JPH09224658A - Production of both jc virus particle and coat protein of jc virus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To produce both a JC virus particle and a coat protein of the JC virus in a high yield and in a short time. SOLUTION: A DNA fragment containing a genetic region of a late protein of a JC virus is prepared and an agnoprotein region arranged between the 277th and the 492nd positions counted from the origin of replication to the downstream side in the genetic region of the late protein of the JC virus is then deleted therefrom to prepare a DNA fragment capable of producing a virus particle. Further, the prepared DNA fragment capable of producing the virus particle is integrated into an expression vector and the resultant expression vector is transduced into a host cell to produce the JC virus particle. The DNA fragment capable of producing the virus particle is preferably a VP231 region DNA fragment containing a VP231 region in the genetic region of the late protein. Each region of the VP23, VP1 and VP3 arranged in the VP231 region is capable of producing a coat protein of the JC virus.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to, for example, a method for producing JC virus particles that cause progressive multifocal leukoencephalopathy, and a method for producing a JC virus outer protein.

[0002]

2. Description of the Related Art A virus has its gene in a particle formed by a protein and expresses its own gene by utilizing the organelle of a host cell. Therefore, the virus is used as a carrier for gene transfer. In the clinical field, viral vectors have been developed for gene therapy, and their safety and usefulness are being investigated.

While such virus research is being conducted, J
The C virus is particularly required to be researched and developed. That is, the JC virus rarely causes progressive multifocal leukoencephalopathy (Progr
essive Multifocal Leukoencephalopathy; PML
That. )cause. As can be seen from the fact that about 70% of the JC virus has its antibody,
It is a virus that has spread all over the world. The JC virus shows high neurotropicity, specifically infects oligodendroglia of the human brain, and is localized in the nucleus. Also, JC
The virus belongs to the genus Polyomavirus and is considered to release the gene by enucleating in the nucleus of the infected cell like SV40 and the like.

[0004]

[Problems to be Solved] However, it is very difficult to culture the JC virus for use in the above research, and the cell lines in which it can be cultured have been limited (Padgett et al.
Lancet, I, 1267-1260, 1971). Also, the study was delayed because high titer virus could not be obtained in a short time. Moreover, there are many unclear points about the pathogenic mechanism of PML. In addition, the pathogenic mechanism of a number of hereditary neurological diseases at the gene level has been clarified, but no attempt has been made to treat it.

In view of such conventional problems, the present invention provides a JC
An object of the present invention is to provide a method for producing a JC virus particle or a JC virus envelope protein, which can produce a virus particle or a JC virus envelope protein in a high yield in a short time.

[0006]

The invention according to claim 1 is a DN containing the late protein gene region of the JC virus shown in FIGS. 1 to 4.
A fragment was prepared, and then, from the above DNA fragment, the agnoprot sequenced between the 277th to the 492nd positions in the late protein gene region, counted from the replication origin to the downstream side.
Ein region is deleted to prepare a virus particle-producing DNA fragment, then the above virus particle-producing DNA fragment is incorporated into an expression vector, and then the expression vector is introduced into a host cell to produce JC virus particles. A method for producing JC virus particles, which comprises:

What is most noticeable in the present invention is to delete the agnoprotein region of the above-mentioned late protein gene region. The above agnoprotei
A virus particle-producing DNA fragment lacking the n region is incorporated into an expression vector, and then introduced into a host cell, and the host cell is propagated to obtain a JC virus particle.
It can be produced in high yield in a short time.

The JC virus particles obtained by the above-mentioned production method are an assembly of constituent proteins of JC virus and have the property of infecting COS7 cells in particular. Therefore, J
The C virus particle is a useful research material in analyzing the JC virus.

Next, a DN containing the above-mentioned late protein gene region
As described in claim 2, in the A fragment, the nucleotide sequence existing between the 883rd to the 2533rd number counted from the replication origin to the downstream side may consist of the nucleotide sequences shown in FIGS. 5 to 7. preferable. As a result, JC virus particles can be produced with a higher yield. It should be noted that the numbers shown in FIGS. 1 to 7 mean the number of bases arranged downstream from the multiple initiation points of the JC virus.

Next, the above virus particle-producing DNA fragment is agglated from the JC virus late protein gene region DNA.
It is a DNA fragment in which the noprotein region has been deleted. This viral particle-producing DNA fragment is, as described in claim 3, counted from the replication origin to the downstream side at the 526th position.
It is preferable to include a VP231 region arranged between # 2 and # 2533. This can further maximize the yield of JC virus particles.

The above-mentioned late protein gene region DNA is shown in FIG.
As shown in FIG. 4, it has an agnoprotein region gene and three viral envelope protein genes. agn
The oprotein region gene is the DNA of JC virus.
It is located at the 277th to 492nd positions counting from the replication origin of the DNA, and its base sequence is as shown in FIGS. 1 and 8.

The outer protein genes of the three viruses have three regions, VP1, VP23 and VP3. These three regions are included in the VP231 region, and each has a function of producing a protein constituting a JC virus outer coat protein. The VP1 region is located between the 1469th and the 2533rd positions counted from the replication origin to the downstream side, and its nucleotide sequence is as shown in FIGS. 3, 4, and 9 to 13. The VP23 region is located at the 526th to 1560th positions counting downstream from the replication origin, and its nucleotide sequence is shown in FIG.
As shown in FIG.

The VP3 region is located from the replication origin to the downstream side from the 883rd to the 1560th position, and its nucleotide sequence is as shown in FIGS. 2, 3 and 14 to 16. VP
The 3 region overlaps with a part of the base sequence of the VP23 region. 8 to 16, the upper row shows the base sequence and the lower row shows the amino acid sequence corresponding to the upper base sequence.

Examples of the JC virus include JC virus.
The virus Tokyo-1 strain is used. This JC virus Tokyo-1 strain is the first cloned JC virus gene in Japan after being isolated and purified by the inventors from an autopsy brain of a PML patient. The JC virus can be isolated and purified by, for example, the method of Padgett et al. (La
ncet, I, 1267-1260, 1971).

As a method for amplifying the above-mentioned late protein gene region DNA of JC virus Tokyo-1 strain, there is a method using a cloning vector. That is, the above-mentioned late protein gene region DNA is inserted and ligated into a cloning vector. As a cloning vector, a vector plasmid using Escherichia coli as a host is preferable because it is easy to handle. As the vector plasmid, for example, pUC1
Those derived from 3, pUC18, pUC19 or pBR322 can be used.

When pUC19 is used as the vector plasmid, it is preferable to select a cloning vector into which the above-mentioned late protein gene region DNA has been inserted and bound by the following method. That is, the above-mentioned late protein gene region DN
PUC19 having A-inserted thereinto was introduced into Escherichia coli DH5a at a temperature of 42 ° C., and isopropylthio-D was added to a medium containing tryptophan, yeast extract, NaCl, ampicillin and agar (hereinafter referred to as LB-Agar agar medium). -Galactoside (hereinafter referred to as IPTG)
And 5-bromo-4-chloro-3-indolyl-b-D-
Add galactoside (hereinafter referred to as X-gal),
Incubate.

PUC19 has an ampicillin resistance gene and a b-D-galactosidase (hereinafter referred to as lacZ) gene. Since the foreign gene JC virus DNA fragment is inserted into the lacZ gene region of pUC19, it is lacZ in recombinant pUC19.
Is not expressed and does not cause a color reaction with the substrate X-gal. Therefore, recombinant pUC19, that is, an Escherichia coli transformant having a cloning vector incorporating the late protein gene region DNA can be easily selected.

The late protein gene region DNA encoding the JC virus gene incorporated into the cloning vector is incorporated into an expression vector. As an expression vector, for example, as shown in FIG. 20, Takebe et al.
be st al., Molecular and Cellular Biology, vol. 8,
466-472, 1988).

The expression vector is introduced into a host cell.
The host cell is not particularly limited, but monkey kidney cell-derived COS7 cells are most preferably used. The reason is that since the JC virus is derived from human brain, it is suitable for expression in the same primate-derived cells as human.

As a method for introducing the expression vector into a host cell, there are, for example, a Hanahan method, a lipofectin method, a transfection by the calcium phosphate method, microinjection, electroporation and the like. By the above method, JC
Viral particles can be easily expressed in high yield in a short time.

Next, as a method for producing the JC virus outer coat protein, for example, as shown in claim 4, as shown in FIGS.
DNA containing the late protein gene region of JC virus shown in
A fragment is prepared and then, in the late protein gene region,
VP23 region DNA containing the VP23 region arranged between the 526th to 1560th positions counting downstream from the replication origin
A fragment and a VP1 region DN including VP1 arranged between the 1469th and 2533rd positions counted downstream from the replication origin
A fragment and a VP3 region DNA fragment containing the VP3 region located between the 883rd and 1560th positions counted downstream from the origin of replication were prepared, and then the VP23 region DN was prepared.
A fragment, the VP1 region DNA fragment, and the VP3 region D
Each of the NA fragment and the VP23 region DNA fragment was inserted into an expression vector, and then the VP1 region DNA fragment was inserted into the expression vector, and the VP3 region DNA fragment was inserted into the expression vector. There is a method for producing a JC virus envelope protein, which comprises introducing it into cells to produce the JC virus envelope protein.

VP23 area, VP1 area, and VP
Region 3 is a gene that produces the JC virus coat protein. Therefore, a VP containing each of these three types of areas
23 region DNA fragment, VP1 region DNA fragment, and VP
By preparing a three-region DNA fragment, individually incorporating these DNA fragments into an expression vector, introducing into a host cell, and proliferating, the JC virus outer peripheral protein
It can be efficiently produced with a high yield.

The JC virus outer coat protein obtained by the above-mentioned production method becomes a useful research material for analyzing JC virus, such as an antigen for producing an anti-JC virus antibody necessary for detection.

VP23 area, VP1 area, and VP
It is preferable that each DNA fragment in the three regions is inserted into a cloning vector, amplified by introducing the cloning vector into a cell line and proliferating, and then incorporated into the expression vector. As a result, a large amount of JC virus envelope protein can be produced more efficiently.
Each of the above DNA fragments can also be amplified by subcloning.

The DNA fragments of the VP23 region, VP1 region, and VP3 region can be prepared, for example, by cutting the above-mentioned late protein gene region DNA with a restriction enzyme. When there is no suitable restriction enzyme recognition site in the late protein gene region DNA of JC virus,
A gene product amplified by PCR using a primer having a restriction enzyme recognition sequence may be used as an insert fragment.

Method for producing the above JC virus particles and JC
The method for producing the viral envelope protein is not only useful for the diagnosis of PML, which is a late viral infection, and for the basic virological research of other JC viruses, but it is also useful for Krebbe.
Disease, metachromatic leukodystrophy, Pelizaeus-Merz
It also provides a genetically engineered material used in gene therapy for neurological diseases caused by inborn errors of metabolism such as Basher disease. Hereinafter, the present invention will be described in more detail with reference to embodiments, but the present invention is not limited to the following embodiments.

[0027]

BEST MODE FOR CARRYING OUT THE INVENTION JC according to an embodiment of the present invention
The method for producing virus particles and JC virus outer coat protein will be described with reference to FIGS. The outline of the production method of this example will be described. As shown in FIGS. 17 and 18, first, the late protein gene region DNA of the JC virus is obtained, and then a part of the late protein gene region DNA is cleaved to carry out the agnoprotein. , VP231, VP1, V
DNA fragments of various regions of P23 and VP3 are obtained, appropriately combined and incorporated into an expression vector, and then introduced into a host cell.

Hereinafter, regarding the method for producing the above-mentioned JC virus particles, S81 to S88 of FIG. 17 and S91 to S91 of FIG.
A detailed description will be given while showing each step of S98.

(Acquisition of Late Protein Gene Region DNA) First, as shown in FIG. 17, JC virus Tokyo-1.
The strain was isolated from the autopsy brain of a PML patient (S81). Then, the separated JC virus was passaged using primary human fetal brain cells (S82). Then, DNA was extracted from the JC virus and purified (S83).

Next, the JC virus DNA was cleaved with the restriction enzyme EcoRI (S84). Next, the cutting D
The NA fragment was transformed into EcoR of the plasmid vector pUC13.
It was inserted and bound to the I site (S85).

Then, this plasmid vector pUC1
The late protein gene region DNA in 3 was amplified by PCR using known primers (S86). The known primer encodes at least part of the late protein gene. Next, the amplified late protein gene region DNA is
Subcloning was carried out using the plasmid vector pUC19 (S87). Next, the late protein gene region DNA is transformed by the transformation method (Hanahan method; Molecular).
cloning 2nd ed. , Vol. 1,198
It was introduced into Escherichia coli DH5a according to 9), and this E. coli was grown (S88).

Then, in S91 shown in FIG. 18, the proliferated Escherichia coli was destroyed to obtain a plasmid vector pUC13 incorporating the late protein gene region DNA.
Then, the plasmid vector pUC13 was cleaved with the restriction enzyme EcoRI to obtain the late protein gene region DNA.
I got

(Cleavage of Late Protein Gene Region DNA) Next, in S92 shown in FIG. 18, the late protein gene region DNA was digested with the restriction enzyme AccI. As a result, DNA fragments of 216 bases (bp), 1065 bases, 1035 bases, and 678 bases were obtained. These DNA fragments were respectively labeled as agnoprotein region DNA fragment, VP1 region DNA fragment, and VP23 region D.
They were designated as NA fragment and VP3 region DNA fragment.

Next, the position of each of the above DNA fragments was determined and the nucleotide sequence was investigated, and the results are shown in FIGS. From these figures, the agnoprotein region DN
The A fragment is located downstream (3 ') from the replication origin of JC virus.
It is composed of 216 bases arranged between the 277th and 492nd positions, counting from the terminal side).

The VP1 region DNA fragment is the above EcoRI.
It is composed of 1065 bases arranged from the 1469th to the 2533rd positions counted from the cleavage site to the downstream side. The VP23 region DNA fragment is counted 1035 downstream from the EcoRI cleavage site and arranged between the 526th to 1560th positions.
It consists of individual bases.

The VP3 region DNA fragment is the above EcoRI.
It is composed of 678 bases arranged from the 883rd position to the 1560th position downstream from the cleavage site. The positional relationship of these various region DNA fragments is shown in FIG. In addition,
In Fig. 4, "........" indicates an unclear base sequence.

(Amplification of agnoprotein domain DNA fragment) The agnoprotein domain DNA fragment was amplified by PCR if necessary.

(Amplification of VP1 region DNA fragment)
The P1 region DNA fragment was amplified by the PCR method using a primer carrying a BamHI recognition sequence at its 5'end and a KpnI recognition sequence at its 3'end. Then,
The amplified VP1 region DNA fragment was used as a blunt end and inserted and ligated into the HincIII recognition site of the plasmid vector pUC19, and it was confirmed that the base sequence of the inserted fragment was not different from the base sequence of the template. PJCTVP1-
It is called pUC.

Then, pJCTVP1-pUC was introduced into Escherichia coli DH5a by the transformation method and propagated. Then, pJCTVP1-pUC was taken out from Escherichia coli DH5a. Then, using the restriction enzyme, pJCTVP1
-A VP1 region DNA fragment was excised from pUC.

(Amplification of VP23 region DNA fragment)
The VP23 region DNA fragment was transferred to AccI / E of pUC19.
It was inserted and ligated into the coRI cleavage site. This is pJCTVP
It is called 23-pUC. Then, pJCTVP23-pU
C was introduced into Escherichia coli DH5a at 42 ° C. by the transformation method (Hanahan method). Then, IPTG and X-gal were added to the LB-Agar agar medium and cultured. Then, pJCTVP23-pUC was taken out from Escherichia coli DH5a and purified. Then, pJCT using restriction enzyme
A VP23 region DNA fragment was excised from VP23-pUC.

(Amplification of VP3 region DNA fragment)
The VP3 region DNA fragment was transferred to AccI / Ec of pUC19.
It was inserted and linked to the oRI cleavage site. This is pJCTVP1
-It is called pUC. Then, pJCTVP3-pUC is changed to 4
E. coli D by transformation (Hanahan method) at 2 ° C
It was introduced into H5a. Then, IPTG and X-gal were added to the LB-Agar agar medium and cultured. Then, take out pJCTVP3-pUC from E. coli DH5a,
Purified. Then, using the restriction enzyme, pJCTVP3-
A VP3 region DNA fragment was excised from pUC.

(Preparation of VP231 Region DNA Fragment) Next, in S93 shown in FIG.
The VP1 region DNA fragment was ligated to the 3'end side (downstream side) of the fragment. As a result, as shown in FIG.
A 31-region DNA fragment was obtained.

(Incorporation into Expression Vector) Next, in S95 shown in FIG. 18, the above various DNA fragments were recloned downstream of the promoter of the animal cell expression vector. As the animal cell expression vector, pcDL-SRa296 was used as shown in FIG.

(Introduction into Host Cell) Next, in S96 shown in FIG. 18, the expression vector incorporating the above various DNA fragments was introduced into monkey kidney cell-derived COS7 cells by the lipofectin method. That is, the expression vector and lipofectamine (trade name, manufactured by Gibco BRL)
The complex was formed and introduced into COS7 cells in a serum-free culture medium (trade name: Opti-MEM, manufactured by GibcoBRL) for about 4 hours. Then, the above culture solution was added to a culture solution containing serum (Eagles' minimum essential containing 10% calf serum).
The medium was replaced with medium (MEM), manufactured by Gibco BRL) and cultured at 37 ° C. in the presence of 5% CO ₂ for 72 hours. As a result, COS7 cells were made to express the proteins derived from the above various DNA fragments.

Next, the obtained various proteins were investigated by the fluorescent antibody method and the immunoprecipitation method. As a result, VP
The proteins derived from the 1, VP23 and VP3 region DNA fragments were all JC virus outer coat proteins (S97). VP
The JC virus envelope proteins obtained by the 1-region DNA fragment, the VP23 region DNA fragment, and the VP3 region DNA fragment differ from each other in that they constitute a specific portion of the envelope.

The protein derived from the VP231 region DNA fragment is
It was a JC virus particle (S98). JC virus particles have the property that they are safe for use in research because they do not have pathogenicity and that they are highly likely to be used as gene transfer vectors in gene therapy. On the other hand, JC virus particles were obtained from the late protein gene region DNA. However, the production amount is
It was significantly less than the amount produced from the NA fragment. The reason is considered to be that agnoprotein inhibits the formation of JC virus particles.

Next, the JC virus particles derived from the VP231 region DNA fragment were observed by an electron microscope, and the photographs are shown in FIGS. 21 and 22. From these photographs, JC virus particles (black particles scattered) are COS.
It can be seen that it is produced in 7 cells.

[0048]

EFFECTS OF THE INVENTION According to the present invention, there are provided a method for producing JC virus particles and a method for producing JC virus envelope proteins, which can produce JC virus particles or JC virus envelope proteins in high yield in a short time. can do.

[Brief description of drawings]

FIG. 1 is an explanatory view (1) showing the position of the late protein gene region DNA of JC virus and its nucleotide sequence in the present invention.

FIG. 2 is an explanatory view (2) showing the position of the late protein gene region DNA of the JC virus and its nucleotide sequence following FIG.

FIG. 3 is an explanatory view (3) showing the position of the late protein gene region DNA of the JC virus and its nucleotide sequence following FIG. 2.

FIG. 4 is an explanatory view (4) showing the position of the late protein gene region DNA of the JC virus and its nucleotide sequence following FIG.

FIG. 5 is the 883rd to 2533rd counted from the replication origin of JC virus DNA to the downstream side in the present invention.
Explanatory drawing (1) which shows the base sequence until the 1st.

FIG. 6 is an explanatory view (2) following FIG. 5, showing the nucleotide sequences from the 883rd position to the 2533rd position counted from the replication origin of JC virus DNA to the downstream side.

FIG. 7 is an explanatory view (3) following FIG. 6, showing nucleotide sequences from the 883rd position to the 2533rd position counted downstream from the replication origin of JC virus DNA.

FIG. 8 is an explanatory view showing the nucleotide sequence and amino acid sequence of an agnoprotein region DNA fragment in the present invention.

FIG. 9 is an explanatory diagram (1) showing the nucleotide sequence and amino acid sequence of the VP1 region DNA fragment in the present invention.

FIG. 10 is an explanatory diagram (2) showing the nucleotide sequence and amino acid sequence of the VP1 region DNA fragment following FIG. 9.

FIG. 11 is an explanatory view (3) showing the nucleotide sequence and amino acid sequence of the VP1 region DNA fragment following FIG. 10.

FIG. 12 is an explanatory diagram (4) showing the nucleotide sequence and amino acid sequence of the VP1 region DNA fragment following FIG. 11.

FIG. 13 is an explanatory view (5) showing the nucleotide sequence and amino acid sequence of the VP1 region DNA fragment following FIG. 12.

FIG. 14 is an explanatory view (1) showing the nucleotide sequence and amino acid sequence of the VP3 region DNA fragment in the present invention.

FIG. 15 is an explanatory view (2) showing the nucleotide sequence and amino acid sequence of the VP3 region DNA fragment following FIG.

FIG. 16 is an explanatory view (3) showing the base sequence and amino acid sequence of the VP3 region DNA fragment following FIG. 15.

FIG. 17 is an explanatory view showing a method for producing JC virus particles and JC virus outer coat protein in the embodiment.

FIG. 18 is an explanatory view showing a method for producing JC virus particles and JC virus outer coat protein, following FIG. 17.

FIG. 19 is an explanatory diagram of various DNA fragments incorporated into an expression vector in the embodiment.

FIG. 20 shows the expression vector (pcD in the embodiment).
Explanatory drawing of L-SR (alpha) 296.

FIG. 21 is an electron micrograph of JC virus particles produced in COS7 cells in an example embodiment (magnification: 1000).
(0x) The drawing substitute photograph shown by.

FIG. 22 is an electron micrograph of JC virus particles produced in COS7 cells in an example embodiment (magnification: 5000).
(0x) The drawing substitute photograph shown by.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Reference number within the agency FI technical display part // (C12P 21/02 C12R 1:91) (72) Inventor Yukiko Shishido Kita-ku, Sapporo-shi, Hokkaido Article 15 West 7-chome, Hokkaido University, Second Department of Pathology (72) Inventor Naoto Kagiyama 2-chome, Asahi-cho, Kariya city, Aichi Aisin Seiki Co., Ltd.

Claims (4)

[Claims] 1. A DNA fragment containing the late protein gene region of JC virus as shown in the following "FIG. 1" to "FIG. 4" is prepared, and then replication is initiated from the above DNA fragment in the late protein gene region. The viral particle-producing DNA fragment is prepared by deleting the agnoprotein region arranged between the 277th and 492nd positions counting from the point to the downstream side,
Next, a method for producing JC virus particles, which comprises incorporating the above virus particle-producing DNA fragment into an expression vector, and then introducing the expression vector into a host cell to produce JC virus particles. [FIG. 1] [FIG. 2] [FIG. 3] 2. The DNA fragment containing the late protein gene region according to claim 1, wherein the nucleotide sequence existing between the 883rd and the 2533rd positions counted downstream from the replication origin is:
A method for producing a JC virus particle, which comprises the base sequence shown in the following "Fig. 5" to "Fig. 7". 5] FIG. 6] 3. The viral particle producing DNA fragment according to claim 1 or 2, wherein the viral particle-producing DNA fragment includes a VP231 region arranged between the 526th and the 2533rd counting from the replication origin to the downstream side. Method for producing JC virus particles. 4. A DNA fragment containing the late protein gene region of JC virus as shown in the following "FIG. 1" to "FIG. 4" is prepared, and then, in the late protein gene region, from the replication origin to the downstream side. A VP23 region DNA fragment containing the VP23 region arranged between the 526th and 1560th counts and a VP1 region DNA fragment containing the VP1 arranged between the 1469th and 2533rd counts downstream from the replication origin When,
A VP3 region DNA fragment containing the VP3 region arranged between the 883rd to 1560th positions counted downstream from the replication origin is prepared, and then the VP23 region DNA fragment, the VP1 region DNA fragment, and the VP3 region are prepared. The DNA fragment and the VP are respectively incorporated into an expression vector, and then the VP
An expression vector incorporating a 23-region DNA fragment, an expression vector incorporating the VP1 region DNA fragment, and an expression vector incorporating the VP3 region DNA fragment are introduced into a host cell to produce a JC virus envelope protein. A method for producing a JC virus outer shell protein, comprising: [FIG. 1] [FIG. 2] [FIG. 3]