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A method for purifying prokaryotic cells expressing virus-like particles

A virus-like particle and purification method technology, applied in the field of virus-like particle purification, can solve the problems of HBcAg purity and the difficulty of meeting quality standards for residual substances, and achieve the effects of easy amplification, short reaction time, high yield and purity

Active Publication Date: 2018-01-02
JIANGSU THERAVAC BIO PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The role of HBcAg in the development of hepatitis B diagnostic reagents and the research of preventive or therapeutic hepatitis B vaccines has been paid more and more attention. Since the late 1970s, full-length or truncated HBcAg has been in Escherichia coli, yeast, and insect systems. Obtain overexpression (Nature282,575-579 (06December1979); doi:10.1038 / 282575a0, Hepatitis Bvirus genes and their expression in E.coliMARK PASEK; Nature296,677-678 (15April1982) Electron microscopy of hepatitis B in sEyn the core antibiotic ..COHEN&J.E.RICHMOND R.E.Lanford,L.Notvall,Virology176(1990)222.), but due to its preparation process, the obtained HBcAg is difficult to meet the quality standards of the Pharmacopoeia in terms of purity and residual substances. Therefore, It is necessary to develop a low-cost preparation and purification process that is suitable for large-scale scale-up production and can meet the standards stipulated in the Pharmacopoeia

Method used

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  • A method for purifying prokaryotic cells expressing virus-like particles
  • A method for purifying prokaryotic cells expressing virus-like particles
  • A method for purifying prokaryotic cells expressing virus-like particles

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1. Analysis and synthesis of HBcAg gene

[0050] There are multiple serotypes and genotypes of hepatitis B virus. In the present embodiment, a total of 9125 strains of HBcAg target gene sequences of various genotypes were obtained from NCBI, wherein the genotype classification is as shown in Table 1 below, using Mega software (Mega5 .0, BorlandDelphi) to analyze the retrieved HBcAg of each genotype, so as to obtain the consensus sequence of each genotype (Consensus sequence), a total of 30 subtypes of HBcAg consensus gene sequence (as shown in Table 1), take the conservativeness greater than 50 % of amino acid categories, the mutation rate of the full-length HBcAg sequences of each genotype was analyzed one by one, and then the consensus sequence analysis (Consensus analysis) was performed again to obtain the final HBcAg full-length consensus sequence of 183 amino acids, which was used as The source of the HBcAg gene in the present invention, the consensus seque...

Embodiment 2

[0056] Example 2. HBcAg expression vector construction and induction identification

[0057] Using the synthesized Con183 nucleotide fragment as a template, Con183F (SEQ.ID No:3) as a forward primer (NdeI restriction site was introduced at the 5' end), and Con183R (SEQ.ID No:4) as a reverse primer (The 3' end introduces an EcoRI restriction site). The PCR reaction was carried out in a PCR instrument (ABI) according to the conditions shown in Table 2.

[0058] SEQ.ID No:3: Forward primer (NdeI): 5'-GGAATTCCATATGGACATTGACCCG-3'

[0059] SEQ.ID No:4: Reverse primer (EcoRI): 5'-CCGGAATTCCTAACACTGGCTTTC-3'

[0060] Table 2PCR reaction conditions

[0061]

[0062] A fragment of about 560bp was obtained after PCR amplification. The amplified product obtained above was connected to a commercially available pMD18-T vector (manufactured by TAKARA Company), and named pMD18-T-Con183, identified by NdeI / EcoRI digestion and sequenced to obtain the enzyme cleavage site added at both e...

Embodiment 3

[0064] Example 3. Fermentation of HBcAg

[0065] Melt the bacteria glycerin tube suspension stored in the -80°C refrigerator at room temperature and shake well, then streak on the LB plate, culture at 37°C for 8-12 hours to grow a single colony and microscopically check for no bacteria, and the bacteria can be used for liquid after rejuvenation. nourish. Select a single colony on the rejuvenation plate and inoculate it into a 500mL Erlenmeyer flask filled with 50mL LB liquid medium, culture at 37°C, 200rpm for 10h, and the OD600 reaches about 3.0 as the first-class seed.

[0066] The primary seeds were inoculated into a 2L Erlenmeyer flask with 300 mL of LB liquid medium at 5% inoculum volume, and cultured at 37°C and 200 rpm for 14 hours until the OD600 reached about 4.0 as the secondary seeds.

[0067] After microscopic examination of the second-level seeds confirms that there is no miscellaneous bacteria, 5% inoculum volume (250ml) is aseptically inoculated into a fermente...

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Abstract

The invention relates to a method for purifying an escherichia coli expressed viroid particle. The purification method comprises a precipitation step, a first chromatography step, a second chromatography step, a concentration step and a third chromatography step, wherein the precipitation step comprises an ammonium sulfate precipitation step; the first chromatography step comprises a molecular screen chromatography step; the second chromatography step comprises a hydroxyapatite chromatographic analysis; the concentration step comprises an ultrafiltration and concentration step; the third chromatography step comprises a heparin affinity chromatography step.

Description

technical field [0001] The invention relates to a method for purifying virus-like particles expressed in prokaryotic cells such as Escherichia coli. Specifically, the present invention relates to a method for purifying virus-like particles expressing hepatitis B virus surface antigen or core antigen in Escherichia coli. Background technique [0002] Virus-like particles or virus-like particles (virus-like particles, VLPs) are hollow particles containing one or more structural proteins of a certain virus, without viral nucleic acid, unable to replicate autonomously, and are identical or similar in shape to real virus particles. Commonly known as pseudo virus. [0003] In recent years, it has been found that the capsid protein genes of most viruses can effectively self-assemble in the eukaryotic expression system and the capsid protein genes of a few viruses in the prokaryotic expression system. This provides convenient conditions for the basic research of viruses and the de...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/04C07K1/36C07K1/18C07K1/34C07K1/22
Inventor 宋翠灵李建强孙洪林顾月周童徐振兴黄红颖程晓晓徐晓威
Owner JIANGSU THERAVAC BIO PHARMA CO LTD
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