Vector of osteoporosis vaccine: expression, identification and purification of Qβ virus-like particles

ZHANG Shudong; ZHOU Jian; TIAN Zhuang; LIU Changzhen; YAO Qi

PDF(982 KB)

Medical Journal of the Chinese People Armed Police Forces ›› 2019, Vol. 30 ›› Issue (11) : 936-940.

Vector of osteoporosis vaccine: expression, identification and purification of Qβ virus-like particles

ZHANG Shudong¹, ZHOU Jian¹, TIAN Zhuang¹, LIU Changzhen², YAO Qi¹

Author information +

History +

Abstract

Objective To optimize expression and purification methods and prepare Qβ-VLPs with correct capsids structure and high purity and productivity.Methods We deleted the sequence of A1 gene sequence after termination codon of Qβ coat protein , and synthesized the gene sequence of Qβ coat protein only. The Qβ coat protein gene sequence was cloned into the pET30a vector plasmid, and the proteins were expressed by BL21(DE3), BL21(DE3) pLysS and Rosetta(DE3) strains. Then, the structure of Qβ-VLPs was verified with SDS-PAGE and transmission electron microscopy. Two different IPTG concentrations and three different methods of bacteria crushing were set respectively to optimize the expression conditions of the protein and methods of bacteria crushing. Finally, the supernatant of Qβ-VLPs was purified by ammonium sulfate precipitation, high-speed centrifugation and gel filtration chromatography.Results The results suggested that 5-6 polymers in SDS-PAGE and correct 25-30 nm capsids under electron microscopy were displayed only through the BL21(DE3)pLysS strain. The production of Qβ-VLPs expressed by IPTG at the concentration of 0.5mM was 2.8 times higher than at the concentration of 0.2mM, and the highest protein separation efficiency could be obtained by ultrasonic crushing of bacteria. The supernatant of Qβ-VLPs was purified and the purity of Qβ-VLPs was about 90%.Conclusions The experiment has explored the preparation strategy of Qβ-VLPs with correct capsids and high purity, which can contribute to the preparation and research of relevant vaccines.

Key words

osteoporosis / Qβ-VLPs / expression / protein purification

Cite this article

EndNote

Ris (Procite)

Bibtex

Download Citations

ZHANG Shudong, ZHOU Jian, TIAN Zhuang, LIU Changzhen, YAO Qi. Vector of osteoporosis vaccine: expression, identification and purification of Qβ virus-like particles[J]. Medical Journal of the Chinese People Armed Police Forces. 2019, 30(11): 936-940

References

[1] Deeks E D. Denosumab: a review in postmenopausal osteoporosis [J]. Drug Aging, 2018, 35(2): 163-173.
[2] Anthony. Romosozumab: first global approval [J]. Drugs, 2019,79(4):471-476.
[3] Leneghan D B, Miura K, Taylor I J, et al. Nanoassembly routes stimulate conflicting antibody quantity and quality for transmission-blocking malaria vaccines[J]. Sci Rep, 2017, 7(1): 3811.
[4] Yin Z, Wu X, Kaczanowska K, et al. Antitumor humoral and T cell responses by Mucin-1 conjugates of bacteriophage Qβ in wild-type mice[J]. ACS Chem Biol, 2018, 13(6):1668-1676.
[5] Basu R, Zhai L, Contreras A, et al. Immunization with phage virus-like particles displaying Zika virus potential B-cell epitopes neutralizes Zika virus infection of monkey kidney cells[J]. Vaccine, 2018, 36(10): 1256-1264.
[6] Spohn G, Schwarz K, Maurer P, et al. Protection against osteoporosis by active immunization with TRANCE/RANKL displayed on virus-like particles[J]. J Immunol, 2005, 175(9): 6211-6218.
[7] Tissot A C, Maurer P, Nussberger J, et al. Effect of immunisation against angiotensin II with CYT006-AngQb on ambulatory blood pressure: a double-blind, randomised, placebo-controlled phase IIa study[J]. Lancet, 2008, 371(9615): 821-827.
[8] Maurer P, Jennings G T, Willers J, et al. A therapeutic vaccine for nicotine dependence: preclinical efficacy, and phase I safety and immunogenicity[J]. Eur J Immunol, 2005, 35(7):2031-2040.
[9] Kozlovska T M, Cielēns I, Dreilinna D,et al. Recombinant rna phage Qβ capsid particles synthesized and self-assembled in escherichia coli[J]. Gene, 1993, 137(1):133-137.
[10] Cui Z, Gorzelnik K V, Chang J Y, et al. Structures of Qβ virions, virus-like particles, and the Qβ-MurA complex reveal internal coat proteins and the mechanism of host lysis[J].Proc Natl Acad Sci U S A, 2017, 114(44):11697-11702.
[11] Singleton R L, Sanders C A, Jones K, et al. Function of the RNA coliphage Qβ proteins in medical in vitro evolution[J]. Methods Protoc, 2018, 1(2): 18.
[12] Zoe A M, Leticia R S, Gloria H A. Exploring the differences between the three pyruvate kinase isozymes from Vibrio cholerae in a heterologous expression system[J]. BMC Research Notes, 2018, 11(1): 527-535.
[13] Bhat P, Pawaskar G M. Expression of bacillus licheniformis chitin deacetylase in E. coli pLysS: sustainable production, purification and characterisation[J]. Int J Biol Macromol, 2019, 131: 1008-1013.
[14] Wei-Ming C, Gen-Hung C, Ching-San C, et al. Cloning, expression and purification of bacillus cereus endochitinase in the escherichia coli AD494(DE3)pLysS expression system[J].Biosci Biotechnol Biochem, 2009, 73(5): 1172-1174.
[15] Rüdiger J, Schwab W. Improving an Escherichia coli-based biocatalyst for terpenol glycosylation by variation of the expression system[J].J Ind Microbiol Biotechnol, 2019,46(8):1129-1138.
[16] Golmohammadi R, Fridborg K. The crystal structure of bacteriophage Q beta at 3.5 A resolution [J].Structure, 1996, 4(5): 543-54.
[17] Wandrey G, Bier C, Binder D, et al. Light-induced gene expression with photocaged IPTG for induction profiling in a high-throughput screening system[J]. Microb Cell Fact, 2016, 15(1): 63.
[18] Leung R C, Robinson M D M, Ajabali A A A, et al. Monitoringthe Disassembly of Virus-like Particlesby 19F-NMR[J]. J Am Chem Soc, 2017, 139(15):5277.
[19] Tian M, Hua Z, Hong S, et al. B cell-intrinsic MyD88 signaling promotes initial cell proliferation and differentiation to enhance the germinal center response to a virus-like particle[J]. J Immunol, 2017, 200(3): 937-948.
[20] Dallenbach K, Maurer P, Röhn T, et al. Protective effect of a germline, IL-17-neutralizing antibody in murine models of autoimmune inflammatory disease[J]. Eur J Immunol, 2015, 45(4):1238-1247.
[21] Moura A, Santos L, Brito C, et al. Virus-like particle display of the α?Gal carbohydrate for vaccination against leishmania infection[J]. ACS Central Sci, 2017, 3(9): 1026-1031.
[22] Araujo R N, Franco P F, Rodrigues H, et al. Amblyomma sculptum tick saliva: α-Gal identification, antibody response and possible association with red meat allergy in Brazil[J]. Int J Parasitol, 2016, 46(3):213-220.