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Recombinant virus-like granule and preparation method and application thereof

A recombinant virus and virus-like technology, applied in the field of biomedicine, can solve problems such as hidden dangers, low-quality vaccines, and easily damaged particle assembly structures, and achieve the effect of efficient secondary assembly

Inactive Publication Date: 2019-04-23
INST OF PROCESS ENG CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this preparation strategy has the following problems: First, the particles assembled in the cells are not uniform in structure and incomplete. There are a large number of dimers, assembly intermediates and even misassembled structures, resulting in low quality, low activity, and easy aggregation and low stability of vaccines after direct purification
The second is that the purification process is easy to destroy the structure of the particle assembly. Since the interaction between the protein of the particle assembly is weak, the interaction with the medium adsorption and elution will destroy the structure of the assembly, depolymerize some particles, and lead to a high purification yield. and significantly reduced activity
The third is that some impurities are not easy to remove, and there are potential safety hazards in directly purified vaccine products. Whether it is for mammalian cells or insect cells, it is easy to wrap host DNA or RNA and host proteins inside when they are assembled in cells. Particles may also bind to host proteins
[0006] CN 101848730 A discloses that the above method is used for the preparation of fusion HBc-VLP, mainly in the presence of denaturing agent urea or guanidine hydrochloride to purify, and then reassemble to form VLP, but the existing method cannot obtain a class similar to the natural structure virus-like HBc particles

Method used

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  • Recombinant virus-like granule and preparation method and application thereof
  • Recombinant virus-like granule and preparation method and application thereof
  • Recombinant virus-like granule and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0132] Example 1 Expression and Identification of Recombinant Hepatitis B Virus Core Antigen

[0133] Whole Gene Synthetic HBc 149 The fragment was inserted into the vector pET28a through BamHI and HindIII sites, named pET28a-HBc 149 , subcloned into competent cells BL21(DE3), HBc 149 The amino acid sequence of the fragment is shown in SEQ ID NO.3. Inoculate to 20L fermenter for fermentation, wait for OD 600 To 5-8 o'clock, add 1mM IPTG induction 4h (thalline growth curve sees Figure 1A ), collected by centrifugation (induced expression identification results see Figure 1B ). Add the bacteriostasis solution (20mM Tris-HCl, 3mM EDTA, 0.5% Triton X-100, 1mM PMSF, pH 8.0) at a ratio of 1:10, and under the pressure of 800MPa, cyclically break through high-pressure homogenization three times, centrifuge Collect the supernatant, i.e. recombinant hepatitis B virus core antigen, transmission electron microscope (TEM) identification results see Figure 1C .

[0134] SEQ ID NO....

Embodiment 2

[0138] Example 2 Effects of Depolymerization Conditions on Protein Yield, Purification, Structure and VLP Extracellular Reassembly

[0139] 1. Effect of depolymerization conditions on protein yield

[0140] The crushed supernatant was precipitated with 1M ammonium sulfate, and denaturants of different types and concentrations were used to depolymerize the protein, and then the protein concentration was measured by the Bradford method (see Table 1 for the protein yield), and the depolymerized solution was identified by SDS-PAGE ( see results figure 2 ).

[0141] Table 1 Effect of different types and concentrations of denaturants on protein yield

[0142] Depolymerization conditions

Protein concentrationmg / ml

protein yield

2M urea

3.64

72.8％

4M urea

4.59

91.8％

6M urea

4.83

96.6％

8M urea

4.94

98.8％

6M GdnHCl

4.99

99.8％

[0143] From Table 1 and figure 2 It can be seen that when the concen...

Embodiment 3

[0152] Example 3 Effect of Extracellular Assembly Conditions on the Assembly of Virus-like Particles of HBc

[0153] The extracellular assembly of virus-like particles is mainly affected by temperature, pH value of solution, surfactant, reaction time and ionic strength, which were investigated in this experiment. Samples depolymerized with 4M urea were purified by Ni FF and dialyzed first into 2M urea and then into the respective assembly buffers.

[0154] The characterization method mainly adopts TSK G4000SWxl with a flow rate of 0.6ml / min. By corresponding with the TEM results, it is determined that the peak time is 9.5min for aggregates, 10.6min for assemblies and 19.4min for unassembled structures.

[0155] 1. The influence of reaction temperature on assembly: the reaction temperature mainly affects the reaction rate, such as Figure 7A As shown, the protein folding speed is too fast at room temperature, and most of the protein is precipitated, but at 4°C, the protein is ...

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Abstract

The invention provides a recombinant virus-like granule and a preparation method and application thereof. The method comprises the following steps that cells expressing the recombinant virus capsid protein are fermented, then fermentation products are collected, a 2-6-M urea solution is adopted for dissolving and depolymerization, and an intact virus-like granule of the recombinant viral capsid protein is obtained through extracellular secondary assembly. According to the preparation method of the recombinant hepatitis B virus core antigen granule, the recombinant capsid protein precipitated by ammonium sulphate is dissolved, depolymerized and purified under relatively mild conditions, and finally extracellular secondary assembling is conducted to form the virus-like granule with a naturalstructure. The method is simple and efficient, and the prepared recombinant hepatitis B virus core antigen granule has the advantages of being complete and uniform in structure and free of host nucleic acid and miscellaneous protein. The granule has the important significance and broad application prospects.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to a recombinant virus-like particle and its preparation method and application, mainly to a recombinant virus-like particle expressed in a soluble form, its preparation method and application, and specifically to a recombinant hepatitis B virus core Antigen particle and its preparation method and use. Background technique [0002] Hepatitis B virus core antigen (HBc) is a single-chain capsid protein, which can independently form a nucleic acid-free icosahedral structure, has a shape similar to that of natural viruses, and has a size of about 28nm. Its main immunodominant region (MIR) is located on the surface of the particle to form spikes, which can effectively present antigens and stimulate strong humoral and cellular immunity. In addition, inserting or replacing a segment of other antigens in its MIR region will not affect its formation of virus-like spherical particle struct...

Claims

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Application Information

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IPC IPC(8): C07K14/02A61K39/295A61K39/29A61K39/145A61K39/125A61K39/12A61K45/00A61K47/46A61K47/69A61P31/14A61P31/16A61P31/20A61P35/00
CPCA61K39/12A61K45/00A61K47/46A61K2039/5258A61K2039/70A61K47/6901A61P31/14A61P31/16A61P31/20A61P35/00C07K14/005C12N2710/20034C12N2730/10123C12N2730/10134C12N2760/16034C12N2770/32334
Inventor 刘永东苏志国张耀
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
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