，AcNPV)是目前研究最为广泛的杆状病毒之一，其基因组大小约为130 kb并含有154个开放阅读框[5]。AcNPV在其感染期内会有2种病毒形式：包埋型病毒，介导病毒在不同宿主之间的传播；芽生病毒(Budded virions，BVs)，介导病毒在同一宿主中不同部位的扩散[6]。BVs中有一种生物学功能较为突出的囊膜糖蛋白Gp64，它在病毒感染的早期和晚期均有表达，早期定位于细胞质，随后迁移至质膜。当杆状病毒通过质膜出芽时获得Gp64，缺失此蛋白时，将无法完成细胞间感染[7]。此外，Gp64糖蛋白也常与外源蛋白进行融合表达，用于表面展示技术，为增强蛋白免疫原性、蛋白的结构解析工作提供了有力支撑[8]。AcNPV的基因表达过程可分为4个阶段：立早期、早期、晚期和极晚期，其中多角体蛋白(Polyhedrin)和P10 (纤维网络相关)蛋白会在极晚期大量富集。由于多角体基因ph (polyhedrin，ph)和p10带有强启动子且编码蛋白为病毒扩增的非必要组分，所以可在删除上述基因的同时插入外源片段，诱导目的蛋白的大量表达[1]。
2 昆虫细胞杆状病毒的受体细胞为昆虫细胞。应用较广的细胞系主要有2种：草地夜蛾细胞系(Spodoptera frugiperda cell line，Sf)，常用Sf21及其分离株Sf9细胞[9]；粉纹夜蛾细胞系(Trichoplusia ni cell line，Tn)，常用商品化的High FiveTM (H5)细胞[10]。目前根据对这几种细胞系的报道及本实验室的工作总结发现：Sf9细胞更适合于重组病毒扩增及包装；Sf21因细胞直径更大，更适用于病毒滴度空斑实验观察；H5细胞更适用于分泌蛋白表达。新细胞系的建立更加丰富了BEVS的功能和应用，例如由Protein Science Corporation生产并已通过FDA认证，成功用于流感疫苗生产的express SF+[11]。由于野生型昆虫细胞难以对目的蛋白进行高甘露糖型糖基化修饰，部分科学家对宿主细胞进行改造，研发出可加强其糖基化复杂程度的MimicTM Sf9[12]。此外，还有通过锚蛋白Vankyrin改造技术延长了细胞生长周期的细胞系VE01/02与可以增加细胞活性及蛋白表达量的BTI-Tnao38等众多细胞系[13-14]。这些细胞均具有对AcNPV极度敏感、适应贴壁/摇瓶/WAVE生物波浪反应器等多种培养方式、适应于无血清培养条件和便于大规模生产等特点。
3 昆虫杆状病毒表达载体系统昆虫杆状病毒表达载体系统是以杆状病毒作为外源基因载体，以昆虫细胞作为宿主进行基因组自我扩增与目的蛋白的表达。昆虫杆状病毒表达载体需要质粒载体和野生型杆状病毒整合成含有外源基因的重组杆状病毒才能实现外源基因的表达。构建重组杆状病毒的关键在于构建穿梭转移载体：将目的基因插入到ph或p10启动子下游的多克隆位点，因转移载体中含有与野生型杆状病毒进行重组的同源序列，所以转移载体与亲本病毒DNA进行重组时将获得携带有外源基因的重组病毒。根据重组发生在昆虫细胞内或细胞外，构建重组杆状病毒可分为细胞内重组和细胞外重组两种方式。
3.1 重组病毒的胞内重组细胞内重组方式即使用转移载体和亲本杆状病毒共感染昆虫细胞，使外源序列通过与亲本杆状病毒基因组之间的同源序列发生重组，从而得到重组病毒。早期重组一般采用野生型杆状病毒，重组率极低。后期利用基因工程手段可在杆状病毒基因组ORF603与ORF1629中加入多个Bsu36Ⅰ酶切位点，从而得到去除了某些复制必需片段的线性亲本杆状病毒，使感染细胞后含有目的片段的重组杆状病毒的比例高达约90%[15]，BaculoGoldTM即采用这种方式。后续部分商业化试剂盒如BacMagicTM与flashBACTM又将必需片段ORF1629删除，并用细菌人工染色体(Bacterial artificial chromosome，BAC)取代ph结构基因，改造后的环状杆状病毒能在大肠杆菌中复制却不能在昆虫细胞中正常传代。当其与携带有目的基因的转移载体共转染昆虫细胞后，转移载体上的外源基因就会组装到ph启动子的下游，移除BAC并恢复ORF1629的基因功能。采用这种方法构建的重组杆状病毒质粒具有稳定性良好、储存时间较长并保持感染昆虫细胞的能力等优势[16]。
3.2 重组病毒的胞外重组通过胞外重组的方式得到含有目的片段的重组杆状病毒可大大提高重组杆状病毒的准确率。经典的Bac-to-Bac系统通过穿梭质粒(pFastBacTM)、辅助质粒(Helper)与杆粒(Bacmid)可在大肠杆菌中进行转座重组，将目的基因导入杆状病毒基因组中，随后再通过蓝白斑筛选技术得到正确的重组杆状病毒，便可直接进行后续的病毒转染实验[6, 17]。此外，Patel等也构建了酵母-昆虫细胞穿梭质粒载体，他们将啤酒酵母的自主复制序列(Autonomously replicating sequence，ARS)和着丝点功能相关序列(Centromeric sequence，CEN)引入杆状病毒基因组的结构基因ph中，使杆状病毒能够在酵母体内进行复制，并能够和相应的转移载体发生重组。这种载体的构建方法最快可以在10 d内得到重组病毒，避免了繁琐的空斑筛选，另外，此方法还能同时分离多个不同的重组子，所以是一个快速而高效的杆状病毒表达载体筛选系统，但这种构建方法的缺点是需要利用蔗糖密度梯度超速离心技术来确保基因组的稳定性[18]。
3.3 昆虫杆状病毒表达载体系统的优化近年来，为提高分泌蛋白的表达量，研究人员陆续开始探索新的技术和应用。例如在杆状病毒基因组中敲除v-cath和chiA基因，它们的缺失会增加分泌及膜靶向蛋白的产生，同时可显著延长宿主细胞的存活期[19-20]，BestBacTM、BacMagicTM、flashBACTM等商品化BEVS均采用此技术。本实验室前期研究也发现，敲除基因后的重组杆状病毒对流感病毒的血球凝集素蛋白(Hemagglutinin，HA)和人类获得性免疫缺陷病毒包膜糖蛋白Gp140的分泌表达具有明显提升作用。此外，有科学家利用病毒锚蛋白(Viral ankyrin/Vankyrin)共表达技术与可持续表达锚蛋白的细胞系进行目的蛋白的表达情况监测，研究结果表明此技术能有效调控病毒感染后的细胞凋亡过程，外源蛋白的表达周期可延长至6 d，同时目的蛋白的产量显著增加，相关载体pAcVE.02/03及细胞系VE-CL02均已商品化[13]。针对种类多样的BEVS，部分研究学者研发出了MultiBacTM与biGBacTM表达体系，它们均可通过多基因片段串联技术表达携带有多个亚基的蛋白复合体，这为解析蛋白质分子结构及相关生物学功能研究提供了很大支持[21-23]。2016年，Martínez-Solís等通过研究启动子相关功能，发现当orf46启动子与ph启动子连用或将p10启动子、OpIE2启动子与第五同源区段(Homologous region 5，hr5)串联使用时，会使目的蛋白的表达量大幅提升[24-25]。2018年，Zhang等向AcNPV相关载体中插入了一段可靶向凋亡蛋白酶1 (Caspase 1)的短发卡型RNA (Short-hairpin RNA)，抑制了细胞凋亡，外源蛋白表达量明显增加[26]。目前，应用较为广泛的商品化BEVS的相关信息见表 1。
| Name | Manufacturer | Baculovirus | Recombination method | Major advantages/disadvantages | Reference |
| Bac-to-Bac | Invitrogen | Circular, contain the lacZ gene | In vitro, transpositional recombination | Permits rapid isolation of recombinant baculovirus/Long-time and work-intensive in recombinant baculovirus screening | [6] |
| BacMagic | Merck Millipore | Circular, delete the v-cath(viral cathepsin L-like gene, v-cath)/ chiA(chitinase gene, chiA) gene | In vivo, homologous recombination | Faster baculovirus production without tedious plaque purification, maximize the expression of secreted protein | [27] |
| BestBac | Expression System | Linear, delete the v-cath/chiA gene | In vivo, homologous recombination | High yield of secreted protein/False positive interference | [27] |
| MultiBac | Geneva Biotech | Circular, contain several expression frame | In vitro, transpositional recombination | Suitable for VLP designs/Recombinant baculovirus is unstable | [28] |
| flashBAC | Oxford Expression Technologies Ltd | Circular, delete the v-cath/chiA/p26/ p10/p74 gene | In vivo, homologous recombination | Faster baculovirus production without tedious plaque purification, maximize the expression of secreted protein | [29] |
| BacMam | Thermo Fisher Scientific | Circular, contain the gateway technology | In vitro, LR recombination reaction | Suitable for multiple expression system/Recombinant baculovirus is unstable | [30] |
相比大肠杆菌、酵母、哺乳动物细胞等表达系统，昆虫杆状病毒表达载体系统具有以下独特优势：1)杆状病毒具有较高的种属特异性，专一寄生于无脊椎动物，安全性高；2)昆虫细胞可对外源蛋白进行翻译后修饰作用(如糖基化、磷酸化、乙酰化等)[31]；3)杆状病毒基因组中的ph/p10基因具有强启动子，可用于完成外源蛋白的高效转录；4)杆状病毒基因组可容纳大片段外源基因，据目前报道最高可容纳25个外源cDNA，具有较好的可塑性[22]，多启动子技术也使得BEVS可以同时表达多个蛋白，非常适合于结构复杂的抗原如病毒样颗粒(Virus-like particle，VLP)的构建[32-33]，相比大肠杆菌和酵母表达系统，BEVS的颗粒组装效率更高；5)相较哺乳动物细胞表达系统，BEVS在动物病毒VLP研究中应用更为广泛，由其生产的疫苗在医药审批程序上有望通过绿色通道获得更快的审批途径。
4 昆虫杆状病毒表达载体系统与疫苗研制自1983年Smith等首次利用BEVS成功表达纯化出人β-干扰素后，BEVS在近30多年来已被进一步优化，广泛用于实验室研究及商业化生产，例如葛兰素史克公司(GlaxoSmithKline，GSK)基于BEVS生产的人乳头状瘤病毒疫苗Cervarix®[34]和Protein Sciences Corporation生产的流感疫苗FluBlok®[35]。其中，VLP是一类不含基因组或不具感染性的蛋白多聚物，VLP和真病毒颗粒的构象与组成成分高度相似并可将抗原表位最大程度地展示在其表面。因VLP不含有病原体的遗传物质，所以其安全性远远高于灭活或减毒疫苗，这使VLP在疫苗研究中更具优势，BEVS因其具有的独特优势在VLP疫苗研究和生产中具有重要应用前景。
4.1 昆虫杆状病毒表达载体系统与商品化疫苗至今为止，利用BEVS生产并已成功上市的疫苗有7种，详细信息见表 2。其中由GSK公司研制的疫苗Cervarix®是利用BEVS生产VLP疫苗的成功代表，Cervarix®为二价苗，主要预防HPV16、18两个型别，它是利用BEVS共表达HPV16、18型别的结构蛋白L1 (L1蛋白为HPV颗粒表面的主要结构蛋白)，再经自组装形成具备免疫原性但丧失感染能力的VLP。一系列临床试验也证实了Cervarix®的安全性、保护效果(除HPV16与18型别，还可对HPV31、33、53与58型别具有一定程度的交叉保护效果)、持久性(有效保护时间≥6.4年)等[36-39]。与其他HPV预防性疫苗Gardasil® (四价，酿酒酵母表达系统)、GARDASIL® (九价，酿酒酵母表达系统)与正处于临床Ⅲ期的馨可宁(二价，大肠杆菌表达系统)相比，Cervarix®具有以下独特优势：1)针对HPV16、18型别，Cervarix®在不同年龄受试组中可诱导机体产生远高于Gardasil®免疫组的抗血清滴度，同时也能诱导机体生成更多的效应T细胞与记忆B细胞[40]；2)相较生产成本低、周期短的大肠杆菌来源的馨可宁，Cervarix®几乎不含有内毒素，产品稳定性更好，批间差更小。
| Disease Human vaccines |
Vaccines’ name | Manufacturer | Antigen | Reference |
| Influenza virus | FluBlok® | Protein Sciences Corporation | HA | [35] |
| Human papillomavirus Animal vaccine |
Cervarix® | GSK | HPV16/18 L1 protein | [36] |
| Classical swine fever | Porcilis® Pesti | MSD Animal Health | E2 protein | [47] |
| BAYOVAC CSF E2® | Bayer AG/Pfizer Animal Health | E2 protein | [48] | |
| Porcine circovirus-2 | CircoFLEX® | B. Ingelheim | PCV2 ORF2 protein | [49] |
| Cirumvent® PCV | Merck Animal Health | PCV2a Cap protein | [50] | |
| Porcilis® PCV | MSD Animal Health | PCV2 ORF2 protein | [51] |
利用BEVS生产亚单位疫苗的成功代表有FluBolk®。针对流感病毒，市面上的灭活、减毒疫苗存在着一些不足之处：利用鸡胚细胞大规模制备疫苗原材料耗时长；成品疫苗中可能存在致病毒株的逃逸突变、含有化学剂福尔马林与致敏原鸡卵清白蛋白的潜在危害等[41]。FluBlok®于2013年由美国食品药品监督管理局批准上市，用以预防流感病毒的大面积爆发。它是由Protein Sciences Corporation利用BEVS研制并已成功上市的世界上第一支针对流感病毒的三价重组疫苗(Recombination HA vaccine，rHA vaccine)，其免疫原由3种不同流感病毒株的HA等量组合而成(3种病毒株分别来自甲型流感病毒中的H1、H3亚型与乙型流感病毒中的Y/V亚系)。临床试验显示相比同剂量的三价灭活疫苗(Trivalent inactivated vaccine，TIV)，rHA vaccine能保证在无明显毒副作用的前提下诱导机体生成更高滴度的抗血清，同时也能更为有效地刺激机体进行相关免疫应答[11, 42-44]。2016年，FDA又通过了Flubolk®四价流感疫苗，同时覆盖了突发率较高的H1、H3亚型与Y/V亚系。
除人用疫苗外，BEVS在兽用疫苗领域也作出了较大贡献，针对猪瘟病与二型猪圆环病毒，已有5种成功上市的预防性疫苗，它们的出现极大程度上缓解了畜牧业的生产成本及养殖风险。前期，欧洲药品管理局针对Porcilis® Pesti与BAYOVAC CSF E2®指导了一系列临床评估实验，结果表明接种过其中任意一种疫苗的小猪均能有效防止猪瘟病毒在体内的繁殖，相比对照组，实验组的小猪生长状态均良好[45-46]。B. Ingelheim与Merck Animal Health针对二型猪圆环病毒所生产的预防性疫苗均为亚单位疫苗，CircoFLEX®、Cirumvent® PCV与Porcilis® PCV都适用于三周龄以上的小猪，针对刚出生的小猪(≥3 d)，可使用Cirumvent® PCV对其进行1 mL剂量的双针免疫策略。
4.2 昆虫杆状病毒表达载体系统与临床研究阶段疫苗除了已被批准上市的数种疫苗，还有很多利用BEVS生产的疫苗候选物正处于临床试验阶段，详细信息见表 3。
| Disease | Manufacturer | Antigen | Development stage | Reference |
| Type Ⅰ diabetes | Diamyd | Glutamate decarboxylase (GAD) | Phase Ⅱ (NCT00435981) |
[52-53] |
| Influenza virus | Novavax | Hemagglutinin (HA) | Phase Ⅱ (NCT03293498) |
[58] |
| Influenza virus | Novavax | Hemagglutinin (HA)、neuraminidase (NA) and matrix 1 (M1) | Phase Ⅱ (NCT01072799) |
[59] |
| Norwalk virus | Ligocyte | Norwalk virus-VLP | Phase Ⅰ (NCT00806962) |
[54] |
| Norwalk virus | Baylor College of Medicine | Norwalk virus-VLP | Phase Ⅰ/Ⅱ (NCT00973284) |
[55] |
| Human parvovirus B19 | National Institute of Allergy and Infectious Diseases/Meridian Life Science | VP1 and VP2 | Phase Ⅰ/Ⅱ (NCT00379938) |
[60] |
| Respiratory syncytial virus | Novavax | Fusion glycoprotein | Phase Ⅰ (NCT01709019) |
[56-57, 61] |
Ⅰ型糖尿病为一种较为常见的自身免疫疾病，其中谷氨酸脱羧酶(Glutamic acid decarboxylase，GAD65)为主要的在研靶标[52]。2005年，Diamyd公司以人GAD65与氢氧化铝联合使用的方式对治疗成年型糖尿病开展了长达5年的试验追踪，结果表明由BEVS生产的GAD65具有很好的安全性并能有效缓解人体内C肽的释放[53]。2017年，Beam等通过贝叶斯分析法对145位受试者进行了数据采集及分析，证实了此疫苗候选物确实能够有效减缓受试者体内C肽的下降速率[52]。
诺瓦克病毒(Norwalk virus)能诱发急性肠胃炎、全身肌肉痛等不良症状，对公民健康造成了严重威胁。Ligocyte公司与Baylor College of Medicine医疗中心利用BEVS生成了Norwalk virus VLP并分别开展了相关临床试验，试验结果表明Norwalk virus VLP具有较好安全性并能有效预防部分病毒的入侵[54-55]。
呼吸道合胞病毒(Respiratory syncytial virus，RSV)是诱发一系列呼吸道疾病的主要病原体之一，相关病例在小孩及老年人中较为常见。2012年，Novavax公司利用BEVS表达出RSV外壳表面的Fusion糖蛋白，后续将其加工成直径约为40 nm的蛋白颗粒疫苗候选物并在棉鼠动物模型上证实了此疫苗能诱发机体产生IgG型中和抗体，此外还能有效抑制RSV在肺部的扩增[56]。随后，Novavax对其安全性及免疫原性开展了Ⅰ期临床试验，相关结果表明受试者接种RSV F蛋白颗粒疫苗候选物后无明显不良症状，并且最快可在7 d后检测到相关免疫应答[57]。
4.3 昆虫杆状病毒表达载体系统与临床前疫苗 4.3.1 昆虫杆状病毒表达载体系统在临床前亚单位疫苗中的研究昆虫杆状病毒表达载体系统已广泛应用于流感疫苗的生产研究，体系较为成熟，该系统对不同型别的基于HA的疫苗候选物均有较好的表达水平。大部分流感亚单位疫苗已处于临床研究阶段。近期针对禽流感病毒H6N1，Hsieh等报道了利用双顺反子昆虫杆状病毒表达载体系统开展的相关研究。他们通过使用pFastBacTM载体与DH10Bac细胞获得了可进行H6N1毒株HA1分泌表达的重组杆状病毒，后续的动物实验也表明此新型亚单位疫苗相较鸡胚细胞来源的减毒病毒疫苗能诱导小鼠体内生成更高滴度的抗血清[62]。这些前瞻性研究为缓解相关行业的生产压力，打开相应疫苗市场作出了贡献。
4.3.2 昆虫杆状病毒表达载体系统在临床前VLP疫苗中的研究轮状病毒(Rotavirus)是造成婴幼儿患严重腹泻病的主要病原体，全球每年因感染轮状病毒而死的儿童大约有60万，其中80%均发生在条件落后的国家[63]。目前，已纳入国家免疫计划的轮状病毒疫苗有单价苗Rotarix® (GSK公司生产)和五价苗Rotateq® (MSD公司生产)，均为减毒人用疫苗[64]。此外也有针对轮状病毒外衣壳表面主要的中和抗原VP4、VP7及其他次要抗原(VP3、VP6与VP1等)设计VLP用以筛选疫苗候选物的相关文献报道。Crawford等利用BEVS表达了VP2/6/7与VP2/4/6/7这两种VLP，实验结果表明它们在颗粒形态与中和能力上都和野生型轮状病毒相似，这为后续阐明轮状病毒入胞机制和研发轮状病毒治疗性疫苗提供了坚实基础[65]。
寨卡病毒(Zika virus，ZIKV)为黄病毒科中的黄病毒属，轻微感染ZIKV时可诱发发热、斑状丘疹等症状，严重时会损害成人神经系统或通过母婴传播造成新生儿小头并发症等严重后果。目前针对ZIKV的疫苗候选物正在积极研制中，暂无可用疫苗[66-67]。2018年Dai等报道通过Bac-to-Bac系统共表达膜前体蛋白PrM/M与包膜蛋白E，构建了ZIKV VLP。实验结果显示ZIKV VLP的免疫原性虽然略低于灭活的ZIKV，但也仍能刺激小鼠体内产生较强的Th1与Th2类免疫反应，激发小鼠体内的T细胞大量富集，这对病毒的清除是至关重要的。此外，ZIKV VLP可刺激小鼠体内产生大量的IgG型中和抗体，这也为后续的疫苗研发提供了新的研究思路[68]。
手足口病是一种在幼童中较为常见并具有高致病性的传染性疾病，主要由肠道病毒71 (Enterovirus 71，EV71)与科萨克病毒(Coxsackieviruses) A16、A6和A10引起。最近，Zhang等利用BEVS构建了EV71/CVA6/CVA10/CVA16四价VLP疫苗候选物并针对其免疫原性、交叉保护效果与持久性等方面开展了相关实验。实验结果表明四价VLP疫苗候选物相较单价VLP疫苗能刺激小鼠体内型特异性的长时间免疫应答，当小鼠被动免疫此疫苗时，能有效预防单种或多种病毒混合物的入侵[69]。
4.4 昆虫杆状病毒表达载体系统表面展示技术在疫苗中的应用BEVS表面展示技术一般通过目的蛋白与病毒衣壳蛋白Gp64融合表达的方式将外源蛋白成功展示在病毒表面，此技术具有可指定展示部分功能性表位、增强蛋白免疫原性等优点，并为药物高通量筛选、临床诊断治疗、蛋白质组学等领域提供了便利。Yoshida等针对疟疾构建了AcNPV- CSPsurf重组杆状病毒，成功将伯氏疟原虫的环子孢子蛋白与杆状病毒外壳上的Gp64融合表达[70]。此外，BEVS表面展示技术在流感病毒、肠道病毒等相关领域也有不少发现，为相关疫苗候选物的开发工作提供了更多选择[71-73]。2017年，Lee等利用Bac-to-Bac系统获得了Bac-RSVM2重组杆状病毒，从而诱导小鼠体内生成了大量CD8+ T细胞[74]。2016年，Sim等利用BEVS构建了rBac-HA重组杆状病毒，相关动物实验已证实rBac-HA能诱导小鼠体内产生较强的免疫应答并对其他亚型的流感病毒具有广谱保护效果[75]。
5 结语经过近40年的发展，昆虫杆状病毒表达载体系统(BEVS)已成为外源重组蛋白表达的有力工具。目前较为前沿的研究中，基于结构生物学对VLP或亚单位疫苗进行设计，再利用BEVS进行抗原制备及相关研究已逐见报道。载体设计的优化、部分基因的敲除/敲入与重组病毒分离技术的简化均加快了疫苗前期的开发流程，此外，阐明杆状病毒如何利用宿主细胞进行自身复制的机制、促进重组杆状病毒在宿主细胞内更为高效地进行目的蛋白的自折叠及糖侧链的加工修饰依旧是扩大BEVS应用前景的关键之处。随着分子生物学技术手段的快速进步与医药行业的蓬勃发展，期望BEVS能借助其独特优势在蛋白制备、疫苗研发、蛋白结构解析及药物设计等方面为人类健康作出更多贡献。
| [1] | Kong M, Zuo H, Zhu FF, et al. The interaction between baculoviruses and their insect hosts. Dev Comp Immunol, 2018, 83: 114–123. DOI: 10.1016/j.dci.2018.01.019 |
| [2] |
Liu YP, Wang FH, Sun ZJ, et al. Progress of studies and application on baculovirus expression vector system.
Chin Bull Entomol, 2006, 43(1): 1–5.
(in Chinese). 刘永平, 王方海, 苏志坚, 等. 昆虫杆状病毒表达载体系统的研究及应用. 昆虫知识, 2006, 43(1): 1-5. DOI:10.3969/j.issn.0452-8255.2006.01.001 |
| [3] | King AMQ, Adams MJ, Carstens EB, et al. Ninth Report of the International Committee on Taxonomy of Viruses. London: Elsevier, 2012. |
| [4] | Blissard GW, Rohrmann GF. Baculovirus diversity and molecular biology. Annu Rev Entomol, 1990, 35: 127–155. DOI: 10.1146/annurev.en.35.010190.001015 |
| [5] | Smith GE, Summers MD, Fraser MJ. Production of human beta interferon in insect cells infected with a baculovirus expression vector. Mol Cell Biol, 1983, 3(12): 2156–2165. DOI: 10.1128/MCB.3.12.2156 |
| [6] | Sokolenko S, George S, Wagner A, et al. Co-expression vs. co-infection using baculovirus expression vectors in insect cell culture: benefits and drawbacks. Biotechnol Adv, 2012, 30(3): 766–781. DOI: 10.1016/j.biotechadv.2012.01.009 |
| [7] |
Feng M, Wu XF. Research progresses in the interaction between baculovirus envelope protein gp64 and host cell surface factors.
Sci Seric, 2014, 40(5): 911–916.
(in Chinese). 冯敏, 吴小峰. 昆虫杆状病毒囊膜蛋白GP64与宿主细胞表面因子互作的研究综述. 蚕业科学, 2014, 40(5): 911-916. |
| [8] | Hu YC, Yao K, Wu TY. Baculovirus as an expression and/or delivery vehicle for vaccine antigens. Expert Rev Vaccines, 2008, 7(3): 363–371. DOI: 10.1586/14760584.7.3.363 |
| [9] | Lee GJ, Lee SH, Lee YJ, et al. Nanostructural characterization of Sf9 cells during virus-like particles generation. Scanning, 2016, 38(6): 735–742. DOI: 10.1002/sca.v38.6 |
| [10] | Krammer F, Schinko T, Palmberger D, et al. Trichoplusia ni cells (High FiveTM) are highly efficient for the production of influenza A virus-like particles: a comparison of two insect cell lines as production platforms for influenza vaccines. Mol Biotechnol, 2010, 45(3): 226–234. DOI: 10.1007/s12033-010-9268-3 |
| [11] | Baxter R, Patriarca PA, Ensor K, et al. Evaluation of the safety, reactogenicity and immunogenicity of FluBlok® trivalent recombinant baculovirus- expressed hemagglutinin influenza vaccine administered intramuscularly to healthy adults 50-64 years of age. Vaccine, 2011, 29(12): 2272–2278. DOI: 10.1016/j.vaccine.2011.01.039 |
| [12] | Legardinier S, Klett D, Poirier JC, et al. Mammalian-like nonsialyl complex-type N-glycosylation of equine gonadotropins in MimicTM insect cells. Glycobiology, 2005, 15(8): 776–790. DOI: 10.1093/glycob/cwi060 |
| [13] | Steele KH, Stone BJ, Franklin KM, et al. Improving the baculovirus expression vector system with vankyrin-enhanced technology. Biotechnol Prog, 2017, 33(6): 1496–1507. DOI: 10.1002/btpr.2516 |
| [14] | Hashimoto Y, Zhang S, Zhang SY, et al. Erratum to: BTI-Tnao38, a new cell line derived from Trichoplusia ni, is permissive for AcMNPV infection and produces high levels of recombinant proteins. BMC Biotechnol, 2012, 12: 12. DOI: 10.1186/1472-6750-12-12 |
| [15] | Kitts PA, Possee RD. A method for producing recombinant baculovirus expression vectors at high frequency. Biotechniques, 1993, 14(5): 810–817. |
| [16] | Hitchman RB, Possee RD, King LA. Baculovirus expression systems for recombinant protein production in insect cells. Recent Pat Biotechnol, 2009, 3(1): 46–54. |
| [17] | Luckow VA, Lee SC, Barry GF, et al. Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli. J Virol, 1993, 67(8): 4566–4579. |
| [18] | Patel G, Nasmyth K, Jones N. A new method for the isolation of recombinant baculovirus. Nucleic Acids Res, 1992, 20(1): 97–104. DOI: 10.1093/nar/20.1.97 |
| [19] | Kaba SA, Salcedo AM, Wafula PO, et al. Development of a chitinase and v-cathepsin negative bacmid for improved integrity of secreted recombinant proteins. J Virol Methods, 2004, 122(1): 113–118. DOI: 10.1016/j.jviromet.2004.07.006 |
| [20] | Ishimwe E, Hodgson JJ, Passarelli AL. Expression of the Cydia pomonella granulovirus matrix metalloprotease enhances Autographa californica multiple nucleopolyhedrovirus virulence and can partially substitute for viral cathepsin. Virology, 2015, 481: 166–178. DOI: 10.1016/j.virol.2015.02.022 |
| [21] | Bieniossek C, Imasaki T, Takagi Y, et al. MultiBac: expanding the research toolbox for multiprotein complexes. Trends Biochem Sci, 2012, 37(2): 49–57. DOI: 10.1016/j.tibs.2011.10.005 |
| [22] | Weissmann F, Petzold G, VanderLinden R, et al. biGBac enables rapid gene assembly for the expression of large multisubunit protein complexes. Proc Natl Acad Sci USA, 2016, 113(19): E2564–E2569. DOI: 10.1073/pnas.1604935113 |
| [23] | Zhang ZG, Yang J, Barford D. Recombinant expression and reconstitution of multiprotein complexes by the USER cloning method in the insect cell-baculovirus expression system. Methods, 2016, 95: 13–25. DOI: 10.1016/j.ymeth.2015.10.003 |
| [24] | Martínez-Solís M, Gómez-Sebastián S, Escribano JM, et al. A novel baculovirus-derived promoter with high activity in the baculovirus expression system. PeerJ, 2016, 4(11): e2183. |
| [25] | Bleckmann M, Schürig M, Chen FF, et al. Identification of essential genetic baculoviral elements for recombinant protein expression by transactivation in Sf21 insect cells. PLoS ONE, 2016, 11(3): e0149424. DOI: 10.1371/journal.pone.0149424 |
| [26] | Zhang XY, Xu KY, Ou YM, et al. Development of a baculovirus vector carrying a small hairpin RNA for suppression of sf-caspase-1 expression and improvement of recombinant protein production. BMC Biotechnol, 2018, 18: 24. DOI: 10.1186/s12896-018-0434-1 |
| [27] |
Liu Q, Ding C. Advances in research of partial functional genes of baculovirus.
Chin J Virol, 2001, 17(2): 183–187.
(in Chinese). 刘强, 丁翠. 杆状病毒部分功能基因的研究进展. 病毒学报, 2001, 17(2): 183-187. DOI:10.3321/j.issn:1000-8721.2001.02.020 |
| [28] | Berger I, Fitzgerald DJ, Richmond TJ. Baculovirus expression system for heterologous multiprotein complexes. Nat Biotechnol, 2004, 22(12): 1583–1587. DOI: 10.1038/nbt1036 |
| [29] | Hitchman RB, Possee RD, Crombie AT, et al. Genetic modification of a baculovirus vector for increased expression in insect cells. Cell Biol Toxicol, 2010, 26(1): 57–68. DOI: 10.1007/s10565-009-9133-y |
| [30] | Keil GM, Pollin R, Müller C, et al. BacMam platform for vaccine antigen delivery//Brun A, Ed. Vaccine Technologies for Veterinary Viral Diseases. New York: Humana Press, 2016: 105-119. |
| [31] | Drugmand JC, Schneider YJ, Agathos SN. Insect cells as factories for biomanufacturing. Biotechnol Adv, 2012, 30(5): 1140–1157. DOI: 10.1016/j.biotechadv.2011.09.014 |
| [32] | Vicente T, Roldão A, Peixoto C, et al. Large-scale production and purification of VLP-based vaccines. J Invertebr Pathol, 2011, 107(S1): S42–S48. |
| [33] | Mena JA, Kamen AA. Insect cell technology is a versatile and robust vaccine manufacturing platform. Expert Rev Vaccines, 2011, 10(7): 1063–1081. DOI: 10.1586/erv.11.24 |
| [34] | Cutts FT, Franceschi S, Goldie S, et al. Human papillomavirus and HPV vaccines: a review. Bull World Health Organ, 2007, 85(9): 719–726. DOI: 10.2471/BLT.00.000000 |
| [35] | Cox MMJ, Patriarca PA, Treanor J. FluBlok, a recombinant hemagglutinin influenza vaccine. Influenza Other Respi Viruses, 2008, 2(6): 211–219. DOI: 10.1111/j.1750-2659.2008.00053.x |
| [36] | Harper DM, Franco EL, Wheeler C, et al. Efficacy of a bivalent L1 virus-like particle vaccine in prevention of infection with human papillomavirus types 16 and 18 in young women: a randomised controlled trial. Lancet, 2004, 364(9447): 1757–1765. DOI: 10.1016/S0140-6736(04)17398-4 |
| [37] | Harper DM, Franco EL, Wheeler CM, et al. Sustained efficacy up to 4.5 years of a bivalent L1 virus-like particle vaccine against human papillomavirus types 16 and 18: follow-up from a randomised control trial. Lancet, 2006, 367(9518): 1247–1255. DOI: 10.1016/S0140-6736(06)68439-0 |
| [38] | Paavonen J, Jenkins D, Bosch FX, et al. Efficacy of a prophylactic adjuvanted bivalent L1 virus-like- particle vaccine against infection with human papillomavirus types 16 and 18 in young women: an interim analysis of a phase Ⅲ double-blind, randomised controlled trial. Lancet, 2007, 369(9580): 2161–2170. DOI: 10.1016/S0140-6736(07)60946-5 |
| [39] | Paavonen J, Naud P, Salmerón J, et al. Efficacy of human papillomavirus (HPV)-16/18 AS04-adjuvanted vaccine against cervical infection and precancer caused by oncogenic HPV types (PATRICIA): final analysis of a double-blind, randomised study in young women. Lancet, 2009, 374(9686): 301–314. DOI: 10.1016/S0140-6736(09)61248-4 |
| [40] | Schwarz TF. Clinical update of the AS04-Adjuvanted human Papillomavirus-16/18 cervical cancer vaccine, cervarix®. Adv Ther, 2009, 26(11): 983–998. DOI: 10.1007/s12325-009-0079-5 |
| [41] | Cox MMJ, Izikson R, Post P, et al. Safety, efficacy, and immunogenicity of Flublok in the prevention of seasonal influenza in adults. Ther Adv Vaccines, 2015, 3(4): 97–108. DOI: 10.1177/2051013615595595 |
| [42] | Karch CP, Burkhard P. Vaccine technologies: from whole organisms to rationally designed protein assemblies. Biochem Pharmacol, 2016, 120: 1–14. |
| [43] | Keitel WA, Treanor JJ, El Sahly HM, et al. Comparative immunogenicity of recombinant influenza hemagglutinin (rHA) and trivalent inactivated vaccine (TIV) among persons ≥65 years old. Vaccine, 2009, 28(2): 379–385. DOI: 10.1016/j.vaccine.2009.10.037 |
| [44] | Treanor JJ, El Sahly H, King J, et al. Protective efficacy of a trivalent recombinant hemagglutinin protein vaccine (FluBlok®) against influenza in healthy adults: a randomized, placebo-controlled trial. Vaccine, 2011, 29(44): 7733–7739. DOI: 10.1016/j.vaccine.2011.07.128 |
| [45] | Uttenthal Å, Le Potier MF, Romero L, et al. Classical swine fever (CSF) marker vaccine: trial I. Challenge studies in weaner pigs. Vet Microbiol, 2001, 83(2): 85–106. DOI: 10.1016/S0378-1135(01)00409-6 |
| [46] | Depner KR, Bouma A, Koenen F, et al. Classical swine fever (CSF) marker vaccine: trial Ⅱ. Challenge study in pregnant sows. Vet Microbiol, 2001, 83(2): 107–120. DOI: 10.1016/S0378-1135(01)00410-2 |
| [47] | van Oirschot JT. Vaccinology of classical swine fever: from lab to field. Vet Microbiol, 2003, 96(4): 367–384. DOI: 10.1016/j.vetmic.2003.09.008 |
| [48] | Moormann RJM, Bouma A, Kramps JA, et al. Development of a classical swine fever subunit marker vaccine and companion diagnostic test. Vet Microbiol, 2000, 73(2/3): 209–219. |
| [49] | Fachinger V, Bischoff R, Jedidia SB, et al. The effect of vaccination against porcine circovirus type 2 in pigs suffering from porcine respiratory disease complex. Vaccine, 2008, 26(11): 1488–1499. DOI: 10.1016/j.vaccine.2007.11.053 |
| [50] | Segalés J. Best practice and future challenges for vaccination against porcine circovirus type 2. Expert Rev Vaccines, 2015, 14(3): 473–487. DOI: 10.1586/14760584.2015.983084 |
| [51] | Blanchard P, Mahé D, Cariolet R, et al. Protection of swine against post-weaning multisystemic wasting syndrome (PMWS) by porcine circovirus type 2 (PCV2) proteins. Vaccine, 2003, 21(31): 4565–4575. DOI: 10.1016/S0264-410X(03)00503-6 |
| [52] | Beam CA, MacCallum C, Herold KC, et al. GAD vaccine reduces insulin loss in recently diagnosed type 1 diabetes: findings from a Bayesian meta-analysis. Diabetologia, 2017, 60(1): 43–49. DOI: 10.1007/s00125-016-4122-1 |
| [53] | Agardh CD, Lynch KF, Palmér M, et al. GAD65 vaccination: 5 years of follow-up in a randomised dose-escalating study in adult-onset autoimmune diabetes. Diabetologia, 2009, 52(7): 1363–1368. DOI: 10.1007/s00125-009-1371-2 |
| [54] | El-Kamary SS, Pasetti MF, Mendelman PM, et al. Adjuvanted intranasal norwalk virus-like particle vaccine elicits antibodies and antibody-secreting cells that express homing receptors for mucosal and peripheral lymphoid tissues. J Infect Dis, 2010, 202(11): 1649–1658. DOI: 10.1086/653023 |
| [55] | Atmar RL, Bernstein DI, Harro CD, et al. Norovirus vaccine against experimental human norwalk virus illness. New Engl J Med, 2011, 365(23): 2178–2187. DOI: 10.1056/NEJMoa1101245 |
| [56] | Smith G, Raghunandan R, Wu YY, et al. Respiratory syncytial virus fusion glycoprotein expressed in insect cells form protein nanoparticles that induce protective immunity in cotton rats. PLoS ONE, 2012, 7(11): e50852. DOI: 10.1371/journal.pone.0050852 |
| [57] | Fries L, Shinde V, Stoddard JJ, et al. Immunogenicity and safety of a respiratory syncytial virus fusion protein (RSV F) nanoparticle vaccine in older adults. Immun Ageing, 2017, 14: 8. DOI: 10.1186/s12979-017-0090-7 |
| [58] | Smith G, Liu Y, Flyer D, et al. Novel hemagglutinin nanoparticle influenza vaccine with Matrix-MTM adjuvant induces hemagglutination inhibition, neutralizing, and protective responses in ferrets against homologous and drifted A (H3N2) subtypes. Vaccine, 2017, 35(40): 5366–5372. DOI: 10.1016/j.vaccine.2017.08.021 |
| [59] | López-Macías C, Ferat-Osorio E, Tenorio-Calvo A, et al. Safety and immunogenicity of a virus-like particle pandemic influenza A (H1N1) 2009 vaccine in a blinded, randomized, placebo-controlled trial of adults in Mexico. Vaccine, 2011, 29(44): 7826–7834. DOI: 10.1016/j.vaccine.2011.07.099 |
| [60] | Bernstein DI, El Sahly HM, Keitel WA, et al. Safety and immunogenicity of a candidate parvovirus B19 vaccine. Vaccine, 2011, 29(43): 7357–7363. DOI: 10.1016/j.vaccine.2011.07.080 |
| [61] | Raghunandan R, Lu HX, Zhou B, et al. An insect cell derived respiratory syncytial virus (RSV) F nanoparticle vaccine induces antigenic site Ⅱ antibodies and protects against RSV challenge in cotton rats by active and passive immunization. Vaccine, 2014, 32(48): 6485–6492. DOI: 10.1016/j.vaccine.2014.09.030 |
| [62] | Hsieh MS, He JL, Wu TY, et al. A secretary bi-cistronic baculovirus expression system with improved production of the HA1 protein of H6 influenza virus in insect cells and Spodoptera litura larvae. J Immunol Methods, 2018, 459: 81–89. DOI: 10.1016/j.jim.2018.06.001 |
| [63] | Dennehy PH. Rotavirus vaccines: an overview. Clin Microbiol Rev, 2008, 21(1): 198–208. DOI: 10.1128/CMR.00029-07 |
| [64] | Renukaradhya GJ, Meng XJ, Calvert JG, et al. Inactivated and subunit vaccines against porcine reproductive and respiratory syndrome: current status and future direction. Vaccine, 2015, 33(27): 3065–3072. DOI: 10.1016/j.vaccine.2015.04.102 |
| [65] | Crawford SE, Labbé M, Cohen J, et al. Characterization of virus-like particles produced by the expression of rotavirus capsid proteins in insect cells. J Virol, 1994, 68(9): 5945–5952. |
| [66] | Driggers RW, Ho CY, Korhonen EM, et al. Zika virus infection with prolonged maternal viremia and fetal brain abnormalities. New Engl J Med, 2016, 374(22): 2142–2151. DOI: 10.1056/NEJMoa1601824 |
| [67] | Adams Waldorf KM, Stencel-Baerenwald JE, Kapur RP, et al. Fetal brain lesions after subcutaneous inoculation of Zika virus in a pregnant nonhuman primate. Nat Med, 2016, 22(11): 1256–1259. DOI: 10.1038/nm.4193 |
| [68] | Dai SY, Zhang T, Zhang YF, et al. Zika virus baculovirus-expressed virus-like particles induce neutralizing antibodies in mice. Virol Sin, 2018, 33(3): 213–226. DOI: 10.1007/s12250-018-0030-5 |
| [69] | Zhang W, Dai WL, Zhang C, et al. A virus-like particle-based tetravalent vaccine for hand, foot, and mouth disease elicits broad and balanced protective immunity. Emerg Microbes Infect, 2018, 7: 94. |
| [70] | Yoshida S, Kondoh D, Arai E, et al. Baculovirus virions displaying Plasmodium berghei circumsporozoite protein protect mice against malaria sporozoite infection. Virology, 2003, 316(1): 161–170. DOI: 10.1016/j.virol.2003.08.003 |
| [71] | Prabakaran M, Velumani S, He F, et al. Protective immunity against influenza H5N1 virus challenge in mice by intranasal co-administration of baculovirus surface-displayed HA and recombinant CTB as an adjuvant. Virology, 2008, 380(2): 412–420. DOI: 10.1016/j.virol.2008.08.002 |
| [72] | Deng L, Mohan T, Chang TZ, et al. Double-layered protein nanoparticles induce broad protection against divergent influenza A viruses. Nat Commun, 2018, 9: 359. DOI: 10.1038/s41467-017-02725-4 |
| [73] | Syed Musthaq S, Madhan S, Sahul Hameed AS, et al. Localization of VP28 on the baculovirus envelope and its immunogenicity against white spot syndrome virus in Penaeus monodon. Virology, 2009, 391(2): 315–324. DOI: 10.1016/j.virol.2009.06.017 |
| [74] | Lee JY, Chang J. Recombinant baculovirus-based vaccine expressing M2 protein induces protective CD8+ T-cell immunity against respiratory syncytial virus infection. J Microbiol, 2017, 55(11): 900–908. DOI: 10.1007/s12275-017-7306-6 |
| [75] | Sim SH, Kim JY, Seong BL, et al. Baculovirus displaying hemagglutinin elicits broad cross-protection against influenza in mice. PLoS ONE, 2016, 11(3): e0152485. DOI: 10.1371/journal.pone.0152485 |