Serological Study to Self-assembled Nanoparticle Influenza Vaccine with the M2e-HA2 Epitopes
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摘要:
目的 构建M2e-HA2串联表位的自组装纳米颗粒通用流感疫苗，并进行免疫血清学检测。 方法 将流感病毒M2e和HA2抗原表位与Ferritin蛋白串联表达，非变性条件下提取并经亲和层析纯化获得纳米自组装颗粒，经Western blot和电镜验证后免疫小鼠。2次免疫后采集小鼠血清，使用Western blot进行抗原性检测，使用ELISA进行抗体水平检测。 结果 成功制备M2e-HA2串联表位的自组装纳米颗粒，将其免疫小鼠后血清中产生高水平的M2e和HA2特异性抗体。 结论 M2e-HA2串联表位的自组装纳米颗粒可以刺激小鼠产生高水平的M2e和HA2特异性抗体，为通用流感疫苗研发奠定基础。 -
关键词:
- M2e-HA2串联表位 /
- 自组装纳米颗粒 /
- 流感疫苗 /
- 血清学
Abstract:Objective To construct the self-assembled nanoparticle influenza vaccine with the M2e-HA2 epitopes, and detect the immunological serology. Methods The M2e and HA2 antigen epitopes of influenza viruses were expressed in tandem with Ferritin protein, and the nanometer self-assembled particles were extracted under the condition of non-denatured and purified by affinity chromatography. The mice were immunized for two times, after the two immunizations, serum of mice was collected, western blot was used for antigenicity detection, and ELISA was used for antibody level detection. Results M2e-HA2self-assembled nanoparticles were successfully prepared, and high levels of M2e and HA2 specific antibodies were produced in the serum of immunized mice. Conclusion M2e-HA2 self-assembled nanoparticles can stimulate the production of high levels of M2e and HA2 specific antibodies in mice, laying the foundation for the development of a universal influenza vaccine. -
Key words:
- M2e-HA2 epitopes /
- Self-assembled nanoparticles /
- Influenza vaccine /
- Serological study
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图 1 M2e-HA2自组装纳米颗粒载体构建
a：M2e片段PCR扩增产物，大小139 bp；b：HA2-ferritin片段PCR扩增产物，大小1 210 bp；C：Hind Ⅲ Ⅰ和Xho Ⅰ酶切验证得到1 296 bp的阳性片段。
Figure 1. Construction of M2e-HA2 self-assembled nanoparticles
图 2 M2e-HA2自组装纳米颗粒表达验证
a：非变性Western blot检测M2e-HA2表达，使用M2抗体，大小在180 kD以上；b：变性Western blot检测M2e-HA2表达，使用M2抗体，大小在48.355 kD。
Figure 2. Validation of expression of M2e-HA2 self-assembled nanoparticles
图 3 M2e-HA2自组装纳米颗粒纯化产物验证
a：非变性PAGE胶考马斯亮蓝染色检测M2e-HA2多聚体，证明产物条带单一；b：非变性Western blot检测M2e-HA2纯化产物，使用M2抗体，大小在180 kD以上；c：变性Western blot检测M2e-HA2纯化产物，使用M2抗体，大小在48.355 kD；d：透射电镜观察M2e-HA2纯化产物，可看到结构稳定大小在100 nm左右的纳米颗粒，标尺大小为200 nm。
Figure 3. Validation of purified products of M2e-HA2 self-assembled nanoparticles
图 4 M2e-HA2自组装纳米颗粒免疫血清学验证
a：变性western blot检测M2e-HA2纯化产物，使用M2e-HA2二免小鼠血清1∶200稀释作为一抗，大小在48.355 kD；b：非变性western blot检测M2e-HA2纯化产物，使用M2e-HA2二免小鼠血清1∶200稀释作为一抗，大小在180 kD以上；c：ELISA检测M2e-HA2二免小鼠血清中特异性抗体水平。分别使用人工合成的M2e多肽片段和HA2多肽片段孵育酶标板检测血清中抗体水平，平均血清稀释度分别为1∶1 066.667 和 1∶1 333.333。
Figure 4. Immune serological validation of M2e-HA2 self-assembled nanoparticles
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[1] Dharmapalan D. Influenza[J]. Indian J Pediatr,2020,87(10):828-832. doi: 10.1007/s12098-020-03214-1 [2] Kim H,R G Webster,R J Webby. Influenza virus:Dealing with a drifting and shifting pathogen[J]. Viral Immunol,2018,31(2):174-183. doi: 10.1089/vim.2017.0141 [3] Yuan L Y,Zhou M,Lv H,et al. Involvement of NEAT1/miR-133a axis in promoting cervical cancer progression via targeting SOX4[J]. J Cell Physiol,2019,234(10):18985-18993. doi: 10.1002/jcp.28538 [4] Graham-Rowe D. Epidemiology:Racing against the flu[J]. Nature,2011,480(7376):S2-3. doi: 10.1038/480S2a [5] Lam T T,Wang J,Shen Y,et al. The genesis and source of the H7N9 influenza viruses causing human infections in China[J]. Nature,2013,502(7470):241-244. doi: 10.1038/nature12515 [6] Deng L,Mohan T,Chang T Z,et al. Double-layered protein nanoparticles induce broad protection against divergent influenza A viruses[J]. Nat Commun,2018,9(1):359. doi: 10.1038/s41467-017-02725-4 [7] Eichelberger M C,Monto A S. Neuraminidase,the forgotten surface antigen,emerges as an influenza vaccine target for broadened protection[J]. J Infect Dis,2019,219(Suppl_1):S75-S80. [8] Dunkle L M,Izikson R. Recombinant hemagglutinin influenza vaccine provides broader spectrum protection[J]. Expert Rev Vaccines,2016,15(8):957-966. doi: 10.1080/14760584.2016.1203261 [9] Wang K,Holtz K M,Anderson K,et al. Expression and purification of an influenza hemagglutinin--one. Step closer to a recombinant protein-based influenza vaccine[J]. Vaccine,2006,24(12):2176-2185. doi: 10.1016/j.vaccine.2005.11.005 [10] Qi M,Zhang X E,Sun X,et al. Intranasal nanovaccine confers homo- and hetero-subtypic influenza protection[J]. Small,2018,14(13):e1703207. doi: 10.1002/smll.201703207 [11] Saelens X. The role of matrix protein 2 ectodomain in the development of universal influenza vaccines[J]. J Infect Dis,2019,219(Suppl_1):S68-S74. [12] Kanekiyo M,Wei C J,Yassine H M,et al. Self-assembling influenza nanoparticle vaccines elicit broadly neutralizing H1N1 antibodies[J]. Nature,2013,499(7456):102-106. doi: 10.1038/nature12202 [13] Liu X,Theil E C. Ferritins:dynamic management of biological iron and oxygen chemistry[J]. Acc Chem Res,2005,38(3):167-175. doi: 10.1021/ar0302336 [14] Stillman T J,Connolly P P,Latimer C L,et al. Insights into the effects on metal binding of the systematic. Substitution of five key glutamate ligands in the ferritin of Escherichia coli[J]. J Biol Chem,2003,278(28):26275-26286. doi: 10.1074/jbc.M207354200 [15] Cho K J,Shin H J,Lee J H,et al. The crystal structure of ferritin from helicobacter pylori reveals unusual conformational changes for iron uptake[J]. J Mol Biol,2009,390(1):83-98. doi: 10.1016/j.jmb.2009.04.078 [16] Ong L L,Hanikel N,Yaghi O K,et al. Programmable self-assembly of three-dimensional nanostructures. From 10,000 unique components[J]. Nature,2017,552(7683):72-77. doi: 10.1038/nature24648 [17] Englander S W,Mayne L,Krishna M M. Protein folding and misfolding:Mechanism and principles[J]. Q. Rev Biophys,2007,40(4):287-326. [18] Pieters B J,van Eldijk M B,Nolte R J,et al. Natural supramolecular protein assemblies[J]. Chem Soc Rev,2016,45(1):24-39. doi: 10.1039/C5CS00157A [19] Chen Y Q,Lan L Y,Huang M,et al. Hemagglutinin stalk-reactive antibodies interfere with influenza virus neuraminidase activity by steric hindrance[J]. J Virol,2019,93(4):e01526. [20] Zhang Y,Ardejani M S,Orner B P. Design and applications of protein-cage-based nanomaterials[J]. Chem. Asian J,2016,11(20):2814-2828. doi: 10.1002/asia.201600769 [21] Rother M,Nussbaumer M G,Renggli K,et al. Protein cages and synthetic polymers:a fruitful symbiosis. For drug delivery applications,bionanotechnology and materials science[J]. Chem Soc Rev,2016,45(22):6213-6249. doi: 10.1039/C6CS00177G -
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