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
View PDF3 Cites 0 Cited by
  • 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 reac

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • 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

Examples

Experimental program
Comparison scheme
Effect test

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....

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Owner INST PASTEUR OF SHANGHAI CHINESE ACADEMY OF SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2023 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap