Method for preparing and purifying oncolytic virus, and recombinant oncolytic rhabdovirus

An oncolytic rhabdovirus and oncolytic virus technology, applied in the field of biomedicine, can solve the problems of low recovery rate of infectious virus particles, limit the later application of oncolytic virus, increase the production cost of virus vaccine, etc. Effect of inactivation rate control and cost reduction

Pending Publication Date: 2020-09-15
FANTASIA BIOPHARMA ZHEJIANG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, virus purification mainly includes PEG concentration, ultracentrifugation concentration, column concentration (ion exchange column, affinity column, molecular sieve, etc.), ultrafiltration concentration, dialysis, etc., although PEG concentration method can obtain a higher virus recovery rate , but the existence of PEG limits the late application of oncolytic virus, such as virus safety evaluation experiments, etc.
Column concentration and ultrafiltration concentration are not suitable for laboratory research because of their high price, and column concentration requires a certain amount of high-salt solution washing, which will cause great inactivation of VSV virus, resulting in the loss of infectious virus particles. Low recovery rate
In general, these operating methods have caused a large amount of virus loss, which greatly increases the production cost of virus vaccines

Method used

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  • Method for preparing and purifying oncolytic virus, and recombinant oncolytic rhabdovirus
  • Method for preparing and purifying oncolytic virus, and recombinant oncolytic rhabdovirus
  • Method for preparing and purifying oncolytic virus, and recombinant oncolytic rhabdovirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Example 1 Vero production cells were infected with inoculated doses of different MOI values, and the changes in the total amount of virus titers obtained from production amplification were compared.

[0078] In Vero cells, replace the original complete medium with opti-MEM, then infect Vero cells with VSV virus at MOI=1, 5, 10, and 20, and replace with complete medium after 1h-3h, and wait until the cells are completely lysed After (about 60h), the supernatant was collected to detect the changes in the prepared virus titer (TCID50) under different original virus inoculation (MOI). The overall experimental process refers to figure 1 The specific implementation described in .

[0079] The specific steps of the above experimental process are as follows:

[0080] 1. Add 2 mL of Vero-E6 cell suspension to each well of a 6-well culture plate to make the cell volume reach 4×10 5 Each well, a total of 5 wells, were incubated at 37°C with 5% CO2 by volume for 16 h.

[0081] 2...

Embodiment 2

[0089] Example 2 Further, in order to enhance the infectivity of the virus to the cells, PBS, DMEM-0, and opti-MEM were specially selected as the solvent (MOI=5) for the dilution of the U400 virus infection to infect the cells,

[0090] In Vero cells, replace the original medium with PBS, DMEM-0, and opti-MEM respectively, then add VSV virus infection at MOI=5 for 2 hours and replace with complete medium, and collect the supernatant after 48 hours to detect the virus produced by the virus strain The titer (TCID50).

[0091] The specific operation steps of detecting the titer of above-mentioned virus are as follows:

[0092] 1. Add 2 mL of Vero-E6 cell suspension to each well of a 6-well culture plate to make the cell volume reach 4×10 5 cells / well, a total of 4 wells were incubated at 37°C with 5% CO2 by volume for 16 h.

[0093] 2. Take the cells in one of the wells to digest and count them. Dilute the U400 virus to 2 mL with PBS, DMEM-0, and opti-MEM for the cells in the r...

Embodiment 3

[0101] Influence of serum concentration on virus titer in embodiment 3 culture medium

[0102] In the process of traditional virus preparation, it will be found that during the amplification process of some viruses, the serum concentration in the medium will seriously affect the increase of virus titer. In order to solve this problem and further increase the virus titer, optimize the virus Amplification process, in Vero cells, replace the original medium with DMEM-0, then VSV virus infects Vero cells according to MOI=5, and after 2h-3h, use volume percentages of 0%, 1.5%, 3%, and DMEM medium prepared with different concentrations of 6% and 9% FBS was used for virus amplification. After the cells were completely lysed, the supernatant was collected to detect the virus titer (TCID50) under different serum concentrations.

[0103] The concrete steps of detecting the titer of above-mentioned virus are as follows:

[0104] 1. Add 2 mL of Vero-E6 cell suspension to each well of a 6...

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Abstract

The invention discloses a method for preparing and purifying oncolytic virus, and a recombinant oncolytic rhabdovirus. According to the preparation method, Vero cells are utilized to prepare oncolyticviruses (especially VSV viruses). Firstly, virus amplification conditions are optimized by utilizing the Vero cells, wherein the optimized conditions include virus infection complex number (MOI), virus diluent, virus amplification liquid, serum concentration during amplification, cell culture volume, virus harvesting time and the like; furthermore, virus purification conditions are optimized, wherein the optimized conditions include centrifugal force, sucrose bottoming centrifugation, centrifugation time, step-by-step centrifugation and the like; the VSV virus particles infect the vero cellsfor 48 h in a 3% FBS culture medium according to a proportion that MOI is equal to 5, and virus liquid is harvested; and after 26000*g centrifugation is conducted for 1 h at a temperature of 4 DEG C,28000*g centrifugation is conducted (30 min to 45 min), and it is determined that the high-titer VSV virus can be stably prepared under the step-by-step centrifugation condition. The preparation method is efficient, stable and cost-saving, provides technical support for subsequent large-scale production of the VSV virus, and can be used for preparing anti-tumor virus drugs.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a method for preparing and purifying an oncolytic virus and a recombinant oncolytic rhabdovirus. [0002] technical background [0003] Vero cells are African green monkey kidney cells, which are a continuous passage cell line. The advantage of this cell is that its quality and exogenous factors are highly controllable, its reproduction speed is fast, it can grow at a high density, and it has the advantages of microcarrier culture and technological production. , such as VSV virus (vesicular stomatitis virus) has little effect on replication, and high and low levels of virus particles can be amplified. Vero cells can be used for virus culture substrate cells in vaccine production. In 1982, Vero cells have been approved by WHO to be used as production substrates for human vaccines, such as the production of polio vaccine and rabies vaccine. [0004] Cancer and conventional cancer therap...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N7/02C12R1/93
CPCC07K14/005C12N7/00C12N2760/20222C12N2760/20232C12N2760/20251
Inventor 秦晓峰吴飞韦治明
Owner FANTASIA BIOPHARMA ZHEJIANG CO LTD
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