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Method for preparing vaccine through transpeptidase shearing and application of vaccine

A technology of transpeptidase and antigen peptide, which is applied in the field of vaccine production technology, can solve the problems of reducing antigen activity, low chemical reaction specificity, and inconsistent antigen conformation

Active Publication Date: 2017-01-18
INST PASTEUR OF SHANGHAI CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the specificity of the chemical reaction is not very high, which may lead to the wrong conformation of the coupled antigen, coupled with the unsatisfactory chemical reaction efficiency [4], which may reduce the activity of the antigen [5], and other limiting factors such as the complexity of the process, this method is not very effective. ideal

Method used

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  • Method for preparing vaccine through transpeptidase shearing and application of vaccine
  • Method for preparing vaccine through transpeptidase shearing and application of vaccine
  • Method for preparing vaccine through transpeptidase shearing and application of vaccine

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preparation example Construction

[0100] Preparation of chimeric antigens

[0101] Currently, exogenous epitopes can be displayed on VLPs through gene fusion and chemical cross-linking. Gene fusion takes a long time, and the production and purification processes of different vaccines are complicated and costly. The method of chemical cross-linking combines non-covalent and covalent binding of exogenous antigen epitopes to the surface of VLPs, which is difficult to control the spatial structure of the antigen and may affect the antigenicity. In addition, the conditions of the chemical method are relatively harsh and the cost is high.

[0102] In the present invention, the hepatitis B virus core antigen (HEPATITIS B CORE ANTIGEN, HBcAg) is used as the vaccine carrier, and the HBcAg is cut off at the position of the antigenic determinant, divided into two peptide segments of N-terminal and C-terminal and expressed in Escherichia coli at the same time, both can Correctly folded virus-like particles (Virus-Like p...

Embodiment 1

[0167] Example 1 Two-step purification of VLPs by ammonium sulfate precipitation enrichment and sucrose gradient separation

[0168] The genes of split HBc N-LPETGG-C and HBc-SP55 and HBc-SP70 used in the present invention were inserted into pET28a vector (purchased from Novagen), and then transformed into BL21plysS for expression. When the OD is 0.6-0.8, add 0.5mM IPTG and induce overnight at 18°C ​​and 250rpm. The bacteria solution was centrifuged at 5000 rpm in a Beckman JLA10,500 rotor at 4°C for 10 min, and then washed once with 50 mM Tris pH7.5 and 150 mM NaCl (NT buffer). Transfer the bacterial solution to a 50ml centrifuge tube and centrifuge at 5000rpm, 4°C, 10min. Discard the supernatant, and the bacterial solution can be stored at -80°C for a long time. 1L of bacterial solution was resuspended with 30ml of NT buffer (50μg / ml of RNaseA was added), and ultrasonically disrupted: work for 5s, stop for 5s, a total of 30min. After the sonicated product was centrifuged ...

Embodiment 2

[0177] Example 2 Ni 2+ Column affinity purification and anion exchange two-step purification of transpeptidase and protein

[0178] The two proteins in the present invention: transpeptidase S-6xHis and GGGS-AD-4-6xHis are both expressed on the pET28a vector, transformed into BL21plysS. The induction conditions were the same as those for VLPs. The same goes for ultrasonic conditions. The centrifuged supernatant after sonication was subjected to Ni 2+ Column affinity purification and ion exchange purification. The buffers used are all NT buffers.

[0179] Ni 2+ Column affinity purification steps

[0180] 1. Add 30ml supernatant to NT buffer to balance Ni 2+ In the column, the shaker in the cold storage was mixed for 30min at a speed of 90rpm.

[0181] 2. Let the unbound Ni 2+ The solution from the column is gravity flowed, collected and labeled as flowthrough.

[0182] 3. Add 10ml of NT buffer containing 25mM imidazole for the first wash, marked as wash 1.

[0183] 4....

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Abstract

The invention provides a method for preparing a vaccine through transpeptidase shearing and application of the vaccine. Experimental results show that the method for preparing the vaccine through transpeptidase shearing is simple, convenient and stable in process; and a prepared VLP (virus-like particle) can efficiently activate protective immunoreactions and generate high neutralizing antibody titer when used as a vaccine.

Description

technical field [0001] The invention belongs to the field of biomedicine, specifically, the invention relates to a novel vaccine production technology, which uses a new antigen display technology to display antigens on the surface of VLPs through protein cleavage to produce vaccines. Background technique [0002] Virus-like particles (VLPs) are an important vaccine tool. VLPs can simulate virus infection and carry virus antigens, which can produce effective immune protection. Plus VLP does not contain the genome of the virus, so it is very safe. In addition, VLPs have a wide range of applications as carriers to display heterogeneous antigens [1]. [0003] At present, there are two main methods for displaying heterogeneous antigens on VLPs: gene fusion expression and chemical coupling [2]. Through gene fusion, the antigen and VLP can be fused and expressed, and the target antigen can be displayed on the surface of the VLP. To produce different vaccines, it takes a long pe...

Claims

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Application Information

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IPC IPC(8): C12N7/04C12N15/62C12N1/21C12N5/10C07K16/10C07K16/08C07K16/18C07K16/12A61K39/00A61K39/04A61K39/245A61K39/125A61P31/14A61P31/22A61P31/06A61P37/02
Inventor 钱志康汤书兵宣宝琴
Owner INST PASTEUR OF SHANGHAI CHINESE ACADEMY OF SCI
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