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Purification method of HBcAg (hepatitis B virus core antigen)-VLP or HBcAg-VLP derivative

A purification method and technology of derivatives, which are applied in the field of hydrophobic chromatography purification of HBcAg-VLP or HBcAg-VLP derivatives, can solve the problems of unfavorable large-scale preparation, low economic value, many steps, etc., and achieve great practical application value and Promotion prospect, good repeatability, stable process effect

Inactive Publication Date: 2019-01-04
INST OF PROCESS ENG CHINESE ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, although a complete HBcAg-VLP purification process has been developed (Expression, purification and characterization of full-length RNA-free hepatitis B core particles, Protein Expression and Purification, 54(2007) 30-37), it involves ion Exchange chromatography, gel filtration chromatography and affinity chromatography, many steps, the yield is very low, less than 10%
However, the steps of this method are too cumbersome and the economic value is low, which is not conducive to large-scale preparation
[0007] Therefore, the current purification method of HBcAg-VLP is still a bottleneck problem that limits its application. How to develop a technology for large-scale preparation of HBcAg-VLP is an urgent task, which is of great significance for expanding its application.

Method used

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  • Purification method of HBcAg (hepatitis B virus core antigen)-VLP or HBcAg-VLP derivative
  • Purification method of HBcAg (hepatitis B virus core antigen)-VLP or HBcAg-VLP derivative
  • Purification method of HBcAg (hepatitis B virus core antigen)-VLP or HBcAg-VLP derivative

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] In this embodiment, the sample solution of HBcAg-VLP is prepared as follows and the pretreatment of the sample solution is carried out

[0066] Plasmid construction and transformation: using the nucleotide sequence of the full-length hepatitis B core antigen, SEQ ID NO.1:

[0067] ATGGACATTGACCCTTATAAAGAATTTGGAGCTACTGTGGAGTTACTCTCGTTTTTGCCTTCTGACTTCTTTCCTTCCGTCAGAGATCTCCTAGACACCGCCTCAGCTCTGTATCGAGAAGCCTTAGAGTCTCCTGAGCATTGCTCACCTCACCATACTGCACTCAGGCAAGCCATTCTCTGCTGGGGGGAATTGATGACTCTAGCTACCTGGGTGGGTAATAATTTGGAAGATCCAGCATCCAGGGATCTAGTAGTCAATTATGTTAATACTAACATGGGTTTAAAGATCAGGCAACTATTGTGGTTTCATATATCTTGCCTTACTTTTGGAAGAGAGACTGTACTTGAATATTTGGTCTCTTTCGGAGTGTGGATTCGCACTCCTCCAGCCTATAGACCACCAAATGCCCCTATCTTATCAACACTTCCGGAAACTACTGTTGTTAGACGACGGGACCGAGGCAGGTCCCCTAGAAGAAGAACTCCCTCGCCTCGCAGACGCAGATCTCAATCGCCGCGTCGCAGAAGATCTCAATCTCGGGAATCTCAATGTTAG.

[0068] Its amino acid sequence SEQ ID NO.2: MDIDPYKEFGATVELLSFLPSDFFFPSVRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLATWVGNNLEDPASRDLVVNYVNTNMGLK...

Embodiment 2

[0077] This example uses ion exchange chromatography to purify HBc-VLP

[0078] The fillers used in this example include conventional DEAE Sepharose FF and Q Sepharose FF anion exchange fillers, and ultra-large pore fillers POROS HQ, POROS 50D and POROS PI.

[0079] In the screening of five kinds of ion-exchange fillers, the supernatant prepared in Example 1 was used as the raw material. After optimizing the pH and elution conditions, the results of yield and final purity were obtained, as shown in Table 1 below. :

[0080] Table 1

[0081]

[0082]

[0083] It can be seen from Table 1 that the best effect of ion exchange chromatography is POROS 50D macroporous anion exchange packing. After purification, the yield of HBc-VLP is 69.64%, and the purity is 37.27%. The effect is equal to or even better than that reported ion-exchange-based purification method.

Embodiment 3

[0085] In this example, the sample solution of HBcAg-VLP was purified using hydrophobic interaction chromatography filler

[0086] In view of the presence of strong hydrophobic regions on the surface of HBc-VLP (structural diagram as shown in figure 1 As shown), hydrophobic chromatography mainly relies on the hydrophobic interaction force to achieve the adsorption and desorption of the target protein, which is milder than the ionic interaction force. Therefore, using the sample solution prepared in Example 1 as the raw material, the ligands of the hydrophobic filler mainly include Butyl-S, Butyl, Octyl and Phenyl. In comparison, Butyl-S Sepharose 6FF and Butyl Sepharose 4FF, two relatively weak hydrophobic fillers, are more suitable for the purification of HBc-VLP. After the sample solution was adjusted to a pH of about pH 7.4 with sodium hydroxide solution, it was fed to the hydrophobicity of Butyl-S Sepharose 6FF or Butyl Sepharose 4FF balanced with 20mM phosphate buffer (p...

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Abstract

The invention provides a purification method for HBcAg (hepatitis B virus core antigen)-VLP or an HBcAg-VLP derivative. The method comprises the following steps: treating a loading solution containingHBcAg-VLP and a loading solution containing the HBcAg-VLP derivative by hydrophobic interaction chromatographic packing, and purified HBcAg or the purified HBcAg-VLP derivative is obtained. Accordingto the purification method, the problems of numerous steps, low yield, expensive consumables and the like of existing methods are solved, purification can be achieved by only one-step hydrophobic chromatography; when a gel filtration refining method is not used for treatment, the yield can reach up to 90% or higher, purity can reach up to about 86%, and the purification effect is greatly improved; if the purification method is supplemented with refining and purification methods such as further gel filtration, the purity can reach 99% or higher. The purification method has good application prospect and very high application value.

Description

technical field [0001] The invention belongs to the field of biochemical separation, and relates to a purification method of HBcAg-VLP or HBcAg-VLP derivatives, in particular to a hydrophobic chromatography purification method of HBcAg-VLP or HBcAg-VLP derivatives. Background technique [0002] Hepatitis B virus core antigen (HBcAg) is the capsid protein of hepatitis B virus. Hepatitis B core antigen virus-like particle (HBcAg-VLP) is composed of 180 or 240 core protein subunits self-assembled and has a particle structure with regular icosahedral symmetry. HBcAg-VLP not only has strong immunogenicity itself, but also can be used as a vaccine carrier, and foreign fragments can be inserted into its C-terminus, N-terminus and immunogenic region (MIR) without affecting the correctness of its assembly. Vaccines based on HBcAg-VLP, such as foot-and-mouth disease vaccine and malaria vaccine, have achieved good results. In addition, HBcAg-VLP can also be used to encapsulate nucleic...

Claims

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Application Information

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IPC IPC(8): C07K14/02C07K1/20C12N15/36C12N15/70C12N1/21C12R1/19
CPCC07K14/005C12N15/70C12N2730/10123C12N2730/10151
Inventor 张松平马小伟苏志国安文琪李正军杨延丽张静静马光辉
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
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