呈现新型冠状病毒RBD抗原病毒样颗粒的表达与鉴定<sup>*</sup>

doi:10.13523/j.cb.2112054

中国生物工程杂志

2022, Vol. 42

Issue (5): 117-123 DOI: 10.13523/j.cb.2112054

新冠肺炎疫苗的研究策略

呈现新型冠状病毒RBD抗原病毒样颗粒的表达与鉴定^*

卢卉双,马家秀,金佳佩,张津,李亚兰,蔡雪飞^**(

)

重庆医科大学感染性疾病分子生物学教育部重点实验室重庆 400016

Expression and Identification of Virus-like Particles Presenting Novel Coronavirus RBD Antigen

LU Hui-shuang,MA Jia-xiu,JIN Jia-pei,ZHANG Jin,LI Ya-lan,CAI Xue-fei^**(

)

Key Laboratory of Molecular Biology of Infectious Diseases of Ministry of Education, Chongqing Medical University,Chongqing 400016,China

全文: PDF(1184 KB) HTML

摘要：

目的:以乙型肝炎病毒核心抗原HBcAg为载体,构建呈现新冠病毒刺突蛋白受体结合域的病毒样颗粒,并鉴定其免疫原性,为新冠病毒疫苗的开发提供新思路。方法:在乙型肝炎病毒核心蛋白氨基酸编码序列第78和81位插入新冠病毒刺突蛋白受体结合域(RBD),并通过柔性linker(G4S)3进行连接,序列优化后将融合基因克隆到原核表达载体pET-28a(+),转化表达菌Rosetta,在自诱导培养基中诱导表达,菌体破碎后经蔗糖密度梯度离心,透析浓缩的方法纯化病毒样颗粒。SDS-PAGE、Western blot、透射电子显微镜检测和鉴定VLPs。将制备的VLPs与佐剂等比例混合经皮下免疫BALB/c小鼠,ELISA检测小鼠血清中特异性抗体,分析该HBc-RBD VLPs的免疫原性。结果:在自诱导培养基中,大肠埃希菌可表达部分可溶的VLPs,经蔗糖密度梯度离心纯化后在透射电子显微镜下可以观察到病毒样颗粒的存在。动物实验表明HBc-RBD VLPs刺激小鼠产生了特异性抗体。结论:在原核表达系统中成功表达了展示RBD抗原的VLPs,并通过小鼠实验初步验证了免疫原性,为新冠病毒疫苗的研发提供了新方向。

关键词： 病毒样颗粒; RBD抗原; 乙肝核心抗原; 免疫原性

Abstract:

Objective: Hepatitis B virus core protein HBc was used as vector to construct virus-like particles expressing novel coronavirus spike protein receptor binding domain RBD, and their immunogenicity was identified, which provides a new idea for the development of COVID-19 vaccines. Methods: The amino acid coding sequence 78 and 81 of hepatitis B virus core protein HBc (1-183 aa) were inserted into novel coronavirus spike protein receptor binding domain RBD and ligated by flexible linker (G4S) 3. After sequence optimization, the fusion gene was cloned into prokaryotic expression vector pET-28a (+) and transformed into expression strain Rosetta. After induced expression in self-inducing medium, the virus-like particles (VLPs) were purified by sucrose density gradient centrifugation. VLPs were detected and identified by SDS-PAGE, Western blot and transmission electron microscope. BALB/c mice were immunized subcutaneously with the prepared VLPs in equal proportion with adjuvant. The specific antibodies in the serum of the mice were analyzed by ELISA to verify the immune effect of HBc-RBD VLPs. Results: Escherichia coli can express partially soluble VLPs in self-inducing medium. VLPs could be observed by transmission electron microscope after purification by sucrose density gradient centrifugation. Mice immunized with HBc-RBD VLPs produced specific antibodies against RBD antigen. Conclusion: VLPs displaying RBD antigen were successfully expressed in prokaryotic expression systems, and their immunogenicity was preliminarily verified by mouse experiment, which provides a new direction for the research and development of novel coronavirus vaccines.

Key words: Virus-like particle(VLP) RBD antigen Hepatitis B core antigen Protein immunogenicity

收稿日期: 2021-12-24 出版日期: 2022-06-17

ZTFLH:

Q814

基金资助: *重庆市科技局技术创新与应用发展重点项目(csts2020jscx-fyzxX0020)

通讯作者: 蔡雪飞 E-mail: caixuefei@cqmu.edu.cn

	服务
	把本文推荐给朋友
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	卢卉双
	马家秀
	金佳佩
	张津
	李亚兰
	蔡雪飞

引用本文:

卢卉双,马家秀,金佳佩,张津,李亚兰,蔡雪飞. 呈现新型冠状病毒RBD抗原病毒样颗粒的表达与鉴定^*[J]. 中国生物工程杂志, 2022, 42(5): 117-123.

LU Hui-shuang,MA Jia-xiu,JIN Jia-pei,ZHANG Jin,LI Ya-lan,CAI Xue-fei. Expression and Identification of Virus-like Particles Presenting Novel Coronavirus RBD Antigen. China Biotechnology, 2022, 42(5): 117-123.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2112054 或 https://manu60.magtech.com.cn/biotech/CN/Y2022/V42/I5/117

图1 HBc-RBD融合基因示意图

图2 重组质粒酶切鉴定

图3 HBc-RBD 蛋白表达的SDS-PAGE 分析

图4 蔗糖密度梯度离心的SDS-PAGE分析

图5 透析浓缩后的蛋白质的SDS-PAGE和Western blot分析

图6 病毒样颗粒的透射电镜图

图7 免疫小鼠的体重和血清中特异性抗体 IgG 的检测

[1]	Jain N K, Sahni N, Kumru O S, et al. Formulation and stabilization of recombinant protein based virus-like particle vaccines. Advanced Drug Delivery Reviews, 2015, 93: 42-55. doi: 10.1016/j.addr.2014.10.023
[2]	Zhu N, Zhang D Y, Wang W L, et al. A novel coronavirus from patients with pneumonia in China, 2019. The New England Journal of Medicine, 2020, 382(8): 727-733. doi: 10.1056/NEJMoa2001017 pmid: 31978945
[3]	Yin Y D, Wunderink R G. MERS, SARS and other coronaviruses as causes of pneumonia. Respirology, 2018, 23(2): 130-137.
[4]	Wrapp D, Wang N S, Corbett K S, et al. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science, 2020, 367(6483): 1260-1263. doi: 10.1126/science.abb2507
[5]	Letko M, Marzi A, Munster V. Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses. Nature Microbiology, 2020, 5 (4): 562-569. doi: 10.1038/s41564-020-0688-y
[6]	Dai L P, Zheng T Y, Xu K, et al. A universal design of Betacoronavirus vaccines against COVID-19, MERS, and SARS. Cell, 2020, 182(3): 722-733.e11. doi: 10.1016/j.cell.2020.06.035
[7]	Guebre-Xabier M, Patel N, Tian J H, et al. NVX-CoV2373 vaccine protects cynomolgus macaque upper and lower airways against SARS-CoV-2 challenge. Vaccine, 2020, 38(50): 7892-7896. doi: 10.1016/j.vaccine.2020.10.064 pmid: 33139139
[8]	Zhang N N, Li X F, Deng Y Q, et al. A thermostable mRNA vaccine against COVID-19. Cell, 2020, 182(5): 1271-1283.e16. doi: 10.1016/j.cell.2020.07.024
[9]	Walsh E E, Frenck R W Jr, Falsey A R, et al. Safety and immunogenicity of two RNA-based COVID-19 vaccine candidates. The New England Journal of Medicine, 2020, 383(25): 2439-2450. doi: 10.1056/NEJMoa2027906
[10]	Peeples L. Avoiding pitfalls in the pursuit of a COVID-19 vaccine. PNAS, 2020, 117(15): 8218-8221. doi: 10.1073/pnas.2005456117 pmid: 32229574
[11]	Pandey S C, Pande V, Sati D, et al. Vaccination strategies to combat novel corona virus SARS-CoV-2. Life Sciences, 2020, 256: 117956. doi: 10.1016/j.lfs.2020.117956
[12]	Pillet S, Couillard J, Trépanier S, et al. Immunogenicity and safety of a quadrivalent plant-derived virus like particle influenza vaccine candidate-two randomized phase II clinical trials in 18 to 49 and ≥ 50 years old adults. PLoS One, 2019, 14(6): e0216533. doi: 10.1371/journal.pone.0216533
[13]	Lee B O, Tucker A, Frelin L, et al. Interaction of the hepatitis B core antigen and the innate immune system. Journal of Immunology (Baltimore, Md: 1950), 2009, 182(11): 6670-6681. doi: 10.4049/jimmunol.0803683
[14]	Dishlers A, Skrastina D, Renhofa R, et al. The hepatitis B virus core variants that expose foreign C-terminal insertions on the outer surface of virus-like particles. Molecular Biotechnology, 2015, 57(11-12): 1038-1049. doi: 10.1007/s12033-015-9895-9 pmid: 26446016
[15]	Mohsen M O, Zha L S, Cabral-Miranda G, et al. Major findings and recent advances in virus-like particle (VLP)-based vaccines. Seminars in Immunology, 2017, 34: 123-132. doi: S1044-5323(17)30038-6 pmid: 28887001
[16]	Pumpens P, Grens E. HBV core particles as a carrier for B cell/T cell epitopes. Intervirology, 2001, 44(2-3): 98-114. pmid: 11509871
[17]	Mechtcheriakova I A, Eldarov M A, Nicholson L, et al. The use of viral vectors to produce hepatitis B virus core particles in plants. Journal of Virological Methods, 2006, 131(1): 10-15. pmid: 16112207
[18]	Ulrich R, Nassal M, Meisel H, et al. Core particles of hepatitis B virus as carrier for foreign epitopes. Advances in Virus Research, 1998, 50: 141-182. pmid: 9520999
[19]	Borisova G, Borschukova Wanst O, Mezule G, et al. Spatial structure and insertion capacity of immunodominant region of hepatitis B core antigen. Intervirology, 1996, 39(1-2): 16-22. pmid: 8957665
[20]	Birkett A, Lyons K, Schmidt A, et al. A modified hepatitis B virus core particle containing multiple epitopes of the Plasmodium falciparum circumsporozoite protein provides a highly immunogenic malaria vaccine in preclinical analyses in rodent and primate hosts. Infection and Immunity, 2002, 70(12): 6860-6870. doi: 10.1128/IAI.70.12.6860-6870.2002 pmid: 12438363
[21]	Clarke B E, Newton S E, Carroll A R, et al. Improved immunogenicity of a peptide epitope after fusion to hepatitis B core protein. Nature, 1987, 330 (6146): 381-384. doi: 10.1038/330381a0
[22]	Ormö M, Cubitt A B, Kallio K, et al. Crystal structure of the Aequorea victoria green fluorescent protein. Science, 1996, 273(5280): 1392-1395. pmid: 8703075
[23]	Kratz P A, Böttcher B, Nassal M. Native display of complete foreign protein domains on the surface of hepatitis B virus capsids. Proceedings of the National Academy of Sciences of the United States of America, 1999, 96(5): 1915-1920.
[24]	Peyret H, Gehin A, Thuenemann E C, et al. Tandem fusion of hepatitis B core antigen allows assembly of virus-like particles in bacteria and plants with enhanced capacity to accommodate foreign proteins. PLoS One, 2015, 10(4): e0120751.
[25]	Donaldson B, Al-Barwani F, Young V, et al. Virus-like particles, a versatile subunit vaccine platform.Subunit Vaccine Delivery. Berlin: Springer, 2015:159-180.
[26]	Walker A, Skamel C, Nassal M. SplitCore: an exceptionally versatile viral nanoparticle for native whole protein display regardless of 3D structure. Scientific Reports, 2011, 1: 5. doi: 10.1038/srep00005

[1]	刘明珠,张良,郭芳,李春,冯旭东. 酵母表面展示体系的构建及在纤维素降解中的应用^*[J]. 中国生物工程杂志, 2022, 42(5): 91-99.
[2]	李江波,郭鸿斌,王诗昆,金蕊,程龙. 利用拆分绿色荧光蛋白检测端粒酶TERT亚基与端粒末端蛋白TPP1的相互作用^*[J]. 中国生物工程杂志, 2022, 42(1/2): 80-87.
[3]	赵强,刘洋,周晶辉,许岗. 大肠杆菌头孢菌素C乙酰酯酶的敲除对头孢菌素C酰化酶应用的影响[J]. 中国生物工程杂志, 2022, 42(1/2): 96-103.
[4]	陈开通,郑文隆,杨立荣,徐刚,吴坚平. 氨基树脂固定化L-苏氨酸醛缩酶及其应用^*[J]. 中国生物工程杂志, 2021, 41(9): 55-63.
[5]	郭芳,张良,冯旭东,李春. 植物源UDP-糖基转移酶及其分子改造^*[J]. 中国生物工程杂志, 2021, 41(9): 78-91.
[6]	郭曼曼,田开仁,乔建军,李艳妮. 噬菌体重组酶系统在合成生物学中的应用^*[J]. 中国生物工程杂志, 2021, 41(8): 90-102.
[7]	陶守松,任广明,尹荣华,杨晓明,马文兵,葛志强. 敲低去泛素化酶USP13抑制K562细胞的增殖^*[J]. 中国生物工程杂志, 2021, 41(5): 1-7.
[8]	董雪迎,梁凯,叶克应,周策凡,唐景峰. 受体酪氨酸激酶对自噬的调控及其研究进展^*[J]. 中国生物工程杂志, 2021, 41(5): 72-78.
[9]	魏子翔,张柳群,雷磊,韩正刚,杨江科. *疏棉状嗜热丝孢菌(Thermomyces lanuginosus)脂肪酶的理性设计提高其活性和温度稳定性*[J]. 中国生物工程杂志, 2021, 41(2/3): 63-69.
[10]	陈中伟,郑璞,陈鹏程,吴丹. 耐热植酸酶突变体的筛选及性质研究 ^*[J]. 中国生物工程杂志, 2021, 41(2/3): 30-37.
[11]	玄美娟,张晓云,高莹,高丽影,吴佳婧,马梅,王艳梅,寇航,路福平,黎明. 大肠杆菌糖酵解途径和三羧酸循环启动子的表征及其应用 ^*[J]. 中国生物工程杂志, 2020, 40(6): 20-30.
[12]	朱衡,张继福,张云,胡云峰. 环氧交联剂和氨基载体固定化海洋假丝酵母脂肪酶^*[J]. 中国生物工程杂志, 2020, 40(5): 57-68.
[13]	苏永君,胡蝶,胡博淳,李闯,文正,章晨,邬敏辰. 定点突变提高环氧化物水解酶AuEH2催化对甲基苯基缩水甘油醚的对映选择性^*[J]. 中国生物工程杂志, 2020, 40(3): 88-95.
[14]	赵晓艳,陈允妲,章雅倩,吴晓玉,王飞,陈金印. **Myxococcus sp.V11海藻糖合酶TreS II分子改造 ^***[J]. 中国生物工程杂志, 2020, 40(3): 79-87.
[15]	朱衡,张继福,张云,孙爱君,胡云峰. 聚乙二醇二缩水甘油醚交联氨基载体LX-1000EA固定化脂肪酶 ^*[J]. 中国生物工程杂志, 2020, 40(1-2): 124-132.

Viewed

Full text

Abstract

Cited

Shared

Discussed