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Method for concentration of virus

A technology of virus concentration and virus, applied in the direction of virus, virus/bacteriophage, double-stranded DNA virus, etc., can solve the problems of harmful infected cells, a lot of labor, hindering operation, etc., and achieve the effect of simple and easy concentration

Inactive Publication Date: 2011-03-02
JAPAN TOBACCO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method requires expensive instruments and a long separation time, and requires a lot of labor in the operation
In addition, there are methods of concentrating viruses by precipitating viruses with ammonium sulfate, polyethylene glycol, etc., but in these methods, there are hidden dangers that hinder subsequent operations because the reagents used or other components for precipitation are harmful to infected cells, etc.
Therefore, there is a problem that the sample must be purified after virus precipitation, requiring a lot of labor
However, in any of the above-mentioned documents, the virus is used in the form of being combined with a carrier, and the virus itself cannot be isolated
Therefore, the use of viruses concentrated by these methods is limited

Method used

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  • Method for concentration of virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0160] Example 1 Exploration of lectins capable of concentrating HHV-6

[0161] Biotinylated 15 lectins (Con A, DBA, LCA, PHA-E4, PNA, RCA120, UEA-I, WGA, ABA, DSA, Lotus, MAM, PHA-L4, SBA, SSA) with cultured HHV-6 solution reaction, investigated whether HHV-6 can be concentrated by binding to tamavidin-bound magnetic beads.

[0162] 1. Making HHV-6 Solution from Cultured T Cells

[0163] Human umbilical cord blood-derived cultured T cells were infected with recombinant HHV-6 expressing EGFP (Patent No. 3923505) to prepare an EGFP-type HHV-6 solution.

[0164] 2. Preparation of tamavidin magnetic beads

[0165] Wash 300 μl of magnetic beads (Dynabeads M-270 Carboxylic Acid, Dynal Company) coated with carboxyl groups on the surface with 300 μl of 0.01N sodium hydroxide for 10 minutes, and then wash 3 times with 300 μl of ultrapure water, each time for 10 minutes. For the washed magnetic beads, add 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) (PIER...

Embodiment 2

[0174] The concentration of HHV-6 in the embodiment 2 saliva

[0175] Concentration of HHV-6 in saliva using lectinated biotin and tamavidin magnetic beads was attempted.

[0176] 1. Collection of saliva

[0177] Saliva from the test subjects was collected using a saliva collection tube (Salivette Cotton, manufactured by Sarstedt). Immediately before saliva collection, the subjects rinsed their mouths with distilled water twice, and then held the cotton core of the saliva collection tube in the oral cavity for 2 minutes to collect saliva.

[0178] 2. Quantification of HHV-6

[0179] First, the concentration of HHV-6 in saliva was quantified.

[0180] For 400 μl of saliva collected as above-mentioned item 1, use BioRobot EZ1 (QIAGEN company) and EZ1 Virus Mini Kit v2.0 (QIAGEN company), according to the protocol of EZ1 Virus MiniHandbook (QIAGEN company), the HHV-6DNA in the saliva is carried out purified.

[0181] The resulting DNA was used for quantitative PCR. In...

Embodiment 3

[0194] Example 3 Concentration of lentiviral vectors using lectins

[0195] (1) concentrated

[0196] As the lentivirus, a lentivirus obtained by recombining the EGFP gene into a lentiviral vector having the HIV base gene and the VSV-G envelope (a gift from Mr. Hiroyuki Miyoshi, RIKEN, RIKEN) was used. The biotinylated lectins used were 15 types of biotinylated lectins manufactured by J-OIL MILLS (Con A, DBA, LCA, PHA-E4, PNA, RCA120, UEA-I, WGA, ABA, DSA, Lotus, MAM, PHA-L4, SBA, SSA).

[0197] First, mix 100 μl of EGFP recombinant lentivirus solution (virus concentration 10 2 / ml TE, and in order to observe the concentration effect, the virus with low titer was used. ), PBS 500 μl, biotinylated lectin 10 μg, incubate at 15° C. for 1 hour (inverted mixing). Next, the tamavidin magnetic beads produced in Example 1 were added to the reaction solution, and incubated again at 15° C. for 1 hour (inverted mixing). Then, the Effendorf tube containing the reaction solution was...

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Abstract

Disclosed is a novel method which enables the concentration of a virus or a viral vector in a solution having a low virus concentration in a more convenient manner. Also disclosed is a kit for use in the method. For concentrating a virus from a virus-containing solution having a low virus concentration with high efficiency, a complicated manipulation, an expensive apparatus or a skilled techniquehas been required. The method can facilitate the concentration of a viral vector while keeping the infectivity of the viral vector. Therefore, the method can be used as a safe and convenient vector concentration technique in the fields of gene therapy or vaccine therapy utilizing a viral vector and others.

Description

technical field [0001] The present invention relates to a new method capable of concentrating viruses, viral vectors while maintaining their infectivity, and a kit for carrying out the method. The present invention relates to a safe and simple technique for concentrating retroviral vectors, herpes virus vectors, etc., vectors useful for gene therapy, vaccines, etc. Background technique [0002] So far, various viral vectors have been developed for the purpose of gene therapy and vaccine therapy, and the results are expected. However, if the concentration of the viral vector is low, the desired effect cannot be sufficiently obtained, so the concentration needs to be adjusted. In the field of gene therapy, the efficiency of gene transfer into cells determines the success or failure of the treatment. Therefore, viral vectors with high gene transfer efficiency are used. However, for retroviral vectors including lentiviral vectors, viruses with high titers have not yet been obt...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/02C12N15/09
CPCC12N2710/16051G01N2333/36C12N7/00C12N15/1003
Inventor 高仓由光市川雅子近藤一博清水昭宏
Owner JAPAN TOBACCO INC
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