Preparation, Purification and Identification of Bacteriophage Qβ Virus-like Particles

doi:10.13523/j.cb.2103034

China Biotechnology

2021, Vol. 41

Issue (7): 42-49 DOI: 10.13523/j.cb.2103034

Preparation, Purification and Identification of Bacteriophage Qβ Virus-like Particles

CHEN Xiu-yue,ZHOU Wen-feng,HE Qing,SU Bing,ZOU Ya-wen(

)

College of Veterinary Medicine, Hunan Agricultural University, Hunan Provincial Key Laboratory of Protein Engineering in Animal Vaccines, Research & Development Center for Animal Reverse Vaccinology of Hunan Province, Changsha 410128, China

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Abstract

Objective: The self-assembly ability of phage Qβ virus-like particles (VLPs),prepared by Escherichia coli (E. coli) prokaryotic expression system was verified. To determine their immunogenicity, New Zealand rabbits were immunized with purified Qβ VLPs, and them ability to enter mammalian cells was also detected.Methods: The pET-28-Qβ-CP plasmid was constructed, and Qβ VLPs were produced by E. coli expression system. Qβ VLPs were purified by sucrose density gradient centrifugation and gel filtration (Sephacryl S-400 column). The particle morphology of Qβ VLPs were observed by transmission electron microscopy (TEM). Antisera were obtained from the rabbits immunized with the VLPs, along with or without adjuvant, and subsequently, antibodies were purified by Protein G resin. The specificity was determined by Western blot. The indirect immunofluorescence assay (IFA) was used to detect the entry of Qβ VLPs into cells.Results: High purity Qβ VLPs were obtained. TEM results showed that substantial VLPs were observed with diameter of about 28 nm. Western blot showed that Qβ VLPs were specifically recognized by anti-rabbit polyclonal antibodies. Adjuvant had no negative effect on the production of antibody. Based on IFA, the results showed that Qβ VLPs entered various mammalian cells.Conclusion: Qβ VLPs were successfully produced in this study, and they would have great potential as carriers to facilitate the development of future vaccine based on Qβ VLPs.

Key words： Bacteriophage Qβ Virus-like particles Polyclonal antibody Cell entry

Received: 15 March 2021 Published: 03 August 2021

ZTFLH:

Q819

Corresponding Authors: Ya-wen ZOU E-mail: yawenzou@stu.hunau.edu.cn

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	Xiu-yue CHEN
	Wen-feng ZHOU
	Qing HE
	Bing SU
	Ya-wen ZOU

Cite this article:

CHEN Xiu-yue,ZHOU Wen-feng,HE Qing,SU Bing,ZOU Ya-wen. Preparation, Purification and Identification of Bacteriophage Qβ Virus-like Particles. China Biotechnology, 2021, 41(7): 42-49.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2103034 OR https://manu60.magtech.com.cn/biotech/Y2021/V41/I7/42

Fig.1 The solubility analysis of Qβ coat protein M: Protein marker. Line 1: total protein of pET-28-Qβ-CP was induced at 28℃. Line 2: the supernatant of pET-28-Qβ-CP was induced at 28℃. Line 3: the precipitation of pET-28-Qβ-CP was induced at 28℃

Fig.2 The results of purification of Qβ VLPs M: Protein marker. Line 1: the supernatant of sucrose density gradient centrifugation. Line 2: the middle layer between the supernatant and 60% sucrose. Line 3: 60% sucrose layer. Line 4: the middle layer between 60% sucrose and 80% sucrose. Line 5: 80% sucrose layer

Fig.3 Qβ VLPs were separated by Sephacryl S-400 molecular sieve gel chromatography (a) Purified BSA by exclusion chromatography (68 kDa) (b) Purification of Qβ VLPs by exclusion chromatography

Fig.4 Transmission electron microscopy morphology of Qβ VLPs

Fig.5 Western blot analysis of rabbit polyclonal antibodies (a) Rabbit negative polyclonal antibody (b) Rabbit anti-Qβ polyclonal antibody (c) Rabbit anti-Qβ polyclonal antibody (Adjuvants were used in immunization)

Fig.6 The results of indirect immuno fluorescence The length of the scale: 50 μm


[1]	Inomata T, Kimura H, Hayasaka H, et al. Quantitative comparison of the RNA bacteriophage Qβ infection cycle in rich and minimal media. Archives of Virology, 2012, 157(11):2163-2169. doi: 10.1007/s00705-012-1419-3

[2]	Furuse K, Osawa S, Kawashiro J, et al. Bacteriophage distribution in human faeces: continuous survey of healthy subjects and patients with internal and leukaemic diseases. The Journal of General Virology, 1983, 64(Pt 9):2039-2043. doi: 10.1099/0022-1317-64-9-2039

[3]	Rumnieks J, Tars K. Crystal structure of the read-through domain from bacteriophage Qβ A1 protein. Protein Science, 2011, 20(10):1707-1712. doi: 10.1002/pro.704

[4]	Tao P, Zhu J G, Mahalingam M, et al. Bacteriophage T4 nanoparticles for vaccine delivery against infectious diseases. Advanced Drug Delivery Reviews, 2019, 145:57-72. doi: 10.1016/j.addr.2018.06.025

[5]	Brown F, Cartwright B. Dissociation of foot-and-mouth disease virus into its nucleic acid and protein components. Nature, 1961, 192:1163-1164. doi: 10.1038/1921163a0

[6]	Blumenthal T, Carmichael G G. RNA replication: function and structure of Qbeta-replicase. Annual Review of Biochemistry, 1979, 48:525-548. pmid: 382992

[7]	Rumnieks J, Tars K. Crystal structure of the maturation protein from bacteriophage Qβ. Journal of Molecular Biology, 2017, 429(5):688-696. doi: S0022-2836(17)30040-2 pmid: 28111107

[8]	Kushnir N, Streatfield S J, Yusibov V. Virus-like particles as a highly efficient vaccine platform: diversity of targets and production systems and advances in clinical development. Vaccine, 2012, 31(1):58-83. doi: 10.1016/j.vaccine.2012.10.083

[9]	Chackerian B, Lenz P, Lowy D R, et al. Determinants of autoantibody induction by conjugated papillomavirus virus-like particles. Journal of Immunology, 2002, 169(11):6120-6126. pmid: 12444114

[10]	Jegerlehner A, Storni T, Lipowsky G, et al. Regulation of IgG antibody responses by epitope density and CD21-mediated costimulation. European Journal of Immunology, 2002, 32(11):3305-3314. pmid: 12555676

[11]	Henry K A, Arbabi-Ghahroudi M, Scott J K. Beyond phage display: non-traditional applications of the filamentous bacteriophage as a vaccine carrier, therapeutic biologic, and bioconjugation scaffold. Frontiers in Microbiology, 2015, 6:755.

[12]	Prisco A, De Berardinis P. Filamentous bacteriophage fd as an antigen delivery system in vaccination. International Journal of Molecular Sciences, 2012, 13(4):5179-5194. doi: 10.3390/ijms13045179

[13]	Beghetto E, Gargano N. Lambda-display: a powerful tool for antigen discovery. Molecules, 2011, 16(4):3089-3105. doi: 10.3390/molecules16043089 pmid: 21490557

[14]	Jafari N, Abediankenari S. Phage particles as vaccine delivery vehicles: concepts, applications and prospects. Asian Pacific Journal of Cancer Prevention, 2015, 16(18):8019-8029. doi: 10.7314/APJCP.2015.16.18.8019

[15]	Fu Y, Li J M. A novel delivery platform based on Bacteriophage MS2 virus-like particles. Virus Research, 2016, 211:9-16. doi: 10.1016/j.virusres.2015.08.022

[16]	Pokorski J K, Hovlid M L, Finn M G. Cell targeting with hybrid Qβ virus-like particles displaying epidermal growth factor. Chembiochem: A European Journal of Chemical Biology, 2011, 12(16):2441-2447. doi: 10.1002/cbic.v12.16

[17]	Jepson C D, March J B. Bacteriophage lambda is a highly stable DNA vaccine delivery vehicle. Vaccine, 2004, 22(19):2413-2419. doi: 10.1016/j.vaccine.2003.11.065

[18]	Lankes H A, Zanghi C N, Santos K, et al. In vivo gene delivery and expression by bacteriophage lambda vectors. Journal of Applied Microbiology, 2007, 102(5):1337-1349. pmid: 17448169

[19]	Bao Q, Li X, Han G R, et al. Phage-based vaccines. Advanced Drug Delivery Reviews, 2019, 145:40-56. doi: 10.1016/j.addr.2018.12.013

[20]	Huang X F, Wang X, Zhang J, et al. Escherichia coli-derived virus-like particles in vaccine development. NPJ Vaccines, 2017, 2:3. doi: 10.1038/s41541-017-0006-8

[21]	Zeltins A. Construction and characterization of virus-like particles: a review. Molecular Biotechnology, 2013, 53(1):92-107. doi: 10.1007/s12033-012-9598-4 pmid: 23001867

[22]	Gatto D, Ruedl C, Odermatt B, et al. Rapid response of marginal zone B cells to viral particles. Journal of Immunology, 2004, 173(7):4308-4316. doi: 10.4049/jimmunol.173.7.4308

[23]	Gatto D, Martin S W, Bessa J, et al. Regulation of memory antibody levels: the role of persisting antigen versus plasma cell life span. Journal of Immunology, 2007, 178(1):67-76. doi: 10.4049/jimmunol.178.1.67

[24]	Jegerlehner A, Maurer P, Bessa J, et al. TLR9 signaling in B cells determines class switch recombination to lgG2a. Journal of Immunology, 2007, 178(4):2415-2420. pmid: 17277148

[25]	Hou B D, Saudan P, Ott G, et al. Selective utilization of Toll-like receptor and MyD88 signaling in B cells for enhancement of the antiviral germinal center response. Immunity, 2011, 34(3):375-384. doi: 10.1016/j.immuni.2011.01.011

[26]	Maurer P, Jennings G T, Willers J, et al. A therapeutic vaccine for nicotine dependence: preclinical efficacy, and phase I safety and immunogenicity. European Journal of Immunology, 2005, 35(7):2031-2040. doi: 10.1002/(ISSN)1521-4141

[27]	Bachmann M F, Jennings G T. Therapeutic vaccines for chronic diseases: successes and technical challenges. Philosophical Transactions of the Royal Society of London Series B, Biological Sciences, 2011, 366(1579):2815-2822.

[28]	Doucet M, El-Turabi A, Zabel F, et al. Preclinical development of a vaccine against oligomeric alpha-synuclein based on virus-like particles. PLoS One, 2017, 12(8):e0181844. doi: 10.1371/journal.pone.0181844

[29]	Chan S K, Du P Y, Ignacio C, et al. Biomimetic virus-like particles as severe acute respiratory syndrome coronavirus 2 diagnostic tools. ACS Nano, 2021, 15(1):1259-1272. doi: 10.1021/acsnano.0c08430

[30]	Liao W H, Hua Z L, Liu C, et al. Characterization of T-dependent and T-independent B cell responses to a virus-like particle. Journal of Immunology, 2017, 198(10):3846-3856. doi: 10.4049/jimmunol.1601852

[31]	Yao L, Li F L, Qu M, et al. Development and evaluation of a novel armored RNA technology using bacteriophage Qβ. Food and Environmental Virology, 2019, 11(4):383-392. doi: 10.1007/s12560-019-09400-5

[32]	Rhee J K, Hovlid M, Fiedler J D, et al. Colorful virus-like particles: fluorescent protein packaging by the Qβ capsid. Biomacromolecules, 2011, 12(11):3977-3981. doi: 10.1021/bm200983k

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