JP2007215466A - Method for preparing recombinant virus originated from reoviridae virus, and dna construct for preparing recombinant rotavirus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a reverse genetics system for viruses belonging to Reoviridae. <P>SOLUTION: This method for producing the recombinant virus comprises the following steps (1) to (4). (1) A step for transfecting a host cell with a DNA construct containing a cDNA encoding the coat protein gene segment of a Reoviridae virus (the first virus). (2) A step for infecting the host cell with the same kind of a virus (the second virus) as that of the first virus as a helper virus. (3) A step for bringing a virus produced from the host cell into contact with an antibody for specifically recognizing and neutralizing the coat protein of the second virus, corresponding to the coat protein encoded in the coat protein gene segment of the first virus. (4) A step for recovering the recombinant virus holding the coat protein gene segment originated from the cDNA. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a reverse genetics system for reoviridae viruses and uses thereof.

Vomiting diarrhea in winter infants due to rotavirus occurs in nearly 100% of infants by age 5 years. Due to its high infectivity, the improvement of hygiene conditions cannot control this virus infection. Globally, approximately 500,000 deaths are reported every year mainly in developing countries. However, because there are many hospitalized cases in developing countries, it is important to prevent infection with this virus from a medical economic viewpoint. It has become a challenge. In addition to vomiting diarrhea, it has been suggested that this virus is directly or indirectly related to encephalitis / encephalopathy, type 1 diabetes, biliary atresia, myositis, sudden infant death, and the like. In adults, immunodeficiency and rotavirus infection in organ transplant patients cause exhaustion due to prolonged diarrhea. In fact, fatal cases in adults have been reported.
RotaShield was developed in 1998 as the world's first vaccine against human rotavirus, but this vaccine was withdrawn from the market in 1998 due to severe side effects (intussusception). Currently, several live vaccines are under development (clinical trial stage), but all of them are focused on preventing the progression of disease, and the aim is to develop vaccines effective in preventing infection. Is not standing. The antigen system of human rotavirus is diverse, and there are about 10 serotypes for both G and P types. Developing a broad and effective vaccine for these many serotypes is extremely difficult.
In many viruses, including poliovirus, reverse genetics systems have been developed that change the character of the virus by artificially modifying the genome. Reverse genetics systems allow artificial manipulation of the viral genome at the cDNA level through site-specific mutation, deletion, insertion, reorganization, and the like. To date, reverse genetics systems in double-stranded RNA viruses (including Reoviridae, Virnaviridae, and Cystviridae) have been developed only for a few segmented double-stranded RNA viruses ( A birnavirus composed of two segmented dsRNAs (Non-patent Documents 1 and 2) and a cystvirus composed of three segmented dsRNAs (Non-patent Document 3), while a reoviridae having 10 to 12 segmented genomes It is extremely difficult to develop a reverse genetics system except for reovirus, and the reovirus reverse genetics system developed by Roner and Joklik (Non-Patent Documents 4-6) is extremely complex. There are no reports of success in this method in other laboratories, and there are reports of applications to other viruses belonging to the family Reoviridae. In addition, an influenza virus system developed by Dr. Palese et al. (Patent Document 1, Non-Patent Documents 7 and 8) is known as a virus reverse genetics system.

Japanese Patent No. 3293620 Mundt, E. & Vakharia, V. N. (1996) Proc. Natl. Acad. Sci. USA 93, 11131-11136. Yao, T. & Vakharia, V. N. (1996) J. Virol. 72, 8913-8920. Olkkonen, V. M., Gottlieb, P., Strassman, J., Qiao, X., Bamford, D. H. & Mindich, L. (1990) Proc. Natl. Acad. Sci. USA 87, 9173-9177 Roner, M. R., Sutphin, L. A. & Joklik, W. K. (1990) Virology 179, 845-852. Roner, M. R., Nepluev, I., Sherry, B. & Joklik, W. K. (1997) Proc. Natl. Acad. Sci. USA 94, 6826-6830. Roner, M. R. & Joklik, W. K. (2001) Proc. Natl. Acad. Sci. USA 98, 8036-8041. Enami, M., Luytjes, W., Krystal, M. & Palese, P. (1990) Proc. Natl. Acad. Sci. USA 87, 3802-3805. Enami, M. & Palese, P. (1991) Virology 185, 291-298.

  An object of the present invention is to provide a reverse genetics system for viruses belonging to the family Reoviridae.

Among viruses belonging to the family Reoviridae, serious diseases are caused in humans. The only common virus is rotavirus, and the development of a reverse genetics system for rotavirus is considered to be extremely important. During rotavirus growth, mRNA transcribed from individual viral genome segments by viral RNA-dependent RNA polymerase is incorporated into the particle and used as a template for the synthesis of negative strands. Double-stranded RNA is synthesized (Chen, D., Zeng, Q.-Y., Wenz, MJ, Gorziglia, M. Estes, MK & Ramig, RF (1994) J. Virol. 68 , 7030-7039., Patton, JT & Spencer, E. (2000) Virology 277, 217-225.). Thus, theoretically, if a cDNA-derived artificial plus-strand RNA corresponding to a viral mRNA is introduced into a host cell, it is packaged in a particle and supplied by a helper virus. Should synthesize a negative strand to produce double-stranded RNA, and finally obtain an infectious virus with cDNA-derived gene segments.
The present inventors paid attention to the outer shell protein of rotavirus while studying for the purpose of developing a reverse genetics system of rotavirus. In other words, a strategy was adopted in which the gene segment encoding the coat protein gene was targeted for recombination, and a recombinant virus was selected using a neutralizing antibody against the coat protein (see the experimental data below for details). ). In summary, (1) a plasmid in which a cDNA encoding the rotavirus coat protein VP4 is placed under the control of a promoter recognized by the DNA-dependent RNA polymerase so that the DNA-dependent RNA polymerase is highly expressed. (2) Infect the host cell with helper rotavirus, and (3) Select by culturing the host cell in the presence of a neutralizing antibody that specifically recognizes VP4 of the helper rotavirus The method of doing is adopted. Then, according to the method, it was experimentally confirmed that a recombinant rotavirus having a VP4 gene segment derived from cDNA can be obtained. In addition, we conducted experiments using not only cDNA corresponding to the natural VP4 gene but also mutated cDNA, and succeeded in obtaining a recombinant rotavirus carrying an artificially mutated VP4 gene segment. The usefulness of the reverse genetics system was confirmed.
According to this reverse genetics system, of course, the VP4 gene segment of rotavirus can be artificially recombined as experimentally shown, but a plasmid containing a foreign gene together with a cDNA encoding VP4. It is also possible to construct a recombinant virus carrying a foreign gene. Thus, this reverse genetics system also makes it possible to use rotavirus as a vector.
On the other hand, as described above, it was shown that the coat protein gene segment is useful as a recombination target in constructing a reverse gene system of rotavirus. From this, it can be said that in addition to the VP4 gene segment, the VP7 gene segment can be used as an effective recombination target.
By the way, viruses belonging to the family Reoviridae have similar genome structures and replication mechanisms. Therefore, it can be said that the reverse genetics system established for rotavirus is widely applicable to viruses of the family Reoviridae.

The present invention is mainly based on the above results and knowledge, and provides a method for preparing a recombinant virus shown below.
[1] A method for preparing a recombinant virus comprising the following steps (1) to (4):
(1) Transfecting a host cell with a DNA construct placed under the control of a promoter recognized by a DNA-dependent RNA polymerase by a cDNA encoding a coat protein gene segment of a reoviridae virus (first virus) (However, the host cell is introduced with a nucleic acid construct expressing a DNA-dependent RNA polymerase corresponding to the promoter before or after the transfection operation);
(2) After step (1), as a helper virus, infecting the host cell with a virus of the same genus as the first virus (second virus);
(3) An antibody that specifically recognizes and neutralizes the outer shell protein of the second virus corresponding to the outer shell protein encoded by the outer shell protein gene segment of the first virus after step (2) And (4) after step (3), recovering the recombinant virus carrying the cDNA-derived coat protein gene segment.
[2] The preparation method according to [1], wherein the first virus is a virus belonging to the genus Rotavirus.
[3] The preparation method according to [1], wherein the first virus is a simian rotavirus and the second virus is a human rotavirus.
[4] The preparation method according to [3], wherein the simian rotavirus is SA11 strain and the human rotavirus is KU strain.
[5] The preparation method according to [3] or [4], wherein the outer shell protein is VP4.
[6] The preparation method according to any one of [1] to [5], wherein the DNA-dependent RNA polymerase is T7 RNA polymerase.
[7] The preparation method according to any one of [1] to [5], wherein the nucleic acid construct is a recombinant vaccinia virus expressing T7 RNA polymerase.
[8] The preparation method according to any one of [1] to [7], wherein the host cell is a COS-7 cell.
[9] The preparation method according to any one of [1] to [8], wherein a plurality of DNA constructs are used as the DNA construct, wherein the type of the outer shell protein encoded by the retained cDNA differs for each DNA construct. .
[10] The preparation method according to any one of [1] to [9], wherein the DNA construct further comprises a sequence encoding a foreign gene.
[11] The preparation method according to any one of [1] to [10], wherein the step (3) is performed in the presence of trypsin and / or chymotrypsin.
[12] The method according to any one of [1] to [11], wherein the step (3) includes any step selected from the group consisting of the following steps (a) to (d): Preparation method:
(A) after step (2), culturing the host cell in the presence of the antibody;
(B) After step (2), culturing the host cell in the presence of the antibody, recovering the produced virus, infecting another host cell (second host cell), and in the presence of the antibody Culturing a second host cell with:
(C) after step (2), recovering the produced virus, infecting another host cell (second host cell), and culturing the second host cell in the presence of the antibody;
(D) After step (2), the produced virus is recovered, infected with another host cell (second host cell), the second host cell is cultured in the presence of the antibody, and the produced virus And infecting another host cell (third host cell) and culturing the third host cell in the presence of the antibody.
[13] The preparation method according to [12], wherein in steps (a) to (d), at least a part of the culturing operation is performed by rotary culture.
[14] The preparation method according to [12] or [13], wherein the second host cell is a MA104 cell or a CV-1 cell.
[15] A T7 RNA polymerase promoter sequence, a cDNA encoding a rotavirus VP4 gene segment, a ribozyme sequence, and a T7 RNA polymerase terminator sequence are arranged in this order from the 5 ′ end to the 3 ′ end. Characteristic DNA construct for preparation of recombinant rotavirus.
[16] The DNA construct according to [15], wherein the ribozyme sequence is a sequence encoding a hepatitis D virus (HDV) ribozyme.
[17] The DNA construct according to [15] or [16], wherein the rotavirus is a simian rotavirus.
[18] The DNA construct according to any of [15] to [17], comprising a cDNA sequence encoding a foreign gene.

The present invention relates to a method for preparing a recombinant virus using a reverse genetics technique. In the preparation method of the present invention, steps (1) to (4) described in detail below are performed. In order to facilitate understanding, an example of the preparation method of the present invention is shown in FIGS.
In addition, matters not specifically mentioned below (conditions, operation methods, etc.) may follow conventional methods, such as Molecular Cloning (Third Edition, Cold Spring Harbor Laboratory Press, New York), Current protocols in molecular biology (edited by Frederick M. Ausubel et al., 1987) (above, with respect to molecular biological techniques), Urasawa, T., Urasawa, S. & Taniguchi, K. (1981) Microbiol. Immunol. 25, 1025-1035. Horimoto, T. & Kawaoka, Y. (1994) J. Virol. 68, 3120-3128. Barclay, W. & Palese, P. (1995) J. Virol. 69, 1275 -1279. (Above, regarding selection by antibody) and the like can be referred to.
1. Step (1)
In this step, a predetermined DNA construct is transfected into the host cell. As the DNA construct, one in which a cDNA encoding an outer shell protein gene segment of a reoviridae virus (first virus) is placed under the control of a promoter recognized by a DNA-dependent RNA polymerase is used.
In the following description, for convenience, “cDNA encoding an outer shell protein gene segment” is also referred to as “outer shell protein cDNA”. Similarly, “DNA-dependent RNA polymerase” is also referred to as “RNA polymerase”. A “promoter recognized by a DNA-dependent RNA polymerase” is also referred to as an “RNA polymerase promoter”.

  The reoviridae is classified into the reovirus genus, rotavirus genus, orbivirus genus, and cortivirus genus as viruses that infect mammals. Viruses belonging to the family Reoviridae have a unique genomic structure called double-stranded RNA divided into multiple segments. In the present invention, rotavirus cDNA (rotavirus) is preferably used, but the present invention is not limited thereto. When rotavirus is selected, a cDNA encoding a gene segment of VP4 (shell spike protein) or VP7 (shell glycoprotein), which is the shell protein, is used.

  It is also possible to use a coat protein cDNA into which a mutation has been introduced (ie, a part of the sequence has been modified). By using such a DNA construct containing cDNA, it is possible to obtain a recombinant virus having a mutation in the outer shell protein. The virus is useful for studying the function of the coat protein, and is expected to be used for vaccines. Mutation can be introduced into the outer shell protein cDNA by a known method such as Kunkel method, Gapped duplex method or the like, or a method analogous thereto. Commercially available mutagenesis kit (eg QuikChange XLsite-directed mutagenesis kit (Stratagene), Mutan-K (TAKARA), LA-PCR in vitro mutagenesis Kit (TAKARA) Mutan-Express Km (TAKARA) )) Can be used to introduce mutations by a simple operation.

  A rotavirus such as a human rotavirus, simian rotavirus, or bovine rotavirus can be used as the first virus. In a preferred embodiment of the present invention, among rotaviruses, simian rotavirus (for example, SA11 strain, RRV strain, etc.) is employed as the first virus. The segment protein gene segment of simian rotavirus generally has high transcription and synthesis efficiency. Therefore, high transcription / synthesis efficiency can be expected by using the outer shell protein cDNA of simian rotavirus. Increasing the efficiency of transcription and synthesis leads to an improvement in the recovery efficiency of the recombinant virus carrying the gene segment derived from the outer shell protein cDNA used.

  In the DNA construct, the coat protein cDNA is placed under the control of a promoter (RNA polymerase promoter) recognized by RNA polymerase. With such a configuration, after being transfected into the host cell, RNA polymerase recognizes the RNA polymerase promoter in the DNA construct, and transcription using the outer shell protein cDNA as a template is performed. For example, if T7 phage-derived T7 RNA polymerase is used as the RNA polymerase, a promoter corresponding to T7 RNA polymerase (ie, a promoter that is recognized by T7 RNA polymerase and promotes transcription of a sequence located downstream thereof) ) Is placed in the DNA construct. A system using T7 RNA polymerase is effective for obtaining high transcription efficiency. In the present invention, not only T7 RNA polymerase but also various RNA polymerases can be used. Examples of RNA polymerase include T3 RNA polymerase and Sp6 RNA polymerase. An example of a promoter sequence recognized by T7 RNA polymerase is shown in SEQ ID NO: 7.

  Typically, one type of DNA construct is used, but two or more types of DNA constructs may be used in combination. When a plurality of DNA constructs are used in this manner, each DNA construct is usually designed so that the type of outer shell protein encoded by the retained cDNA differs for each DNA construct. If multiple DNA constructs are used, a recombinant virus can be obtained in which multiple gene segments are derived from the DNA construct.

In one embodiment of the present invention, the DNA construct further comprises a sequence encoding a foreign gene. By using such a DNA construct, a recombinant virus in which a foreign gene is incorporated can be obtained. The recombinant virus thus obtained can be used as a vector for expressing a foreign gene, and is expected to be applied to a means for supplying a foreign protein to intestinal cells, for example. On the other hand, if the DNA construct is designed so that the expression product of the foreign gene binds to the outer shell protein, it is possible to obtain a recombinant virus having antigenicity different from that of the helper virus (second virus). Such recombinant viruses are expected to be used in vaccines.
A DNA construct may be designed and constructed so as to include sequences encoding a plurality of foreign genes.

  The DNA construct of the present invention can be constructed using a known plasmid or the like. For example, in the case of a DNA construct using the T7 RNA polymerase promoter, the target DNA construct can be constructed by performing the necessary genetic manipulation (insertion of outer shell protein cDNA, etc.) using the T7 expression vector as the backbone. . A wide variety of T7 expression vectors are provided available. Examples of available T7 expression vectors include commercially available pBluescriptII SK (+/-) (Stratagene), pCRII (Invitrogen), pGEM (Promega), pET (Novagene), pcDNA (Invitorgen). it can. Examples of vectors that can be used to construct the DNA construct of the present invention when using an RNA polymerase other than T7 RNA polymerase include pBluescriptIISK (+/−), pCRII, and pGEM.

By the way, a nucleic acid construct that expresses a DNA-dependent RNA polymerase (RNA polymerase) corresponding to the RNA polymerase promoter incorporated in the DNA construct is introduced into the host cell before or after the transfection operation. Therefore, as a result of transfection of the DNA construct and introduction of the nucleic acid construct, viral mRNA (plus strand) is generated in the host cell using the cDNA encoding the outer protein gene segment in the DNA construct as a template by RNA polymerase supplied by the nucleic acid construct. ) Is transcribed.
Preferably, the nucleic acid construct is introduced before the transfection operation of the DNA construct. As a result, an environment in which viral mRNA is transcribed quickly and efficiently can be created.

Reoviridae viral mRNAs have the characteristic of having a cap structure at the 5 'end and no poly A at the 3' end. These structures have been found to be important for efficient genome replication and translation. Considering this fact, if an attempt is made to efficiently obtain a recombinant virus having a gene segment derived from the coat protein cDNA in the DNA construct of the present invention, a virus produced by transcription from the DNA construct of the present invention It is preferable to provide an environment in which a capping enzyme acts efficiently on mRNA. Therefore, in a preferred embodiment of the present invention, a sequence encoding a capping enzyme is placed in a nucleic acid construct used for supplying RNA polymerase in a state where it can be expressed. By introducing such a nucleic acid construct, RNA polymerase and a capping enzyme are supplied into the host cell. As a result, mRNA transcribed from the DNA construct of the present invention can be efficiently matured.
As a nucleic acid construct capable of simultaneously supplying RNA polymerase and a capping enzyme, a recombinant vaccinia virus that has been genetically modified to express RNA polymerase can be used. For example, if a recombinant vaccinia virus that expresses T7 RNA polymerase is used as shown in the experimental examples described below, mature viral mRNA can be produced at a high rate from the DNA construct of the present invention, and the desired recombinant virus can be recovered. Efficiency can be improved.
Note that the capping enzyme may be supplied by a contract different from the nucleic acid construct of the present invention. For example, the capping enzyme can be supplied by separately introducing an expression vector carrying a gene encoding the capping enzyme into a host cell.

  The type of host cell is not particularly limited as long as it can function as a host for the DNA construct and nucleic acid construct of the present invention. For example, COS-7 cells, MA104 cells, CV-1 cells, 293T cells, Vero cells and the like can be used as host cells. COS-7 cells can be said to be suitable host cells because of high transfection efficiency and easy handling. On the other hand, MA104 cells are suitable host cells because of their high rotavirus growth rate.

2. Step (2)
After step (1), a step of infecting host cells with a virus belonging to the same gene as the first virus (second virus) as a helper virus is performed. For example, when rotavirus is used as the first virus, rotavirus is also used as the second virus. However, the first virus and the second virus need to differ at least with respect to the outer shell protein in order to enable selection by an antibody in the subsequent step (3). Therefore, for example, a combination of simian rotavirus and human rotavirus can be adopted as a combination of the first virus and the second virus. As described above, the first virus is preferably a simian virus from the viewpoint of transcription / synthesis efficiency. In view of this, a preferred embodiment employs a combination in which the first virus is a simian rotavirus and the second virus is a human rotavirus (for example, human rotavirus KU strain). You may decide to employ | adopt the combination of two types of viruses which differ by the level of a strain | stump | stock, such as using the combination of two strains of simian rotavirus, and the combination of two strains of human rotavirus.
In addition to wild-type or mutant viruses that exist in nature, recombinant viruses that have been artificially modified can also be used as the second virus.

  In the case of a system using rotavirus (that is, when rotavirus is used as the first virus and the second virus), trypsin or chymotrypsin is usually used to promote infection of the second virus (helper virus) into the host cell. Infection is carried out in the presence of. It is considered that the outer shell protein VP4 needs to be cleaved at a specific position for infection of rotavirus host, and trypsin and the like cleave VP4 necessary for this infection.

  In the host cell in which step (2) has been performed, the outer shell protein gene segment derived from the outer shell protein cDNA in the DNA construct transfected in step (1) and the other gene segment derived from the second virus (The target recombinant virus) is constructed (see FIG. 5). On the other hand, a virus having only a gene segment derived from the second virus is also constructed. Therefore, in the subsequent step (3), the target recombinant virus is selected from the thus constructed viruses.

3. Step (3)
In the present invention, an antibody is used to select the target recombinant virus. Specifically, an antibody that specifically recognizes and neutralizes the outer shell protein of the second virus corresponding to the outer shell protein encoded by the outer shell protein cDNA in the DNA construct used in step (1), Contact with the virus produced from Thus, in the present invention, the target recombinant virus is selectively propagated by neutralizing a virus having an outer shell protein derived from the second virus (helper virus) (see FIG. 6). For example, an antibody that selectively neutralizes VP4 of human rotavirus when cDNA encoding VP4 of simian rotavirus is used as the coat protein cDNA used in the DNA construct and human rotavirus is used as the second virus (Neutralizing antibody) is used.

The contact between the virus and the neutralizing antibody is performed, for example, by any of the following steps (a) to (d).
Step (a): After step (2), the host cells are cultured in the presence of neutralizing antibodies.
Step (b): After culturing host cells in the same procedure as in step (a), the produced virus is collected and infected with another host cell (second host cell). Subsequently, the second host cell is cultured in the presence of a neutralizing antibody.
Step (c): After step (2), the produced virus is recovered and infected with another host cell (second host cell). Subsequently, the second host cell is cultured in the presence of a neutralizing antibody.
Step (d): After the second host cell is cultured in the same procedure as in step (c), the produced virus is recovered and infected with another host cell (third host cell). Subsequently, the third host cell is cultured in the presence of a neutralizing antibody.

  In steps (b) to (d), the virus can be recovered by recovering the culture solution and / or cell disruption solution after culture. On the other hand, by adding (inoculating) the collected culture solution and / or cell disruption solution to the host cells (second host cell, third host cell), the virus can be infected into the host.

  In steps (a) to (d), it is preferable to carry out at least a part of the culturing operation by rotary culture. “Rotating culture” as used herein refers to culturing while rotating the culture container after seeding the cells in a culture container such as a test tube. Rotational culture can create a state in which the amount of the culture solution that contacts the cell surface is reduced (a state in which the degree of wetness of the cell surface decreases) as well as the transfer of the culture solution occurs. Expected to improve infection efficiency. A dedicated device for rotational culture is commercially available (for example, using Tytech Co., Ltd., Rotator RT-550, culture holder for test tubes), and using this commercially available device, carry out rotational culture here. Can do. The operation method may follow the instruction manual attached to the apparatus. For example, the rotation speed can be adjusted so that the time required for one rotation of the culture vessel is within a range of 30 seconds to 2 minutes.

As the second host cell and the third cell in the above steps, for example, MA104 cells, CV-1 cells, 293T cells, Vero cells and the like can be used.
In the step (d), an example in which the host cell is changed to two stages and the selection operation is performed is shown. Of course, the host cell may be changed to three stages.
In addition, when culturing a host cell, a second host cell, or a third host cell, subculture is performed as necessary. In order to enhance the effect of selection with a neutralizing antibody, subculture is preferably performed in the presence of a neutralizing antibody.

  Particularly in the case of a system using rotavirus, it is preferable to activate the virus with trypsin or chymotrypsin during the infection operation in order to promote infection of the virus to the host cells. Even in the case of a system using reovirus or the like, activation with trypsin or the like is effective in improving the infectious titer. To activate the virus with trypsin or the like, trypsin is added to the virus solution to activate the virus, and the host cells (second host cell and third host cell) are added with trypsin or the like added to the culture solution. Culture may be performed. In addition, it is preferable to add trypsin etc. during culture as needed.

The form and origin of the neutralizing antibody are not particularly limited as long as it has an activity of specifically recognizing and neutralizing the target outer shell protein. The neutralizing antibody may be any of a polyclonal antibody, an oligoclonal antibody (a mixture of several to several tens of antibodies), and a monoclonal antibody. As a polyclonal antibody or an oligoclonal antibody, an anti-serum-derived IgG fraction obtained by animal immunization, or an affinity-purified antibody using an antigen can be used. The neutralizing antibody may be an antibody fragment such as Fab, Fab ′, F (ab ′) ₂ , scFv, or dsFv antibody.
In order to obtain high selection efficiency by causing only the necessary neutralization reaction, it is preferable to use a monoclonal antibody as the neutralizing antibody. It is also effective to improve the selection efficiency to use two or more neutralizing antibodies in combination.

  Neutralizing antibodies can be prepared using immunological techniques, phage display methods, ribosome display methods, and the like. Preparation of a polyclonal antibody by an immunological technique can be performed by the following procedure. An antigen (target outer shell protein or a part thereof) is prepared, and this is used to immunize animals such as mice and rabbits. Specific examples of the antigen include purified rotavirus expressing the target coat protein, rotavirus VP4, VP5 *, VP7, VP8 * or a part thereof. The antigen consisting of the coat protein can be prepared by purification from a virus or, if the sequence has been identified, by a method using a gene recombination technique. In order to enhance the immunity-inducing action, an antigen bound with a carrier protein may be used. As the carrier protein, KLM (Keyhole Light Hemocyanin), BSA (Bovine Serum Albumin), OVA (Ovalbumin) and the like are used. The carbodiimide method, the glutaraldehyde method, the diazo condensation method, the MBS (maleimidobenzoyloxysuccinimide) method, etc. can be used for the coupling | bonding of carrier protein. On the other hand, an antigen obtained by expressing a target outer shell protein (or a part thereof) as a fusion protein with GST, β-galactosidase, maltose-binding protein, histidine (His) tag or the like can also be used. Such a fusion protein can be easily purified by a general method.

Immunization is repeated as necessary, and blood is collected when the antibody titer sufficiently increases, and serum is obtained by centrifugation or the like. The obtained antiserum is affinity purified to obtain a polyclonal antibody.
On the other hand, a monoclonal antibody can be prepared by the following procedure. First, an immunization operation is performed in the same procedure as described above. Immunization is repeated as necessary, and antibody-producing cells are removed from the immunized animal when the antibody titer sufficiently increases. Next, the obtained antibody-producing cells and myeloma cells are fused to obtain a hybridoma. Subsequently, after this hybridoma is monoclonalized, a clone that produces an antibody having high specificity for the target protein is selected. The target antibody can be obtained by purifying the culture medium of the selected clone. On the other hand, the desired antibody can be obtained by growing the hybridoma to a desired number or more, then transplanting it into the abdominal cavity of an animal (for example, a mouse), growing it in ascites, and purifying the ascites. For purification of the culture medium or ascites, affinity chromatography using protein G, protein A or the like is preferably used. Alternatively, affinity chromatography in which an antigen is immobilized may be used. Furthermore, methods such as ion exchange chromatography, gel filtration chromatography, ammonium sulfate fractionation, and centrifugation can also be used. These methods can be used alone or in any combination. In addition, even ascites without purification, it can be used as a reagent for enzyme antibody method (ELISA) for neutralizing viruses.

4). Step (4)
In this step, the target recombinant virus selectively propagated in step (3) is collected. It can be performed by recovering the culture solution and / or cell disruption solution after the culture. However, when plaque purification is performed using CV-1 or the like in step (3), the target recombinant virus is recovered from the formed plaque. The recovered recombinant virus is purified as necessary. The virus can be purified by appropriately combining fractional centrifugation, density gradient ultracentrifugation (using Nycodenz, cesium chloride, etc.), column chromatography, and the like.

  In the following, we report that the genome consists of 10 to 12 double-stranded RNAs and succeeded in developing a reverse genetics system for rotavirus belonging to the family Reoviridae. This system utilizes T7 RNA polymerase from recombinant vaccinia virus to supply artificial viral mRNA into the cytoplasm. A VP4 gene segment is synthesized from the full-length VP4 mRNA of the simian rotavirus SA11 strain transcribed from cDNA in the cytoplasm by superinfection with a helper virus (human rotavirus KU strain), and the KU strain based Transfectant virus (recombinant virus) is constructed. Infectious rotavirus transfectant introduced with a silent mutation that disrupts both or one of the two restriction enzyme cleavage sites as a gene marker in the SA11 strain VP4 genome in addition to a transfectant virus having a genuine SA11 strain VP4 gene segment We have also succeeded in building three types of factants.

1. Materials and Methods (1) Virus Human rotavirus KU strain (Urasawa, S., Urasawa, T., Taniguchi, K. & Chiba, S. (1984) Arch. Virol. 81, 1-12.) And simian rotavirus SA11 strain (SA11-L2) (Taniguchi, K., Nishikawa, K., Kobayashi, N., Urasawa, T., Wu, H., Gorziglia, M. & Urasawa, S. (1994) Virology 198, 325- 330.) was pretreated with trypsin (10 μg / ml, Sigma) and grown in the presence of trypsin (1 μg / m) using MA104 cells. Recombinant vaccinia virus rDIs-T7pol (Ishii, K., Ueda, Y., Matuo, K., Matsuura. Y., Kitamura, T., Kato, K., Izumi, Y., Someya, K., Ohsu, T., Honda, M. & Miyamura, T. (2001) Virology 302, 433-444.) Was distributed by Professor Yoshiharu Matsuura (Institute for Microbial Diseases, Osaka University). The virus was engineered to express T7 RNA polymerase and was propagated in chicken embryo fibroblasts.

(2) Cell culture and infection
COS-7 (monkey kidney epithelial cells), MA104 cells, and CV-1 cells were cultured in Eagle basal medium (MEM) supplemented with 5% fetal bovine serum.
Monolayer cells were inoculated with rotavirus pretreated with trypsin (10 μg / ml) at 37 ° C. for 30 minutes. After adsorption at 37 ° C. for 1 hour, the infected cells were washed with MEM (incomplete medium) not containing fetal calf serum, and then cultured in incomplete medium containing trypsin (1 μg / m) until collection. The culture solution (including cells) was subjected to freeze-thaw treatment (three times) and then centrifuged at low speed to remove cell debris. The supernatant was used for experiments.

(3) Construction of authentic VP4 gene cDNA of SA11 strain (Fig. 2)
The culture solution was disrupted with a disruption solution (1% SDS, 0.1% 2-mercaptoethanol, 60 mM EDTA), and extracted with phenol-chloroform to obtain double-stranded RNA (dsRNA) of SA11 virus. The cDNA of VP4 gene of SA11 virus is avian myeloblastosis virus as described above (Taniguchi, K., Kojima, K. & Urasawa, S. (1996) J. Virol. 70, 4125-4130.) Synthesized from virion dsRNA using reverse transcriptase (Seikagaku Corporation) and primer 1 (SEQ ID NO: 1, FIG. 1). Perform PCR using KOD-plus (Toyobo) and primers 1 and 2 (SEQ ID NOs: 1 and 2) containing the base sequence of the T7 promoter and the base sequence corresponding to the 5 ′ end sequence of the VP4 gene of SA11 virus, A cDNA encoding the full-length VP4 gene (SEQ ID NO: 8, 2.4 kb, GenBank Accession No. D16345) was amplified. After digestion with HindIII, the amplified cDNA was distributed by Dr. Zenji Matsuura and originally created by Dr. Conzelmann KK (Max-von Petterkofer Institute and Gene Center, Ludwig-Maximillians-University, Germany) pX8dT (Schnell, MJ, Mebatsion, T. & Conzelmann, KK (1994) EMBO J. 13, 4195-4203.) Were ligated to the HindIII and SmaI sites. The plasmid pT7 / VP4 (SA11) prepared here is located at the position between the T7 RNA polymerase promoter and the hepatitis D virus (HDV) ribozyme (further downstream, the T7 RNA polymerase terminator is located). Contains the long VP4 gene cDNA.

(4) Introduction of genetic markers into the rotavirus genome (Figure 2)
Manipulation of the VP4 gene was performed using the plasmid pT7 / VP4 (SA11). Using the QuikChange XLsite-directed mutagenesis kit (Stratagene) according to the attached instructions and artificially introducing nonsense mutations, unique PstI and HaeII sites (SA11 VP4 bases Nos. 1,364 and 1,569) ) Was destroyed. Briefly, pT7 / VP4 (SA11) using primers 3 and 4 (SEQ ID NOs: 3 and 4) and primers 5 and 6 (SEQ ID NOs: 5 and 6) which add mutations to the base sequence of PstI and HaeII sites of SA11. Was amplified. In order to remove pT7 / VP4 (SA11) used as a template, E. coli competent cells were transformed with the PCR product after digestion with DpnI. The resulting plasmids pT7 / VP4 (SA11) -ΔPstI and pT7 / VP4 (SA11) -ΔHaeII have silent mutations at the PstI site and the HaeI site, respectively. Furthermore, plasmids pT7 / VP4 (SA11) -ΔPstI and ΔHaeII were prepared in the same manner as described above using pT7 / VP4 (SA11) -ΔHaeII as a template and primers 3 and 4 (SEQ ID NOs: 3 and 4). . This has mutations in both PstI and HaeII sites. All newly prepared plasmids were confirmed to be appropriate clones by confirming their base sequences.

(5) Reverse genetics (Figs. 5 and 6)
COS-7 cells were grown to such an extent that they were buried about 80% in a 60 mm petri dish. COS-7 cells infected with rDIs-T7pol at a multiplicity of infection (MOI) 3 of 1 hour ago using TransIT-1 (Mirus), 5 μg of pT7 / VP4 (SA11), pT7 / VP4 ( SA11) -ΔPstI, pT7 / VP4 (SA11) -ΔHaeII or pT7 / VP4 (SA11) -ΔPstI, ΔHaeII were transfected. At 20 hours after transfection, the cells were washed twice with incomplete medium, followed by infection with the KU strain (the VP4 gene sequence of KU strain is shown in SEQ ID NO: 9, GenBank Accession No. M21014) as a helper virus. Superinfection was performed at a multiplicity of 3. At 24 hours after infection with helper virus, a transfectant virus having SA11 cDNA-derived VP4 gene in the culture was selected by the following method.
In order to select a transfectant virus having a VP4 gene derived from SA11 cDNA, the collected virus was activated with trypsin and then inoculated into MA-104 cells cultured in a monolayer in a 60 mm petri dish. After 1 hour of adsorption, the infected cells are washed twice with incomplete medium, and trypsin (1 μg / ml) and an anti-VP4 neutralizing monoclonal antibody that specifically neutralizes P [8] type helper virus KU strain ST-1F2 (Taniguchi, K., Morita, Y., Urasawa, T. & Urasawa, S. (1987) J. Virol. 61, 1726-1730.) And YO-2C2 (Taniguchi, K., Urasawa, S & Urasawa, T. (1985) J. Gen. Virol. 66, 1045-1053.). When the cytopathic effect produced by rotavirus appeared, the cultured cells were collected and subjected to subculture. Transfectant virus thus removed was passaged 3 times with MA104 cells and then plaque purified with CV-1 cells (Taniguchi, K., Morita, Y., Urasawa, T. & Urasawa, S. (1987) J. Virol. 61, 1726-1730.).
The neutralizing monoclonal antibodies ST-1F2 and YO-2C2 were prepared as follows (for details, see Sapporo Medical Journal 56 (1) 71-85 (1987), Taniguchi, K., Morita, Y. , Urasawa, T. & Urasawa, S. (1987) J. Virol. 61, 1726-1730., Taniguchi, K., Urasawa, S. & Urasawa, T. (1985) J. Gen. Virol. 66, 1045 -1053.) As the immunogen, purified human rotavirus YO strain (G serotype 3) and ST-3 strain (G serotype 4) (provided by Dr. RGWyatt of NIH) were used. First, 0.1 ml of purified virus solution (about 10 ⁸ p · f · u / ml) was inoculated intraperitoneally on BALB / c mice on the 1st, 30th and 45th days. On the 48th day, spleen cells of the immunized mouse and mouse bone marrow P3-X63-Ag8 · 653 cells were fused using polyethylene glycol 1540. On the 20th to 30th day after the fusion, the anti-HRV neutralizing activity in the hybridoma culture supernatant was searched by the indirect fluorescent antibody method to confirm the presence or absence of neutralizing monoclonal antibody (N-mAb) production. The hybridoma determined to produce N-mAb was cloned at least twice by the limiting dilution method. Finally, 10 ⁷ hybridomas were intraperitoneally inoculated into BALB / c mice pretreated with prestain to obtain ascites. Rabbit antiserum against mice IgG, IgM, IgG1, IgG2a, IgG2b and IgG3 (Miles Yeda, Rehorot, Israel) and 25-fold concentrated supernatant of hybridoma culture were used to determine the isotype of the immunoglobulin produced by each hybridoma And performed by the Ouchterlony method. By the above method, a hybridoma producing neutralizing monoclonal antibody ST-1F2 against KU strain and a hybridoma producing YO-2C2 were obtained.

(6) Identification of cDNA-derived VP4 gene in recovered virus Genomic dsRNA of recovered virus was extracted from the culture solution. Using the precipitated virion dsRNA, (1) polyacrylamide electrophoresis (PAGE), (2) RT-PCR restriction enzyme digestion analysis, and (3) base sequencing were performed. In PAGE, dsRNA was electrophoresed on a 2 mm-thick 10% acrylamide gel at 20 mA and room temperature for 16 hours and judged by silver staining. For RT-PCR restriction enzyme digestion analysis, primer 1 (SEQ ID NO: 1) is used, dsRNA is converted to cDNA using SuperScript II RT (Invitrogen), and full-length VP4 gene is converted to primers 1 and 2 (SEQ ID NOs: 1, 2). Amplified with The PCR product was digested with PstI or HaeII, and the resulting fragments were separated by electrophoresis using a 1.25% agarose gel. The gel was stained with ethidium bromide. For the nucleotide sequence determination, the DYEnamiic ET terminator cycle sequencing kit (Amersham Biosciences) was used, and the sequence of the PCR product was directly determined by an ABI PRISM 310 automatic sequencer.

2. Results (1) SA11 VP4 gene cDNA structure (Figure 2)
To produce infectious transfectants with new gene segments derived from cDNA, Dr. Palese et al. (Enami, M., Luytjes, W., Krystal, M. & Palese, P. (1990) Proc. Natl. Acad. Sci. USA 87, 3802-3805., Enami, M. & Palese, P. (1991) Virology 185, 291-298.) Reverse genetics using helper virus first developed with influenza virus The method was partially modified. In this technique, since the majority of viruses in the culture solution are helper viruses, a strong selection system is required to obtain a transfectant virus containing a cDNA-derived gene. Therefore, it has no effect on the growth of viruses with VP4 types other than P [8], such as SA11 strain, and specifically neutralizes P [8] type human rotaviruses, such as KU strain. A selection system using neutralizing monoclonal antibody against VP4 spike protein was constructed. That is, by subculturing in the presence of such neutralizing antibody, the transfectant virus containing the VP4 gene segment derived from SA11 cDNA is selectively propagated in the background of the helper virus KU strain. Decided to let. This strategy also shows that rotavirus with SA11 VP4 shows significantly better growth than the original human rotavirus (Greenberg, HB, Kalica, AR, Wyatt, RG, Jones, RW, Kapikian, AZ & Chanock, RM (1981) Proc. Natl. Acad. Sci. USA 78, 420-424.).
First, the SA11 virus full-length VP4 gene placed between the T7 RNA polymerase promoter and the hepatitis D virus (HDV) ribozyme (a T7 RNA polymerase terminator is placed downstream of the hepatitis D virus (HDV) ribozyme). ) Was cloned (FIG. 2). The vector pT7 / VP4 (SA11) thus obtained produces a 2,362-base positive strand RNA having the correct 5 ′ end and 3 ′ end corresponding to the ends of the authentic SA11 VP4 mRNA.

(2) Transfection of cDNA encoding full-length SA11 VP4 gene and recovery of virus having VP4 genome derived from cDNA (Figs. 5 and 6)
Transcription plasmid pT7 / VP4 (SA11) was transfected into COS-7 cells infected with recombinant vaccinia virus one hour ago to supply T7 RNA polymerase. On the first day after transfection, the transfected cells were superinfected with KU virus as a helper virus, and the culture was further continued for 24 hours. After adding the collected culture medium and two types of neutralizing antibodies ST-1F2 and YO-2C2 that specifically neutralize KU strain helper virus to the culture medium of MA104 cells cultured in the test tube, Was transferred to a rotary incubator (Rotator RT-550, manufactured by Taitec Co., Ltd.), and MA104 cells were cultured under conditions of 2 minutes / rotation. Eight days after infection, MA104 cells showed cytopathic effect (CPE), suggesting the generation of a transfectant virus incorporating the cDNA-derived VP4 gene.

(3) RNA analysis of recovered virus (Figure 3)
After purifying the virus obtained in the selection experiment, plaques were amplified in MA104 cells. Virion dsRNA was extracted from this sample and analyzed by PAGE. FIG. 3 shows the pattern of dsRNA from SA11 (lane 1) used for VP4 gene cloning, the KU strain used for helper virus (lane 3), and a mixture of both viruses (lane 5). The VP4 gene dsRNA in lanes 2 and 4 moves to the same position (lane 1) as the corresponding dsRNA of SA11, and the mobility is based on the mobility of the VP4 gene segment of the KU strain, which is a helper virus (lane 3). It was late. Furthermore, the nucleotide sequence of the RT-PCR fragment showed that the recovered virus VP4 gene was derived from SA11 (data not shown). As described above, infectious rotavirus transfectant (KU // rVP4 (SA11) virus) containing cDNA-derived VP4 gene segment is obtained as a result of cDNA transfection and subsequent superinfection with helper virus. It was shown that.

(4) Introduction of site-specific mutations into the genome of infectious rotavirus Next, four site markers as gene markers in the coding region of the VP4 gene in plasmid pT7 / VP4 (SA11) were obtained by site-specific mutagenesis using PCR. Silent mutation was introduced. The four mutations in cDNA are those that change base No. 1,365 from T to A, those that change base No. 1,368 from A to C, those that change base No. 1,569 from G to A, and bases No. 1,572 is changed from G to A, and the PstI site of base No. 1,364 and the HaeII site of base No. 1,569 are destroyed. The newly prepared plasmids pT7 / VP4 (SA11) ΔPstI, pT7 / VP4 (SA11) ΔHaeII, and pT7 / VP4 (SA11) -ΔPstI, ΔHaeII are respectively represented as PstI site, HaeII site, and both sites. Has a mutation to destroy. KU // rVP4 (SA11) -ΔPstI, KU // rVP4 () was transfected by transfection of COS-7 cells that had already been infected with recombinant vaccinia virus with plasmids with the respective mutations, followed by superinfection with helper virus. Transfectant viruses designated SA11) -ΔHaeII and KU // rVP4 (SA11) -ΔPstI, ΔHaeII grew in the presence of helper virus-specific neutralizing monoclonal antibodies. Virion dsRNA was extracted from the culture, and PAGE and RT-PCR were performed on the entire VP4 nucleotide sequence. As a result of PAGE, the mobility of the fourth gene segment of the recovered virus was the same as the mobility of the corresponding gene segment of the SA11 strain (data not shown). As a result of RT-PCR restriction enzyme digestion analysis, the 2,386 base pair product of KU // rVP4 (SA11) was digested with PstI into a 1,392 base pair fragment and a 994 base pair fragment, and digested with HaeII, 1,597 base pairs. A pair of fragments and a fragment of 789 base pairs were obtained (FIG. 4A). On the other hand, the corresponding DNA fragment from the newly recovered transfectant virus was not cleaved with PstI and / or HaeII as expected (FIG. 4A). In addition, direct silencing of the PCR product certainly showed these silent mutations in the recovered viral genome (FIG. 4B). Thus, infectious rotaviruses with site-specific mutations in the genome were recovered by transfection of cloned cDNA and subsequent superinfection of helper virus in a system using T7 RNA polymerase of recombinant vaccinia virus. Succeeded in doing.

3. As described above, we have succeeded in constructing the world's first rotavirus reverse genetics system. The means presented here utilize the infection of recombinant vaccinia virus expressing T7 RNA polymerase and the superinfection of helper virus. For viruses belonging to the family Reoviridae, Roner and Joklik reported a reverse genetics system that generates reovirus transfectants (Roner, MR, Sutphin, LA & Joklik, WK (1990) Virology 179, 845- 852., Roner, MR, Nepluev, I., Sherry, B. & Joklik, WK (1997) Proc. Natl. Acad. Sci. USA 94, 6826-6830., Roner, MR & Joklik, WK (2001) Proc Natl. Acad. Sci. USA 98, 8036-8041.). However, the system is extremely complicated and requires extremely complicated preparations such as preparation of temperature-sensitive mutant strains and preparation of transformed cells that stably express a specific viral protein encoded by the gene segment to be manipulated. For this reason, there are no reports of use in other laboratories or reports of application to other reoviridae viruses. On the other hand, Dr. Palese et al. (Enami, M., Luytjes, W., Krystal, M. & Palese, P. (1990) Proc. Natl. Acad. Sci. USA 87, 3802-3805., Enami , M. & Palese, P. (1991) Virology 185, 291-298., Japanese Patent No. 3293620) applied a reverse genetics system using a conventional T7 RNA polymerase expression system to rotavirus Is. By using the recombinant vaccinia virus T7 RNA polymerase, artificial viral mRNA mimicking wild-type rotavirus mRNA can be expressed to a very high degree in the cytoplasm, and all replication cycles occur in the cytoplasm. It is convenient for the development of the reverse genetics system of rotavirus.
Rotavirus mRNA has a cap structure at the 5 ′ end and no poly A at the 3 ′ end (Imai, M., Akatani, K., Ikegami, N. & Furuichi, Y. (1983) J. Virol. 47, 125-136., McCrae, MA McCorquodale, JG (1983) Virology 126, 204-212.). These structures are important for efficient genome replication and translation. (Patton, JT, Wentz, M., Xiaobo, J. & Ramig, RF (1996) J. Virol. 70, 3961-3971., Wentz, MJ, Patton, JT & Ramig, RF (1996 ) J. Virol. 70, 7833-7841., Chizhikov, V. & Patton, JT (2000) RNA 6, 814-825.). In the construct used in this experiment, the authentic 5 'and 3' end structures were maintained by the capping enzyme encoded by vaccinia virus and the hepatitis D virus ribozyme sequence inserted in the transcription plasmid, respectively (Fig. 2). CDNA mRNA derived from cDNA transcribed in the cytoplasm using recombinant vaccinia virus T7 RNA polymerase is used, and helper virus co-infection rescues infectious rotavirus containing new gene segments derived from cDNA . In order to select such a transfectant virus, we selected a system that requires the rescue virus to have a VP4 gene similar to the simian rotavirus SA11. Growth of viruses containing this gene is not restricted in the presence of neutralizing monoclonal antibodies that selectively neutralize VP4 from helper virus (human rotavirus KU strain). In this experiment, the KU // rVP4 (SA11) virus having a genuine SA11 VP4 gene derived from the plasmid pT7 / VP4 (SA11) was successfully recovered for the first time (FIG. 2). The KU // rVP4 (SA11) virus shows the expected RNA pattern (Fig. 3) and the nucleotide sequence of the VP4 gene (Fig. 4), almost the same as the infectious titer of the SA11 virus used for cloning of the VP4 gene. Proliferated (data not shown). Differences in growth between KU // rVP4 (SA11) and the parental KU virus were predicted from previous findings that VP4 protein contributes significantly to infectivity of rotavirus (Patton, JT & Spencer, E (2000) Virology 277, 217-225., Kalica, AR, Flores, J. & Greenberg, HB (1983) Virology 125, 194-205., Offit, PA, Blavat, G., Greenberg, HB & Clark, HF (1986) J. Virol. 57, 46-49.).

  We also disrupted certain restriction enzyme sites as gene markers in the VP4 genes from plasmids pT7 / VP4 (SA11) ΔPstI, pT7 / VP4 (SA11) ΔHaeII, and pT7 / VP4 (SA11) -ΔPstI, ΔHaeII We succeeded in obtaining three transfectant viruses with silent mutations (FIG. 2). The resulting mutant viruses KU // rVP4 (SA11), KU // rVP4 (SA11) ΔPstI, KU // rVP4 (SA11) ΔHaeII and KU // were obtained by RT-PCR restriction enzyme cleavage analysis and sequencing of virion dsRNA. The existence of corresponding mutations in rVP4 (SA11) -ΔPstI, ΔHaeII was verified (FIG. 4). These mutations are not expected to change the biological properties of the KU // rVP4 (SA11) virus having a genuine SA11 virus-derived VP4 gene without causing amino acid changes in the VP4 protein. In fact, all rescued mutant viruses showed the same growth as KU // rVP4 (SA11) virus (data not shown). Thus, the usefulness of the reverse genetics system developed with rotavirus was confirmed.

If mutations that change the biological properties of rotavirus can be introduced, detailed functions of all viral proteins including nonstructural proteins can be clarified. Research on untranslated regions of the genome will be possible by site mutation. This will provide a better understanding of the regulatory signals present in the viral RNA. In addition, this reverse genetics system will provide an opportunity to identify attenuated markers within the rotavirus genome and will facilitate the development of attenuated recombinant viruses that can be used as vaccine vector candidates.
The approach presented here may be useful for dsRNA transcription, studying the signals involved in replication and packaging, and studying the function of viral proteins.

The reverse genetics system provided by the present invention enables the preparation of viruses belonging to the family Reoviridae, in which the outer shell protein gene segments have been artificially manipulated. Therefore, a recombinant virus with altered properties (for example, antigenicity) can be prepared using the present invention. In particular, the present invention is considered effective as a means of preparing attenuated recombinant viruses that can be used as vaccines.
On the other hand, it is also possible to construct a recombinant virus carrying a foreign gene using the present invention. Thus, the present invention also makes it possible to use reoviridae viruses as vectors.
In addition, according to the present invention, it becomes possible to artificially manipulate the viral genome, and it is expected to contribute to detailed analysis on the growth and pathogenicity of reoviridae viruses.

The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims.
The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

The table | surface which shows the PCR primer used for construction of a plasmid. It is described in the order of primer, base sequence position, base sequence (restriction enzyme site). Restriction enzyme sites are underlined. Also, italic indicates the T7 RNA polymerase promoter sequence. The bold letters indicate the base sequence substituted with the VP4 gene of SA11 virus. The lower case letter in primer 2 indicates a non-viral base sequence added to the 5 'end of the primer. Structure of a transcription plasmid encoding the full-length VP4 gene derived from SA11. Plasmid pT7 / VP4 (SA11) contains the T7 RNA promoter, the authentic full-length VP4 gene of SA11, the hepatitis D virus ribozyme, and the T7 RNA terminator. The VP4 gene is sandwiched between the T7 RNA promoter and the hepatitis D virus ribozyme. Manipulation of the VP4 gene by silent mutation (mutation position is indicated by an arrowhead below the base sequence) was performed using pT7 / VP4 (SA11). Mutant plasmids pT7 / VP4 (SA11) ΔPstI, pT7 / VP4 (SA11) ΔHaeII, and pT7 / VP4 (SA11) -ΔPstI, ΔHaeII are, in turn, disrupted to unique PstI sites, unique HaeII sites, and both. including. A numerical value shows the base position in the base sequence of SA11 VP4 gene. pT7, ribozyme, and TT7 indicate a T7 RNA polymerase promoter, a hepatitis D virus ribozyme, and a T7 RNA polymerase terminator, respectively. Results of polyacrylamide electrophoresis of dsRNA extracted from rescued VP4 gene transfectants. Lane 1, dsRNA from SA11; Lanes 2 and 4, dsRNA from mutant virus with VP4 gene of KU // rVP4 (SA11); Lane 3, dsRNA of helper virus KU strain; Lane 5, SA11 and KU dsRNA blend. The numbers on the left indicate the order of SA11 genomic dsRNA. Site-specific mutation introduced into rescued VP4 gene transfectant. The full-length VP4 gene of each virus was amplified by RT-PCR to obtain a 2,386 base product. (A) The amplified fragments were digested with PstI or HaeII and separated on a 1.25% agarose gel. KU // rVP4 (SA11) (lanes 1, 2, 3), KU // rVP4 (SA11) ΔPstI (lanes 4, 5, 6), KU // rVP4 (SA11) ΔHaeII (lanes 7, 8, 9), KU // rVP4 (SA11) -ΔPstI, ΔHaeII (lanes 10, 11, 12). A fragment of 2,386 bases (lanes 1, 4, 7, 10) was digested with PstI (lanes 2, 5, 8, 11) or HaeII (lanes 3, 6, 9, 12). M, 1kb ladder marker. (B) The base sequence was directly determined for a fragment of 2,386 bases, and site-specific mutations introduced into the genome of infectious VP4 gene transfectants were confirmed. The figure which shows the preparation method of the recombinant rotavirus using the system of reverse genetics. The figure which shows selection operation of the recombinant rotavirus in the system of reverse genetics.

Claims (18)

A method for preparing a recombinant virus comprising the following steps (1) to (4):
(1) Transfecting a host cell with a DNA construct placed under the control of a promoter recognized by a DNA-dependent RNA polymerase by a cDNA encoding a coat protein gene segment of a reoviridae virus (first virus) (However, the host cell is introduced with a nucleic acid construct expressing a DNA-dependent RNA polymerase corresponding to the promoter before or after the transfection operation);
(2) After step (1), as a helper virus, infecting the host cell with a virus of the same genus as the first virus (second virus);
(3) An antibody that specifically recognizes and neutralizes the outer shell protein of the second virus corresponding to the outer shell protein encoded by the outer shell protein gene segment of the first virus after step (2) And (4) after step (3), recovering the recombinant virus carrying the cDNA-derived coat protein gene segment.   The preparation method according to claim 1, wherein the first virus is a virus belonging to the genus Rotavirus.   The preparation method according to claim 1, wherein the first virus is a simian rotavirus and the second virus is a human rotavirus.   The preparation method according to claim 3, wherein the simian rotavirus is SA11 strain and the human rotavirus is KU strain.   The preparation method according to claim 3 or 4, wherein the outer shell protein is VP4.   The preparation method according to claim 1, wherein the DNA-dependent RNA polymerase is T7 RNA polymerase.   The preparation method according to any one of claims 1 to 5, wherein the nucleic acid construct is a recombinant vaccinia virus expressing T7 RNA polymerase.   The preparation method according to claim 1, wherein the host cell is a COS-7 cell.   The preparation method according to any one of claims 1 to 8, wherein a plurality of DNA constructs are used as the DNA construct, wherein the type of outer shell protein encoded by the cDNA to be retained is different for each DNA construct.   The preparation method according to claim 1, wherein the DNA construct further comprises a sequence encoding a foreign gene.   The preparation method according to any one of claims 1 to 10, wherein the step (3) is carried out in the presence of trypsin and / or chymotrypsin. The preparation method according to any one of claims 1 to 11, wherein the step (3) comprises any step selected from the group consisting of the following steps (a) to (d):
(A) after step (2), culturing the host cell in the presence of the antibody;
(B) After step (2), culturing the host cell in the presence of the antibody, recovering the produced virus, infecting another host cell (second host cell), and in the presence of the antibody Culturing a second host cell with:
(C) after step (2), recovering the produced virus, infecting another host cell (second host cell), and culturing the second host cell in the presence of the antibody;
(D) After step (2), the produced virus is recovered, infected with another host cell (second host cell), the second host cell is cultured in the presence of the antibody, and the produced virus And further infecting another host cell (third host cell) and culturing the third host cell in the presence of the antibody.   The preparation method according to claim 12, wherein in steps (a) to (d), at least a part of the culturing operation is carried out by rotary culture.   The preparation method according to claim 12 or 13, wherein the second host cell is a MA104 cell or a CV-1 cell.   A T7 RNA polymerase promoter sequence, a cDNA encoding a rotavirus VP4 gene segment, a ribozyme sequence, and a T7 RNA polymerase terminator sequence are arranged in this order from the 5 ′ end to the 3 ′ end. A DNA construct for the preparation of recombinant rotavirus.   16. The DNA construct according to claim 15, wherein the ribozyme sequence is a sequence encoding a hepatitis D virus (HDV) ribozyme.   The DNA construct according to claim 15 or 16, wherein the rotavirus is a simian rotavirus.   The DNA construct according to any one of claims 15 to 17, comprising a cDNA sequence encoding a foreign gene.