LV15190B - Method for obtaining empty and packed with nucleic acids capsids of virus-like particles of hbc-protein - Google Patents

Abstract

The invention relates to the production of nutritional virus-like particles consisting of HBcG protein of the HBVG genotype and capable of packaging nucleic acids after purification from a non-specific filling. The method includes HBc particle extraction using an E. coli expression system, capsid purification and blank particle dissociation reabsorption in process. Demonstration of RNA-phage f2sus11 dsRNA packaging.

Description

DESCRIPTION OF THE INVENTION

BACKGROUND OF THE INVENTION The present invention relates to male particle technology.

Virus-like particles (VVD) are viral protein-generated particles that are produced in prokaryotic or eukaryotic systems by expressing certain viral structural proteins and are capable of assembling into multimeric structures in these systems, resembling either viruses themselves, e.g. , or certain morphogenetic structures such as yeast-derived hepatitis В virus (HBV) HBs protein particles and bacterial and yeast HBV HBc protein-like particles - capsids. An important characteristic of VVD is their high viral-like immunogenicity, which underlies the widespread use of VVD in immunological and recombinant vaccine studies. The range of VVD objects studied has a wide range of applications, and their practical application has now led to the development and introduction of HBV and recombinant human papillomavirus (HPV) vaccines. Further research into VVD involves modifying VVD for presentation of epitopes and packaging of functional substances - nucleic acids, fragments, proteins or magnetic particles - to form transfer vectors for use in gene therapy, drug therapy, and thermotherapy and immunocorrection. [1,2].

[003] One of the currently structurally best-studied VVDs that exhibits high foreign insertion capacity and immunogenicity of chimeric structures is HBV HBc-formed VVD [3,4]. HBV genome of D-genotype (subtype ayw), its HBc gene cloned and expressed also in Latvia [5,6]; The HBc protein of HBV D-genotype has 183 amino acids in length. One feature of the HBV G-genotype is the HBc protein extended by 12 amino acids at the N-terminus [7].

The present invention relates to the use of the HBV G genotype HBc protein for the production of fully purified recombinant VVD (HBcG capsids) and further use in the encapsulation of nucleic acids (dsRNA).

[005] The level of VVD synthesis (expression) and the ability to get rid of both impurities and encapsulated VVD structures are essential for the purification of VVD from cellular impurities. The invention utilizes an E.coli expression system to obtain high VVD results [8]. For the extraction and purification of VVD from external impurities, the invention employs a protein purification scheme involving extraction in the presence of urea, precipitation with ammonium sulfate, gel filtration and ion exchange chromatography. Several methods are described for removing impurities encapsulated in capsids, which may or may not involve disassembly of the capsids into subunit dimers. Treatment of HBc VVD with micrococcal nuclease allows cleavage of the nucleic acid packaged in the capsid and particles, depending on the structure of HBc (amount of Arg domains), whether or not disrupted; the assembly of disrupted structures in the capsids, in turn, is stimulated by various polyanions [9]. However, it should be noted that impurities in the nuclease used can cause problems in this technology. Ribonuclease treatment can also cause similar problems [10]. Incubation of HBc VVDs at extremely high pH, which leads to hydrolysis of packaged nucleic acids and destruction of capsids, but without disruption to dimers, has also been described [11].

The invention utilizes disassembly of capsids to dimers by incubation in GuHCl / LiCl solution (dissociation of capsids and precipitation of nucleic acids) followed by separation of dimers by chromatography and their re-association in capsids [12].

[007] All of these methods allow the packaging of material of different contents, depending on the task being set up - nucleic acid, CpG oligonucleotides, longer DNA fragments or plasmids, as well as, in many cases, proteins and other natural materials. . It should be noted that double-stranded RNA (dsRNA) has not been used for packing HBc capsules, except that tRNAs and other RNAs used are largely double stranded. The HBc protein has been represented in these studies by HBc from HBV genotypes A and D, while HBc particles of HBV G genotype have not been used in the studies. Structural differences in the G-genotype HBc protein allow for the different behavior of G-genotype HBc virus-like particles in dissociation-reassociation processes used to release capsids from packaged material. The invention is based on the ability of HBV G-genotype HBc virus-like particles to produce high yields using an efficient particle producer (E. coli) as well as high yields of dimer-resolved particles. HBcG particles purified from external and internal impurities are stable and retain their ability to pack phage f2susl 1 dsRNA [13] fragments for a long time under conditions of reduced ionic strength in the environment.

DISCLOSURE OF THE INVENTION The invention is an expression vector for the HBc protein (HBcG) of the hepatitis virus G genotype (HBV-G), pHBc45p, consisting of the plasmid pBR327 replicon, promoter Ptrp, HBV-G HBc gene (HBcG), and ampicillin ne. resistance marker genes (Ap ^R and Km ^{R, respectively} ). Recombinant HBV-G HBc virus-like particles (HBcG-VVD) are produced by host cells of E.coli K.802 or E.coli BL21 transformed with pHBc45p.

Another aspect of the invention is a method of obtaining high purity HBcG-VVD which utilizes HBV-G HBc protein dimers to obtain fully purified HBcG-VVD.

[010] Another aspect of the invention is the filling of the resulting virus-like particles with dsRNA fragments of RNAfaga f2susl 1.

[011] The invention is further illustrated in the drawings.

Figure 1: Schematic diagram of HBV G genotype (HBV-G) HBc protein expression vector pHBc45p.

rep (pMBl) - replication ori, Ap - (ampicillin) resistance gene, NEO - neomycin kanamycin resistance gene, Ptrp - E.coli Ptrp promoter, HBcAg45p Genotype G HBV-G HBc gene.

Figure 2:

Distribution of the mixture of particles obtained by dissociation of HBcG virus-like particles (HBcG-VVD) by gel filtration (Sepharose4FF).

From left: peak 1 - undissociated HBcG-VVD, peak 2 - HBcG dimers.

Figure 3:

Analysis of dimer-resolved HBcG virus-like particles (HBcG-VVD). SDS-PAGE,

Figure 4

Analysis of dimer-resolved HBcG virus-like particles (HBcG-VVD).

NAGE: 1.2% / TAE agarose gel, visualization with EtBr / UV (alternately) and Coomasie Blue (right), 1 - DNA marker (Fermentas # SM1331), 2 - native HBcG-VVD, 3 reassociated, nucleic acid free HBcG-VVD,

Figure 5

Analysis of dimer-resolved HBcG virus-like particles (HBcG-VVD).

EM ''

Figure 6

Analysis of dimer-resolved HBcG virus-like particles (HBcG-VVD).

DLS.

Figure 7;

Analysis of HBcG virus-like particles (HBcG-VVD) filled with f2susl 1 dsRNA. SDS-PAGE,

Figure 8

Analysis of HBcG virus-like particles (HBcG-VVD) filled with f2susl 1 dsRNA.

NAGE: 1.2% / TAE on agarose gel, visualization with EtBr / UV (left) and Coomasie Blue (right): 1 - marker (Fermentas & SM1331), 2 - native HBcG-VVD, 3 - f2susl 1 dsRNA filled particles

Figure 9

Analysis of HBcG virus-like particles (HBcG-VVD) filled with f2susl 1 dsRNA.

EM ''

Figure 10

Analysis of HBcG virus-like particles (HBcG-VVD) filled with f2susl 1 dsRNA.

DLS. '

Figure 11

Nucleotide sequence of the HBc gene of the hepatitis G virus (HBV-G) genotype G.

(GenBank Accession JQ707660), Expression vector

The expression vector pHBc45p is a modified plasmid pBR327, which is shown schematically.

Figure 1.

[013] The Gene Genotype HBV HBc Protein Gene Sequence (GenBank Accession JQ707660) expressed using the vector pHBc45p is depicted in Figure 11.

Г014] 2. Producer

The HBc G-genotype HBc virus-like particles (HBcG-VVD) are produced by E.coli strain K802 or E.coli strain BL21 containing the HBc expression vector pHBc45p, E.coli K802 / pHBc45p and E.coli BL21 / pHBc45p, respectively.

Г015] 3. Cultivating the Producer

Producer cells are cultured in either M9-cas tryptophan-free medium or tryptophan-rich medium 2TYP. M9-Cas Medium: Standard M9 Saline Medium with Additives - 10 G / L Casamic Acid (Casamino Acids, BD, USA), 2 g / L Glucose, 50 mg / L Ampicillin Na (Ap) and Kanamycin Sulphate (Km) 10 mg / L); Composition of 2TYP Medium: Standard 2TY Medium with Additives - phosphates KH2PO4 3.47 g / L and K2HPO4 18.8 g / L, glucose 2 g / L, Ap 50 mg / L and Km 10 mg / L. Culture conditions: Cells are cultured in flasks on a rotary shaker under reduced aeration - 20-25% flask volume, shake rate 150-200 rpm, culture t - 37 ° C, culture time 18 h.

Example 1

Culturing of E.coli BL21 / pHBc45p in 2TYP medium.

Cultivation conditions: 230 mL medium, 1 L flask, 37 ° C, Multitron (Infors AG, Switzerland) shaker incubation at 200 rpm, 18 h culture, OD540 10.4. Total volume 3.0 l, biomass yield 28.0 g. HBcG protein expression level - 3-5% of total cellular protein (after SDS-PAGE analysis with Coomasie Blue G250).

[017] 4. Preparation of HBcG Virus-like Particles

HBcG virus-like particles (HBcG-VVD) are obtained using biomass obtained as described in section 3. The VVD is purified in the following consecutive steps: biomass disintegration, VVD extraction, clarification, impurity precipitation with 10% ammonium sulfate, clarification, HBc precipitation with 35% ammonium sulfate, extraction, clarification, gel filtration, ion exchange. Fractions are analyzed on SDS-PAGE and NAGE gels. The final product is analyzed by DLS (dynamic light scattering) and electron microscopy (EM).

Example 2 Purification of VVD.

g of HBcG-VVD-containing biomass (frozen at -20 ° C) is suspended in 4 volumes (32 ml) of lysis buffer (50 mM Tris / HCl pH 8.0 / 5 mM EDTA / 0.5 mM PMSF / 150 mM NaCl / 0). 1% TritonX100 / 5 mM DTT) and disintegrated in a high pressure machine (FrechPress, GlenMills Inc, USA) at 20,000 psi for three cycles. Total target protein in cell lysate ~ 220 mg (SDS-PAGE) or 20% of total protein in lysate. Protein extraction was then followed by addition of urea to 0.5 M and 1.55 ml of DNaseI solution (2 mg / ml) and MgCl 2 to 10 mM in a rotator for 30 min at + 4 ° C. Clarification is accomplished by centrifugation of the lysate at 10,000 rpm (13,000 g) for 30 min at + 4 ° C; yield 214 mg of target protein. Add 4.74 ml of saturated ammonium sulphate solution (final conc. 10% saturation) to the supernatant, incubate for 1 h in a rotator at + 4 ° C. Clarify at 10,000 rpm (13,000g) for 30 min at + 4 ° C. HBc protein was precipitated with ammonium sulphate up to 35% saturation by adding 18 ml of saturated ammonium sulphate solution and incubating in a rotator for 1 h at +4 ° C. Precipitate is collected after centrifugation at 10,000 rpm (13,000 g) for 30 min at + 4 ° C; ~ 15 mg of the target protein remains in the supematant. Precipitate dissolution is performed with 13 ml PBS + 0.5 M urea / 0.5 mM PMSF / 0.1% TritonX100 / 5 mM DTT for 30 min at +4 ° C using a rotator, followed by clarification at 10,000 rpm (13000g). 30 min at + 4 ° C; Amount of HBc in extract ~ 210 mg in insoluble fraction ~ 0.4 mg. Gel filtration is then performed on a Sepharose4FF 320 ml column by transferring the fractions automatically onto a 60 ml ion exchange column (Fractogel DEAE (M), equilibrating the columns with PBS and 5 mM DTT. Fractions are analyzed by SDS-PAAG and NAGE, yielding 133 mg or 16.6 mg of HBc capsid per 1 g of biomass.

[019] 5. Preparation and purification of HBcG dimers

HBcG protein dimers are obtained using purified HBcG-VVD as described

4 and incubation in a dissociation solution containing Guanidine hydrochloride GuHCl and lithium chloride LiCl [8]. After incubation, the dissociation mixture was centrifuged to remove nucleic acid and capsid aggregates. The supernatant, in turn, is subjected to gel filtration, separating and collecting the fractions containing the dimers (Fig. 2).

Example 3

Preparation of HBcG dimers.

For HBcG-VVD preparation obtained as described in Example 4, Example 4 (volume 165 ml, protein content 133 mg), the buffer was changed using a G25 column (from PBS with 0.5 M KCl / 5 mM DTT to 20 mM PB / 5 mM DTT); protein is concentrated with Amicon Ultra-15 100K filters (Millipore Inc., USA) to conc. 10 mg / ml, total volume 15 ml. Add GuHCl to 1.5 M, 50 mM Tris / HCl (pH 7.5), LiCl to 0.5 M and DTT to 5 mM. Protein end conc. - 2 mg / ml. Incubate for 16 h at +4 ° C in a rotator. Clarification at 10,000 rpm (13,000g) for 30 min at + 4 ° C. 15 ml of supernatant is applied to a Superose6IV2Q ml gel filtration column equilibrated with 1.5 M GuHCl / 50 mM Tris / HCl pH 7.5 / 5 mM DTT. The fractions containing the dimers are collected. Total volume 18 ml, HBc dimer conc. 1.5 mg / ml.

[021] 6. Reassociation of HBcG dimers in capsids

The reseaction of the dimer obtained as described in paragraph 5 into virus-like particle-capsids is carried out by dialysis of the dimer-containing solution against buffer PBS, which involves self-association of the dimers in capsules, analyzed by SDS-PAGE and NAGE gels, electron microscopy. (Zetasizer Nano ZS instrument, Malvem Instruments Ltd, UK), which measures the hydrodynamic size of particles in nanometers (Figure 3).

Example 4

Reaction of HBcG capsids from dimers.

HBcG dimer-containing fractions (18 mL, conc. 1.5 mg / mL) are dialyzed against 6 liters of PBS buffer (14 kD cellulose membrane permeability, Sigma Aldrich, Cat.No D9777-100FT). Clarify at 10,000 rpm (13,000g) for 30 min, +4 ° C, and analyze material by SDS-PAAG, NAGE, electron microscopy, and DLS. The result is a 16 ml capsule solution, protein conc. 1.5 mg / ml.

[023] 7. Packaging of dsRNA in Reassociated HBcG Virus-like Particles - Packaging of f2susll dsRNA in Reassociated HBcG Capsules Obtained as described in Claim 6 is achieved by incubating the reassociated capsid in a medium with low ionic strength; after incubation, the material is concentrated and analyzed as described in section 6 (Figure 4). DsRNA fragments of the RNA-phage f2susll replicative form obtained as described in reference [13] are used as dsRNA sources.

Example 5 Packaging of dsRNA from dimers in reassociated HBc capsids.

To 4.5 mg capsid (1.5 mg / ml) is added 4.5 mg dsRNA in water (0.167 mg / ml), total volume 30 ml. Incubate for 16 h at room temperature. The mixture is concentrated to 3 ml. Add 50 μΐ benzonase (Benzonase 25 U / μΙ, Novagen, Cat.No D00168369), incubate for 16 h at room temperature. Packaged nucleic acids are purified from the nucleases and unpackaged nucleic acids by gel filtration on a Superdex200 '20 ml column equilibrated with PBS. The material obtained shall be analyzed as described in point 6. Yield: 5 ml capsule containing material with HBc conc. 0.9 mg / ml in PBS medium. Store at + 4 ° C.

References

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Claims (5)

Claims 1. HBV G-genotype (HBV-G) HBc protein (HBcG) expression vector pHBc45p, composed of plasmid pBR327 replicon, promoter Ptrp, HBV-G HBc gene (HBcG), ampicillin and neomycin-kanamycin resistance marker genes (Ap ^R , respectively). and Km ^R ), The producer of recombinant HBV-G HBc virus-like particles (HBcG-VVD), which according to claim 1 is a vector of E. coli K802 or E. coli BL21 transformed with the vector pHBc45p. 3. A process for the preparation of high purity HBcG-VVD, characterized in that HBV-G HBc protein dimers are used to obtain fully purified HBcG-VVD. A method for obtaining dsRNA-filled HBcG virus-like particles (HBcG-dsRNA), characterized in that the HBcG protein-like virus particles obtained according to claim 3 are filled with dsRNA fragments of RNA-phage f2sus11. Virus-like particles obtained according to claim 3 or 4.