JPH08187086A - Purification of viral gene - Google Patents

Abstract

PURPOSE: To quickly and easily purify the gene by filtering a virus-contg. solution through a porous hollow fiber membrane with specific average pore diameter into a concentrated virus solution followed by denaturation of the viral protein in the solution to decompose it into a gene and protein. CONSTITUTION: A virus-contaminated or virus-contg. solution is filtered through a porous hollow fiber membrane (e.g. made of cuprammonium regenerated cellulose) 10-200nm in average pore diameter and 10-100μm in thickness capable of trapping virus particles to prepare a concentrated virus solution, which is then spiked with a protease to denaturate the viral protein in the virus in the solution to effect decomposition the virus protein into a viral gene and viral, protein, thus easily purifying in a short time the viral gene useful in the genetic engineering, genetic therapy and viral test areas by skipping complicated operations as much as possible.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for purifying viral genes, and more particularly to a method for purifying viral genes (DNA and RNA) used in the fields of genetic engineering, gene therapy and virus testing.

[0002]

2. Description of the Related Art In recent years, with the progress of genetic engineering technology, in the fields of nucleotide sequence analysis and gene therapy of viral genes, viral vectors are prepared by gene recombination, and further, PCR method (Polym) for amplifying viral genes is carried out.
A virus test using the erase chain reaction method) has been carried out, and thus purification of viral genes has become necessary.

As a method for concentrating and purifying viruses from an aqueous solution, a method of separating the particles according to the size of particles by combining low-speed centrifugation and high-speed centrifugation or polyethylene glycol (PE
A method of precipitating and concentrating a virus using a polymer such as G) and removing high concentration of PEG by dialysis has been performed. However, the former has a problem that an expensive centrifuge is required and it takes a long time to centrifuge, and the latter has a problem that dialysis takes time and a coexisting protein is precipitated together with the virus. In addition, there is a problem because the operation is complicated when using a virus having a high degree of risk.

On the other hand, as a method of separating and purifying viral genes, a method of protein denaturation of a virus solution by phenol extraction, a treatment with a surfactant such as sodium lauryl sulfate (SDS) or sarcosyl, and a protease K are used. There is a method using such a proteolytic enzyme, an alkali denaturation treatment using an alkali metal hydroxide aqueous solution, and a heat treatment, and in some cases, these treatments are combined.

There is a problem that performing the virus gene separation / purification process after the virus concentration / purification process from the aqueous solution further complicates the operation because these processes are independent systems. In order to solve such a problem, for example, in Japanese Unexamined Patent Publication No. 3-180182, in a method for purifying a gene of a phage in a microbial culture solution, a microbial cell is a membrane filter having a pore size of 0.45 μm to 0.22 μm. After removal by filtration with a filter, the filtrate is treated with proteolytic enzyme, etc. to decompose and denature the phage protein, and then filtered through an ultrafiltration membrane having a molecular weight cut-off of 20,000 to 1,000,000 to obtain proteins and impurities. To remove the
After obtaining the digene or removing the microbial cells with a filter, the filtrate is filtered through an ultrafiltration membrane to remove coexisting small molecular weight substances, and the phage is purified on the ultrafiltration membrane. A method of processing by processing or the like is disclosed.

However, in the method of filtering with an ultrafiltration membrane, clogging easily occurs, so that the filtration rate is slow,
There was a problem that the processing capacity was not high, and as a result, a large proportion of large proteins, bacterial fragments, and free naked genes derived from bacterial cells that were mixed with other than viral genes were contaminated. .

[0007]

SUMMARY OF THE INVENTION An object of the present invention is to purify highly pure viral genes after concentrating viruses in a short time while avoiding complicated operations from a large amount of protein aqueous solution as much as possible.

[0008]

[Means for Solving the Problems] The present inventors have accomplished the present invention as a result of diligent efforts to solve the above problems. That is, the present invention comprises a step of obtaining a concentrated virus solution by filtering a solution containing a virus or containing a virus through a porous hollow fiber membrane having an average pore size of 10 nm or more and 200 nm or less, and a virus in the concentrated virus solution. Is a method for purifying a viral gene, which comprises a step of denaturing the viral protein to decompose it into a viral gene and a viral protein. The contents will be described below.

The porous hollow fiber membrane used for filtration in the present invention has a mean pore diameter and a pore diameter measured by the method described below in relation to the size of the virus to be removed in order to capture the virus from the protein solution and allow the protein to permeate. Is 10n
It is necessary to be in the range of m to 200 nm. 10n
If it is less than m, the protein permeability is remarkably reduced, and 200 nm
If it exceeds, the virus removal performance is significantly reduced.

Further, an appropriate pore size is selected according to the size of the target virus particle. That is, it is advisable to select a filter having a pore size equal to or smaller than the virus particle size. JP-A-61-254202, JP-A-61-2
As the copper-ammonia method regenerated cellulose porous hollow fiber membranes represented by JP-A-547027 and JP-A-4-371221, filters having an average pore diameter of 15, 35 and 75 nm are commercially available. In addition, the virus contaminated solution is a cell lysate,
When a large amount of large contaminants such as cell culture medium and blood are mixed, it is filtered in advance with a filter having a pore size of 35 nm to 0.45 μm to remove the large amount of large contaminants, and then filtered for virus concentration. If this is done, the time of the filtration process can be shortened, and a virus of higher purity can be purified.

The porous hollow fiber membrane used for filtration in the present invention has a film thickness capable of capturing viruses of 10 μm or more and 100 μm or more.
m or less. If it is less than 10 μm, the virus removal rate is lowered, and if it exceeds 100 μm, the protein permeability is lowered. Here, good virus removal performance means that the logarithmic removal performance (LRV) of Japanese encephalitis virus (particle diameter of about 45 nm) is 4 or more, and high protein permeability means that albumin permeability is 90%, for example. It means that it is above. LRV is a value defined as follows.

LRV = log ₁₀ (N _o / N _f ) N _o ; virus concentration in the original solution before filtration N _f ; virus concentration in the filtrate after filtration The porous hollow fiber membrane used for filtration in the present invention is a membrane. It is preferable to have a layered structure in the thickness direction.

The porous hollow fiber membrane used for filtration in the present invention has an average porosity of 0.3 to 0.6 in the thickness direction in order to reduce clogging and maintain a high permeation rate. It is preferable. Here, the porosity (P _r ) is calculated by the following method. P _r = 1-ρ _a / ρ _p P _r : Porosity (-) ρ _a : Apparent density when swollen in water ρ _p : Polymer density (1.561 g in the case of cellulose)
/ Cm ³ ) The material of the porous hollow fiber membrane used for filtration in the present invention includes polyvinylidene fluoride, regenerated cellulose, cellulose, cellulose acetate, polysulfone, vinyl chloride, polyester, polymethylmethacrylate.
Glycine, acrylonitrile, or nylon can be used. Of these, hydrophilic polymers are preferably used to remove virus particles in the protein aqueous solution, and regenerated cellulose is particularly preferable from the viewpoint of filtration performance and protein permeability. As a result of investigating the correlation regarding the adsorption between the protein and the polymer material, it is considered that the hydrophilic material is more preferable as the material of the present invention because the protein adsorption is smaller, and regenerated cellulose is particularly preferable.

In the method of the present invention, the filtration pressure is 0.1.
It is preferably -1 atm. If the pressure is less than 0.1 atm, the protein permeability will increase, but the virus removability tends to decrease. Furthermore, by washing the filter after virus filtration with a buffer such as PBS, it is possible to further remove contaminating proteins and dissolved naked DNA. In order to effectively remove free DNA / RNA, DNA / RNA degradation reaction may be performed in advance by adding DNase / RNase to the sample before filtration.

In the present invention, the viral protein is denatured
The method of decomposition can be carried out while the virus is trapped on the hollow fiber membrane, or the concentrated virus solution can be recovered from the hollow fiber membrane. Examples of the method performed while the virus is captured on the hollow fiber membrane include a method using a proteolytic enzyme, a method using a surfactant, an alkali denaturation treatment, and a heat treatment. When the concentrated virus liquid is recovered from the membrane, in addition to the above method,
Processing can also be performed. In some cases, these processes may be combined. By these treatments, the viral protein is denatured and the viral gene is separated from the viral protein.

Next, a buffer solution such as TE buffer solution (Tr
The virus gene solution can be obtained by washing the hollow fiber membrane with is-HCl buffer EDTA) or the like for filtration. A PCR reaction can be performed using the solution, or an ultrafiltration method or an ion exchange method can be performed in order to remove a small amount of coexisting proteins and improve the purification degree of viral genes. it can. If necessary, this viral gene solution can be concentrated by a conventional ethanol precipitation treatment.

[0017]

【Example】

[0018]

Example 1 Escherichia coli phage ΦX17 was added to a supernatant obtained by removing cell fractions from a cell culture solution cultured with hybridoma cells.
I spiked 4. This solution was filtered at a pressure of 200 mmHg using a copper ammonia method regenerated cellulose porous hollow fiber membrane (BMM) having an average pore diameter of 35 nm, a film thickness capable of capturing viruses of 35 μm, and an average porosity of 0.5, and the following ( The transmittance of protein, released DNA, and ΦX174 was calculated by the formula (1). The amount of protein in the solution was measured by Bio-Rad Protein Assay Kit (Bio-Rad), the amount of released DNA was measured by Threshold Hold Total DNA Assay System (Molecular Devices), and the concentration of ΦX174 was measured by the plaque method. . Table 1 shows the above results.

Permeability = concentration in filtrate / concentration in original solution (1)

[0020]

[Table 1]

On the filter which captured the virus, proteolytic treatment was carried out with proteolytic enzyme Protease k.
Further, the filter was washed with PBS, and the solution was treated with a primer having a sequence specific to the viral gene to give P
CR reaction (Saki, RK et al, Scien
ce 239 487-491 (1988)). At the same time, a commercial standard of viral genes (Takara Shuzo)
PCR reaction was also performed with the same primer. Each sample was subjected to electrophoresis according to a standard method. Lane 1
Is a molecular weight marker obtained by cleaving pUC18 with a restriction enzyme HaeIII, lane 2 is obtained by subjecting the sample purified from Example 1 to PCR reaction, and lane 3 is
A commercially available standard product was subjected to PCR reaction. The result is shown in FIG. As a result, it was found that the present method can easily purify a viral gene with high purity.

[0022]

INDUSTRIAL APPLICABILITY According to the present invention, the virus in the protein solution can be easily concentrated in a short time in a large amount with a high purity using a filtration filter, and then the viral gene can be purified.

[Brief description of drawings]

FIG. 1 is a diagram showing an electrophoretic pattern of a viral gene purified according to Example 1 of the present invention, together with a viral gene purified by a conventional method and an electrophoretic pattern of a commercially available viral gene.

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Claims (5)

[Claims] 1. A step of obtaining a concentrated virus solution by filtering a solution containing a virus or containing a virus through a porous hollow fiber membrane having an average pore diameter of 10 nm or more and 200 nm or less, and the virus in the concentrated virus solution. A method for purifying a viral gene, which comprises the step of denaturing the viral protein to decompose the viral protein into a viral protein. 2. The method for purifying a viral gene according to claim 1, wherein the hollow fiber membrane has a thickness of 10 μm or more and 100 μm or less in the membrane for capturing virus particles. 3. After carrying out the filtration on a hollow fiber membrane, the viral protein is denatured on the hollow fiber membrane to decompose into viral genes and viral proteins, and the hollow fiber membrane is washed with an aqueous solution to obtain viral genes. The method for purifying a viral gene according to claim 1 or 2, wherein the virus is recovered. 4. The method for purifying a viral gene according to claim 1, wherein the concentrated viral liquid is recovered from the hollow fiber membrane, and then the viral viral protein is denatured in the solution to decompose into a viral gene and a viral protein. 5. The method for purifying a viral gene according to claim 1, wherein the hollow fiber membrane has an average porosity in the membrane direction of 0.3 or more and 0.6 or less.