Patent Application
20160215271
The present invention relates to a method for producing culture containing virus-like particles by using the baculovirus-insect cell expression system.
Norovirus is a virus that causes acute gastroenteritis such as vomiting and diarrhoea. Norovirus causes food poisoning from eating oysters and the like, and orally infects humans via feces or vomited material.
It is known that particles called as virus-like particles (VLPs) that resemble viral particles are formed when a baculovirus vector incorporated with the structural protein coding region of norovirus genome is expressed in insect cells. Though VLPs resemble viral particles in appearance, they do not contain virus genome and is not infectious.
Norovirus vaccine has been recently developed using VLPs as antigens, and patent applications concerning it have been filed (Japanese Translation of PCT International Application Publication Nos. JP-T-2010-505766 and JP-T-2011-530295). In order to produce such norovirus vaccine, it is important that VLPs can be efficiently produced by using the baculovirus-insect cell expression system.
Conventionally, when protein was produced by using the baculovirus-insect cell expression system, the cells were collected after about 3 days from the start of the cultivation, disrupted, and extracted to purify the protein from the cell extract. For example, Patent Literature 1 discloses a method for producing hemagglutinin of influenza virus. In this method, the cells were collected after 72 hours from the start of the cultivation, and the cell pellet was disrupted with homogenizer in presence of a surfactant (Example 5).
Cell contents are released to the culture solution by death of the cells when they are cultivated for a longer time, so the process for disrupting the cells can be omitted. However, protease released from the dead cells may decompose a target protein, so cultivation for such a long time has not been performed.
Patent Literature 1: WO96/37624
When VLPs are produced by the above conventional method, the process for disrupting the cells must be performed to collect the VLPs. However, a surfactant that is used in the disrupting process may have bad effect on the structure of the VLPs. Further, the disrupting process itself is complicated.
The object of the present invention is to solve the problems of the conventional production method for VLPs and to provide a more efficient means for producing VLPs.
As the result of intensive studies for solving the above-described problems, the present inventors have found that even in the presence of proteases released from the dead cells VLPs are virtually not affected by the proteases. Thus, the cells can be cultivated for a long time and VLPs can be collected without the disrupting process.
Further, the present inventors have found that the purity of VLPs in the culture supernatant is high because contaminant proteins are decomposed by the proteases released from dead cells when the cells are cultivated for a long time.
The present invention has been completed based on the above findings.
Specifically, the present invention provides the following (1) to (5).
(1) A production method for culture containing virus-like particles, comprising: transforming insect cells with baculovirus vector containing a viral nucleic acid sequence; and cultivating the insect cells, wherein the culture is obtained after continuing cultivating the insect cells until the viability of the insect cells reaches 10% or less.
(2) The production method according to (1), wherein the cultivation period of the insect cells is 5 days or more.
(3) The production method according to (1) or (2), wherein the virus is a virus belonging to the Family Caliciviridae.
(4) The production method according to (1) or (2), the virus is norovirus.
(5) The production method according to (4), wherein the viral nucleic acid sequence is a nucleic acid sequence encoding a norovirus structural protein VP1.
This specification includes the contents as disclosed in the specification and/or drawings of Japanese Patent Application (Patent Application No. 2013-146242), which is a priority document of the present application.
In the production method of the present invention, VLPs can be obtained without the process for disrupting the cells, so effect of a surfactant that is used in the disrupting process can be excluded. Further, high-purity VLPs can be obtained because substances such as proteins derived from the host cell are decomposed during the process of cultivation.
Hereinafter, the present invention will be described in detail.
The production method for culture containing virus-like particles of the present invention comprises: transforming insect cells with baculovirus vector containing a viral nucleic acid sequence; and cultivating the insect cells, wherein the culture is obtained after continuing cultivating the insect cells until the viability of the insect cells reaches 10% or less.
The type of a virus to be used is not particularly limited. For example, such viruses as non-enveloped viruses that have the shape of a regular icosahedron can be used. Examples of such viruses include circovirus, parvovirus, polyomavirus, papillomavirus, adenovirus, picornavirus, astrovirus, birnavirus, reovirus, hepevirus, nodavirus, anellovirus, calicivirus, and the like. Calicivirus (viruses belonging to the family Caliciviridae) is preferably used among these. Norovirus is the most preferable.
Norovirus in the present invention means a virus that belongs to the family Caliciviridae and the genus Norovirus. Norwalk virus is the only species of the genus Norovirus at present. However, if a new virus belonging to this genus is found in the future, it is included in “Norovirus” in the present invention.
A virus-like particle in the present invention means a hollow particle that has only viral outer shell without viral genome inside the shell.
The cultivation may continue until the viability of the insect cells reaches 10% or less, but the cultivation may continue until the lower viability, for example, 9% or less, 7% or less, 5% or less, 3% or less, or 1% or less. The viability can be measured by using a method such as trypan blue staining method, AlamarBlue staining method, or MTT method. In addition, devices that automatically analyze whether a cell is alive or dead are commercially available, so the viability may be measured by using such devices.
Although specific cultivation period may be determined in accordance with the cell viability, the viability usually reaches 10% or less at about 5 days after the start of cultivation (the infection of baculovirus), so cultivation for 5 days or more is preferable. However, cultivation period may be 6 days or more, 7 days or more, or 8 days or more so that viability may become lower. The upper limit of the cultivation period is not particularly limited, but the cultivation period is preferably within 10 days.
In a conventional method, after cultivation, the cells collected from the culture were disrupted and extracted to obtain the target protein (VLPs) from the cell extract. However, in the present invention, VLPs can be obtained without the processes for disrupting and extracting the cells because the VLPs are released to the culture.
Except for the cultivation period and the unnecessity of the disrupting process described above, the production method of the present invention can be performed in the same manner as the known production method for a specific protein by using the baculovirus-insect cell expression system (for example, the method described in International Publication WO96/37624).
The nucleic acid sequence of the virus to be used is not particularly limited as long as it can form a VLP. For example, if the virus is norovirus, a nucleic acid sequence encoding VP1 can be used. Further, if the virus is norovirus, not only a nucleic acid sequence encoding VP1 but also a nucleic acid sequence encoding VP2 may be used.
The nucleic acid sequences of the virus to be used are published in such a database as GenBank. For example, registered numbers of the sequences of norovirus include M87661 of Norwalk virus, L07418 of Southampton virus, U04469 of Desert Shield virus, AB042808 of Chiba virus, U07611 of Hawaii virus, U70059 of Snow Mountain virus, AY032605 of Maryland virus, AB031013 of Seto virus, AF145896 of Camberwell, X86557 of Lordsdale virus, AJ004864 of Grimsby virus, U22498 of Mexico virus, AY502023 of Houston virus, and AY652979 of Parris Island.
The baculovirus vector which can be used includes pFastBac vector (Invitrogen) and BD BaculoGold (BD Biosciences).
The insect cell which can be used includes Sf9 cell and HighFive cell.
The cultivation of transformed insect cells can be performed according to the cultivation method generally used in the cultivation of insect cells. The medium which can be used include PSFM medium, SF900II medium, and SF900III medium. The cultivation temperature is preferably 20 to 30° C., and more preferably 26 to 28° C.
The present invention will be explained more specifically with reference to the following Examples. However, the present invention is not limited thereto.
(1) Experimental Methods
1 Preparation of Recombinant Baculovirus
A cDNA encoding VP1, whose amino acid sequence is shown in SEQ ID NO.: 1, of a norovirus strain classified as GII-4, which was collected from an inpatient in University of Tampere, was incorporated into a transfer plasmid (pFastBac, Invitrogen). Then, the transfer plasmid incorporated with the cDNA was introduced into DH10Bac (Invitrogen), which is an Escherichia coli having a baculovirus genome, and the cDNA encoding the norovirus VP1 was incorporated into the baculovirus genome DNA by homologous recombination. The baculovirus genome DNA was extracted from the Escherichia coli, purified, and introduced into insect cells (expresSF+cells, Protein Sciences). The insect cells were cultivated, and from the culture supernatant, recombinant baculovirus was obtained.
2 Cultivation to Express the GII-4 VP1
The recombinant baculovirus (MOI=1) was added to the insect cells (expresSF+cells) (1×106/mL). The cells (expresSF+cells) were cultivated in PSFM medium at 27° C. After the infection of baculovirus, 1 ml of the culture solution was collected everyday and centrifuged at 5000×g for 5 minutes to separate the culture supernatant from the cell pellet.
3 Electrophoresis
The culture supernatant was mixed with 250 μL of 5×DB (300 mmol/L Tris-HCl pH 6.8, 50% glycerol, 10% SDS, 0.5% Bromo Phenol Blue, 500 mmol/L DTT). The cell pellet was dissolved in 1250 μL of 1×DB, which is prepared by diluting 5× DB to 5 times with distilled water. After both were heated at 95° C. for 5 minutes, 10 μL of them were applied on each lane and electrophoresed at 200 V for 35 minutes.
The electrophoresed gel was soaked in fixing solution (25% methanol, 10% acetic acid, 10% trichloroacetic acid (TCA)) and shaken for 5 minutes. Then, it was stained by soaking in CBB stain solution (0.1% CBB, 7.7 mmol/L ethanol, 1.75 mmol/L acetic acid) and shaking for 1 hour, and destained with destaining solution (10% acetic acid) overnight. The gel image was obtained by using a scanner. SeeBlue prestained standard (Invitrogen) was used as a molecular weight marker.
(2) Experimental Results
Results of electrophoresis are shown in
As shown in the figure, while the amount of GII-4 VP1 in the culture supernatant, which was detected as a band of about 60 k Da, increased with the cultivation days, the amount of GII-4 VP1 in the cell pellet decreased. This suggests that the VLPs released to the culture supernatant increased by the increase of the dead cells with the cultivation days. Thus, it is believed that an enough amount of VLPs can be obtained without the process for disrupting the cells if the cultivation continues for about five days.
Further, the culture supernatant cultivated for 6 days contains a great amount of the GII-4 VP1, so it is believed that the proteases released from dead cells hardly act on the GII-4 VP1.
Further, the signal intensity ratio of GII-4 VP1, which was detected as a band of about 60 k Da, to the sum of the signal intensity of all bands in each lane was measured by using a densitometer. As a result, while the ratio of the cell pellet cultivated for 3 days was 28%, the ratio of the culture supernatant cultivated for 6 days was 62%. Thus, the culture supernatant cultivated for 6 days contained less impurity than the cell pellet cultivated for 3 days.
The cells (expresSF+cells) were cultivated by using a bioreactor. PSFM medium was used as a medium, and the cultivation temperature was 27° C. The culture solution was collected at just before the infection of the baculovirus expressing GII-4 VP1 and at 1-6 days after the infection. The viable cell number, the total cell number, the cell diameter, and the viability were measured by an automated cell viability analyzer (Vi-CELL XR, Beckman Coulter).
The temporal change of viable cell number and total cell number was shown in
As shown in these figures, the viability of cells is rapidly reduced after 3 days from the start of the cultivation.
All the publications, patents, and patent applications cited in the present specification are incorporated into the present specification by reference in their entirety.
Since the present invention is useful as production of vaccines against viruses such as norovirus, it can be utilized in an industrial field such as pharmaceutical industry.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2013-146242 | Jul 2013 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2014/063424 | 5/21/2014 | WO | 00 |
