JP2005102702A - Recombinant sendai virus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a sendai virus vector sufficiently available in the field of gene therapy and the like by establishing a sendai virus reconstitution system with good manufacturing efficiency and enabling gene manipulation of the sendai virus. <P>SOLUTION: A method for reconstituting sendai virus particles by introducing a sendai virus genome into a host cell expressing all initial replication genes is developed. Gene manipulation of the sendai virus thereby allows to effectively use the sendai virus as a vector. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a recombinant Sendai virus and a method for producing the same.

  Sendai virus is also called HVJ (Hemagglutinating virus of Japan), and is classified into parainfluenza virus type 1 belonging to Paramyxoviridae and Paramyxovirus genus.

  Sendai virus particles are polymorphic, have an envelope with a diameter of 150 to 200 nm, and have genomic RNA (hereinafter referred to as “(−) strand RNA”) that does not serve as a template for translation. Sendai virus is historically known as an industrially useful virus, and is particularly widely used for the production of cell heterokaryons and hybrid cells, that is, cell fusion. In addition, as a material for membrane-fusible liposomes, development as a vector for gene therapy is also in progress. Furthermore, Sendai virus is also used as an inducer for various interferons.

  In classification according to the form of genomic nucleic acid, Sendai virus belongs to the group of (-) single-stranded RNA viruses of (-) strand RNA viruses of RNA viruses. RNA viruses are classified into three groups: dsRNA virus (double stranded RNA virus), (+) strand RNA virus, and (-) strand RNA virus. The dsRNA virus group includes reovirus, rotavirus, plant reovirus, and the like, and has a plurality of segmented linear dsRNA genomes. The (+) strand RNA viruses include poliovirus, Sindbis virus, Semliki Forest virus, Japanese encephalitis virus, etc., and have one (+) strand RNA as a genome, and this RNA genome is simultaneously converted into mRNA. Can function, and can produce proteins necessary for replication and particle formation depending on the translation function of the host cell. In other words, the genomic RNA itself of the (+) strand RNA virus has the ability to propagate. In this specification, “propagating power” means “infectious particles or complex equivalents thereof after the nucleic acid is introduced into the cell by infection or an artificial technique and then replicated in the cell. "The ability to form and propagate one after another to another cell". Sindbis virus classified as (+)-strand RNA virus and Sendai virus classified as (-)-strand RNA virus have infectivity and transmission ability, but adeno-associated virus (Adeno) classified as parvoviridae -associated virus) is infectious, but has no transmission power (adenovirus co-infection is required for virus particles to form). In addition, Sindbis virus-derived (+)-strand RNA artificially transcribed in vitro has a propagating power (forms virus particles when introduced into cells), but is artificially transcribed in vitro. Sendai virus RNA thus produced has no ability to propagate (+) and (−) strands (does not form virus particles when introduced into cells).

  In recent years, vectors derived from viruses have been used as vectors for gene therapy. In order to use a virus as a vector, it is necessary to establish a method for reconstructing virus particles. ("Reconstruction of viral particles" means to artificially create viral genome nucleic acid and to produce the original virus or recombinant virus in vitro or in cells.) This is because the virus particles must be reconstructed from the virus genome into which the foreign gene has been incorporated by genetic manipulation in order to introduce it into the virus vector. If a virus reconstitution technique is established, it is possible to produce a virus in which a desired foreign gene is introduced into the virus, a desired gene of the virus is deleted, or inactivated.

  In addition, if a virus reconstitution system is constructed and genetic manipulation of the virus becomes possible, it will be a great tool for genetic analysis of the virus function. Genetic analysis of viral functions is extremely important from a medical standpoint such as prevention and treatment of diseases. For example, if the replication mechanism of viral nucleic acid is elucidated, the development of an antiviral agent based on the replication of nucleic acid that causes less damage to the host cell by utilizing the difference from the replication mechanism of the nucleic acid in the host cell. Is possible. In addition, by elucidating what functions the protein encoded by the viral gene has, it would be possible to develop an antiviral agent targeting a protein involved in virus particle infectivity and virus particle formation ability. Moreover, it can be expected that by improving a gene related to membrane fusion ability, a better membrane fusion liposome can be prepared and used as a gene therapy vector. In addition, as represented by interferon, infecting a virus activates a gene related to the virus resistance of a host gene and may exhibit virus resistance. With regard to activation of such host genes, important knowledge will be obtained by genetic analysis of virus functions.

  Reconstitution of DNA viruses using DNA as a genomic nucleic acid has been carried out relatively early. For example, purified genomic DNA itself such as SV40 (J. Exp. Cell Res., 43, 415-425 (1983)) It is possible to carry out by introducing into the cells.

  The reconstruction of RNA viruses using RNA as genomic nucleic acid was preceded by development in (+) strand RNA viruses. This is because genomic RNA simultaneously functions as mRNA. For example, in Poliovirus, it was already reported in 1959 that the purified RNA itself has transmission ability (Journal of Experimental Medicine, 110, 65-89 (1959)). In addition, Semliki forest virus (SFV) reports that it is possible to reconstruct the virus by introducing the cDNA into the cell by using the DNA-dependent RNA transcriptional activity of the host cell. (Journal of Virology, 65, 4107-4113 (1991)).

  Furthermore, gene therapy vectors have been developed using these reconstitution techniques [Bio / Technology, 11, 916-920 (1993), Nucleic Acids Research, 23, 1495-1501 (1995), Human Gene. Therapy, 6, 1161-1167 (1995), Methods in Cell Biology, 43, 43-53 (1994), Methods in Cell Biology, 43, 55-78 (1994)].

  However, as described above, although Sendai virus has many advantages that can be used as an industrially useful virus, a reconstitution system has not been established because it is a (−) strand RNA virus. It was. This is because the virus particle reconstitution system via the viral cDNA was extremely difficult.

  As described above, (-)-strand RNA virus may not be generated even when (-) strand RNA virus RNA (vRNA; viral RNA) or its complementary RNA (cRNA; complementary RNA) is introduced into the cell alone. It has been revealed. This is a crucial difference from the (+) strand RNA virus. In addition, Japanese Patent Laid-Open No. 4-21377 describes “a cDNA corresponding to the genome of a negative-strand RNA virus and a method for producing an infectious negative-strand RNA virus”, but the experimental contents of the publication are described as they are. "EMBO.J., 9, 379-384 (1990)" has revealed that the experiment is not reproducible, and the author has completely withdrawn the content of the paper (EMBO. J., 10, 3558 (1991)). Therefore, it is clear that the technique described in Japanese Patent Application Laid-Open No. 4-21377 does not correspond to the prior art of the present invention.

  (-) Strand RNA virus reconstitution system has been reported for influenza viruses (Annu. Rev. Microbiol., 47, 765-790 (1993), Curr. Opin. Genet. DEV., 2, 77-81). (1992)). Influenza virus is a (-) strand RNA virus composed of an 8-segment genome. According to these reports, an exogenous gene was inserted into one of these cDNAs in advance, and RNA transcribed from all eight cDNAs containing the exogenous gene was pre-associated with a virus-derived NP protein to produce RNP and did. Reconstitution was established by supplying these RNPs and RNA-dependent RNA polymerase into the cells. As for (-) single stranded RNA virus, there is a report on virus reconstitution from cDNA in rabies virus belonging to Rhabdoviridae (J. Virol., 68, 713-719 (1994)).

  Therefore, the (-) strand RNA virus reconstitution technology has basically become known, but in the case of Sendai virus, the virus cannot be reconstituted even if this method is applied as it is. It was. In addition, the report that virus particles were reconstituted in rhabdoviruses was only confirmed by marker gene expression, RT-PCR, etc., and was not sufficient from the viewpoint of production. Furthermore, conventionally, for the purpose of supplying intracellular factors necessary for reconstitution, viruses such as natural virus and recombinant vaccinia virus are supplied to cells simultaneously with the nucleic acid of the virus to be reconstituted. Therefore, there is a problem that it is not easy to separate the desired virus that has been reconstructed from those harmful viruses.

  An object of the present invention is to establish a Sendai virus reconstitution system with high production efficiency, to enable Sendai virus gene manipulation, and to supply a Sendai virus vector that can be sufficiently practically used in the field of gene therapy and the like.

  First, the present inventors applied cDNA or Sendai virus minigenome derived from Sendai virus DI particles (see defective interfering particle / EMBO.J., 10, 3079-3085 (1991)) for application to Sendai virus reconstitution tests. Various studies were carried out using the cDNA. As a result, we found efficient conditions for the ratio of the amount of cDNA introduced into cells, the cDNA group related to transcriptional replication, and the recombinant vaccinia virus, which is a T7 RNA polymerase expression unit. The present inventors further obtained both the (+) strand and the (−) strand of the full-length Sendai virus cDNA so that (+) strand or (−) strand Sendai virus RNA is biosynthesized in the cell. Plasmids were constructed and introduced into cells expressing cDNAs for transcriptional replication. As a result, we succeeded in reconstructing Sendai virus particles from Sendai virus cDNA for the first time. For the purpose of efficient particle reconstitution by the present inventors, it is appropriate that the form of cDNA introduced into the cell is circular rather than linear, and (−) strand RNA is transcribed inside the cell. It was newly found that the particle formation efficiency is higher when (+)-strand RNA is transcribed in the cell than in the cell.

  Furthermore, the present inventors have found that Sendai virus can be reconstituted even when a recombinant vaccinia virus that is a T7 RNA polymerase expression unit is not used. That is, when Sendai virus full-length RNA transcribed in a test tube was introduced into cells, and the cDNA of the initial transcript replication enzyme group was transcribed under the control of the T7 promoter, virus particles were reconstituted. This indicates that it is possible to create recombinant Sendai virus without using any helper virus such as vaccinia virus by constructing cells that express all the initial transcriptase groups. Yes. The cells that express all the initial transcriptase groups are described in “J. Virology, 68, 8413-8417 (1994)”, and can be created by those skilled in the art with reference to the description. . The cell described in this document is a cell derived from 293 cells having NP, P / C, and L on the chromosome among Sendai virus genes, and these cells are NP, P / C, , L expresses three proteins.

  From many examples of viral vectors, if the virus particles can be efficiently reconstituted from nucleic acids, the desired viral genes can be recombined, foreign genes can be inserted, or the desired viral genes can be inactivated. Obviously, this can be easily done by those skilled in the art. That is, it is obvious to those skilled in the art that the first successful reconstitution of Sendai virus particles in the present invention means that the present invention has enabled Sendai virus genetic manipulation.

That is, the present invention includes the following.
(1) a recombinant Sendai virus having a propagation ability, which retains a genome containing a desired foreign gene or in which a desired gene is deleted or inactivated;
(2) The recombinant Sendai virus according to (1), wherein one or more functional protein genes are modified,
(3) The recombinant Sendai virus according to (1) or (2), which has a foreign gene that can be expressed in a host,
(4) RNA containing RNA contained in the recombinant Sendai virus according to any one of (1) to (3),
(5) RNA containing cRNA of RNA contained in the recombinant Sendai virus according to any one of (1) to (3),
(6) (a) DNA containing a template cDNA capable of transcribing the RNA described in (4) or (5), and (b) using the DNA as a template in a test tube or cell in (4) or (5) A kit comprising a unit capable of transcribing the described RNA;
(7) (a) a host that expresses Sendai virus NP protein, P / C protein and L protein (each protein may be a protein having equivalent activity); and (b) described in (4) or (5) A kit comprising RNA,
(8) Introducing the RNA described in (4) or (5) into a host that expresses Sendai virus NP protein, P / C protein and L protein (each protein may be a protein having equivalent activity). A method for producing the recombinant Sendai virus according to any one of (1) to (3),
(9) (a) a host expressing NP protein, P / C protein and L protein of Sendai virus, (b) a template cDNA capable of transcribing RNA or cRNA according to any of (4) or (5) (C) a kit comprising three members capable of transcribing the RNA according to (4) or (5) in a test tube or in a cell using the DNA as a template, and (10) an NP protein of Sendai virus, In a host expressing P / C protein and L protein, a DNA containing a template cDNA capable of transcribing the RNA according to (4) or (5), and using the DNA as a template in a test tube or in a cell (4) or A method for producing the recombinant Sendai virus according to any one of (1) to (3), comprising introducing a unit capable of transcribing the RNA according to (5),
(11) A method for producing an exogenous protein comprising a step of infecting a host with the recombinant Sendai virus according to (3) and recovering the expressed exogenous protein,
(12) A culture solution or chorioallantoic fluid containing an expressed foreign protein, which can be obtained by introducing the recombinant Sendai virus according to (3) into a host and collecting the culture broth or chorioallantoic fluid, and ( 13) Expression of a protein encoded by the exogenous gene incorporated in a Sendai virus vector, comprising the exogenous gene arranged downstream of the promoter in a direction in which the antisense RNA of the encoded protein is transcribed and the promoter DNA for.

  The recombinant Sendai virus vector of the present invention is, for example, transcribed in vitro in a recombinant cDNA encoding a recombinant Sendai virus vector genome produced by genetic engineering to produce a recombinant Sendai virus genomic RNA, The RNA can be obtained by introducing it into a host that simultaneously expresses Sendai virus NP protein, P / C protein and L protein (each protein may be a protein having equivalent activity). As another method, the Sendai virus vector of the present invention comprises (i) a recombinant cDNA encoding a genetically engineered recombinant Sendai virus vector genome, and (ii) RNA in a cell using the DNA as a template. A transcribable unit can be obtained by introducing a Sendai virus NP protein, P / C protein, and L protein (each protein may be a protein having the same activity) into a host that simultaneously expresses. In this case, for example, (i) may be DNA that is connected downstream of a specific promoter, and (ii) is DNA that expresses a DNA-dependent RNA polymerase that acts on the specific promoter.

  In the recombinant Sendai virus of the present invention, Sendai virus used as a material before inserting a desired foreign gene or deleting or inactivating the desired gene is a strain classified as parainfluenza type 1. For example, Z strain (Sendai virus Z strain), Fushimi strain (Sendai virus Fushimi strain) and the like can be mentioned. Incomplete viruses such as DI particles, synthesized oligonucleotides, and the like can also be used as part of the material.

  Moreover, as long as the recombinant Sendai virus of the present invention retains its transmission ability, any exogenous gene inserted into any site of RNA contained in the recombinant, and any genomic gene deleted or It may be modified. Examples of the foreign gene to be inserted include genes encoding various cytokines and genes encoding various peptide hormones that can be expressed in the host. In order to express a desired protein, a foreign gene encoding the desired protein is inserted. In Sendai virus RNA, it is desirable to insert a sequence having a multiple of 6 between the R1 sequence (5′-AGGGTCAAAGT-3 ′) and the R2 sequence (5′-GTAAGAAAAA-3 ′) ( Journal of Virology, Vol. 67, No. 8, (1993) p.4822-4830). Expression efficiency The expression level of the inserted foreign gene can be controlled by the position of gene insertion and the RNA base sequences before and after the gene. For example, in Sendai virus RNA, it is known that the closer the insertion position is to the NP gene, the greater the expression level of the inserted gene. The host for expressing the desired protein may be any cell as long as it is infected with recombinant Sendai virus. Examples thereof include cultured mammalian cells and chicken eggs. A foreign gene product can be efficiently produced by infecting these hosts with a recombinant Sendai virus in which an exogenous gene that can be expressed is incorporated, and collecting the expressed foreign gene product. The expressed protein can be recovered by a conventional method, for example, from a culture solution when a cultured cell is used as a host, or from urine serum when a chicken egg is used as a host.

  When a foreign gene is incorporated into a plasmid where (-) strand Sendai virus RNA is biosynthesized, the foreign gene is transcribed in the direction in which the antisense RNA of the protein encoded by the foreign gene is transcribed. It needs to be inserted downstream. A protein encoded by the exogenous gene incorporated in a Sendai virus vector, which includes the exogenous gene arranged downstream of the promoter in such a direction that the antisense RNA of the encoding protein is transcribed and the promoter. “DNA for expression” was first made available by the present invention and is part of the present invention.

  In addition, for example, genes that are involved in RNA replication of some Sendai viruses may be modified in order to inactivate genes involved in immunogenicity or increase RNA transcription efficiency or replication efficiency. Specifically, for example, at least one of NP protein, C / P protein, and L protein, which are replication factors, can be modified to enhance or weaken transcription and replication functions. HN protein, which is one of the structural proteins, has both hemagglutinin activity and neuraminidase activity, which are hemagglutinin. If the former activity can be reduced, for example, blood It may be possible to improve the stability of the virus in it, and it is also possible to regulate the infectivity, for example by modifying the activity of the latter. It can also be used to improve membrane fusion liposomes by fusing the reconstituted Sendai virus with artificial liposomes encapsulating the desired drug or gene by modifying the F protein involved in membrane fusion. is there.

  The present invention has made it possible to introduce point mutations and insertions at arbitrary positions in genomic RNA, and this is highly expected to accumulate genetic knowledge of viral functions at an accelerated pace. . For example, if the replication mechanism of viral RNA is elucidated, an antiviral agent that acts on the replication of nucleic acids with less damage to the host cell will be developed using the difference from the replication mechanism of the nucleic acid derived from the host cell. Is possible. In addition, by elucidating what functions the protein encoded by the viral gene has, it would be possible to develop an antiviral agent targeting a protein involved in virus particle infectivity and virus particle formation ability. Specifically, for example, it can be used for analysis of antigen-presenting epitopes of F protein and HN protein that can be antigen molecules on the cell surface. In addition, when a virus is activated and a gene related to virus resistance of a host gene is activated and exhibits virus resistance, activation of such a host gene is also important by genetic analysis of virus function. Knowledge will be gained. Since Sendai virus has an interferon-inducing effect, it is used in various basic experiments. It is also conceivable to produce a non-viral interferon inducer by analyzing the region necessary for this induction. The technology of the present invention can also be used for vaccine development. A live vaccine can be produced by inoculating a recombinant chicken Sendai virus whose gene has been artificially modified into a developing chicken egg, and the knowledge obtained in this way can be used for other (−) strand RNA viruses such as It can also be applied to viruses with a high need for vaccines, such as measles virus and mumps virus. Furthermore, the present invention makes it possible to use a recombinant Sendai virus as a vector for gene therapy. Since the viral vector of the present invention is derived from Sendai virus, it is highly safe, and since the viral vector retains the transmission power, it is expected that a large therapeutic effect can be obtained even with a small amount of administration. In addition, when treatment is completed and it is necessary to inhibit viral vector growth, or during treatment, RNA-dependent RNA polymerase inhibitors can be administered to specifically identify viral vector growth without damaging the host. Can be deterred.

  EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

[Example 1] Preparation of Sendai virus transcription unit pUC18 / T7 (-) HVJRz.DNA and pUC18 / T7 (+) HVJRz.DNA
Plasmid pUC18 / T7 (-) HVJRz.DNA was prepared by inserting the T7 promoter, Sendai virus cDNA designed to transcribe (-) strand RNA, and DNA holding the ribozyme gene in this order into the pUC18 plasmid. In addition, a plasmid pUC18 / T7 (+) HVJRz.DNA was constructed by inserting the T7 promoter, Sendai virus cDNA designed to transcribe (+) strand RNA, and DNA holding the ribozyme gene in this order into the pUC18 plasmid. did. The structures of pUC18 / T7 (-) HVJRz.DNA and pUC18 / T7 (+) HVJRz.DNA are shown in FIG. 1 and FIG.

[Example 2] Sendai virus reconstitution experiment from cDNA Add 2,000,000 LLC-MK2 cells treated with normal trypsin to a plastic petri dish with a diameter of 6 cm and 2 ml of MEM medium (MEM + FBS 10%), and CO. _25%, were cultured for 24 hours under the conditions of 37 ° C.. After removing the culture medium and washing with 1 ml of PBS, 0.1% of a recombinant vaccinia virus expressing T7 polymerase, vTF7-3, prepared to have a moi / multiplicity of infection of 2 was obtained. The suspension in ml PBS was added. Every 15 minutes, the petri dish was shaken so that the virus solution spread throughout, and the infection was carried out for 1 hour. The virus solution was removed and washed with 1 ml PBS. A medium containing a cDNA solution was added to the petri dish. The medium containing the cDNA solution was prepared as follows.

  Take the nucleic acids listed in the table (including plasmids expressing factors necessary for Sendai virus replication, including pGEM-L, pGEM-P / C, and pGEM-NP) in a 1.5 ml sampling tube and add HBS (Hepes buffered saline; 20 mM) Hepes pH 7.4, 150 mM NaCl) was added to bring the total volume to 0.1 ml. (-) Or (+) cDNA in the table indicates plasmid pUC18 / T7 (-) HVJRz.DNA or pUC18 / T7 (+) HVJRz.DNA itself, / C remains circular, / L indicates restriction enzyme MluI It shows that it introduce | transduced into the cell after linearizing by.

  On the other hand, 0.07 ml of HBS and 0.03 ml of DOTAP (Boehringer Mannheim) were prepared in a polystyrene tube, and the nucleic acid solution was transferred to this polystyrene tube. In this state, it was allowed to stand for 10 minutes. To this, cell culture medium (2 ml MEM + FBS 10%) was added. Furthermore, rifampicin (Rifampicin) and cytosine arabinoside C (Cytosin arabinoside C / Ara C), which are inhibitors of vaccinia virus, were added to the final concentrations of 0.1 mg / ml and 0.04 mg / ml, respectively. . As a result, a medium containing the cDNA solution was prepared.

The petri dish was cultured for 40 hours at 5% CO _{2 at} 37 ° C. The cells in the petri dish were scraped using a rubber policeman, transferred to an Eppendorf tube, centrifuged at 6,000 rpm for 5 minutes to precipitate only the cellular components, and again suspended in 1 ml of PBS. A part of this cell solution was used as it was or diluted and inoculated into 10-day-old embryonated chicken eggs. This cell solution was diluted with PBS to the number of cells shown in Table 1, and 0.5 ml inoculated eggs were cultured at 35 ° C. for 72 hours, then transferred to 4 ° C. and left overnight. The egg chorioallantoic fluid was collected as a virus solution using a syringe and an injection needle.

  The recovered virus solution was measured for HAU (hemmaglutinin unit) and PFU (plaque forming unit) by the method described below.

  The measurement of HAU was performed as follows. The chicken blood was centrifuged at 400 × g for 10 minutes, and the supernatant was discarded. The remaining precipitate was suspended in PBS 100 times the amount of the precipitate, and this was further centrifuged at 400 × g for 10 minutes, and the supernatant was discarded. This operation was repeated twice more to prepare a 0.1% blood cell solution. The virus solution was diluted 2-fold by a serial dilution method, and 0.05 ml each was dispensed into a 96-well titer plate. To this titer plate, 0.05 ml of blood cell solution was further dispensed, mixed gently by shaking lightly, and allowed to stand at 4 ° C. for 40 minutes. Thereafter, erythrocyte aggregation was observed with the naked eye, and among the aggregates, the dilution rate of the virus solution with the highest dilution rate was indicated as HAU.

The measurement of PFU was performed as follows. CV-1 cells were grown in a monolayer on a 6-well culture plate. The culture plate medium was discarded, and 0.1 ml of the virus solution diluted 10-fold by serial dilution was dispensed to each well in each culture plate and infected at 37 ° C. for 1 hour. During infection, 2xMEM without serum and 2% agar were mixed at 55 ° C, and trypsin was added to a final concentration of 0.0075 mg / ml. After 1 hour of infection, the virus solution was removed, 3 ml of medium mixed with agar was added to each well in each culture plate, and incubated at 37 ° C. for 3 days under 5% CO ₂ conditions. 0.2 ml of 0.1% phenol red was added and incubated at 37 ° C. for 3 hours, and then removed. The number of uncolored plaques was counted and the virus titer was evaluated as PFU / ml.

HAU、PFUをともに示したサンプルを超遠心で沈渣とした後、再浮遊して20%〜60%のショ糖密度勾配遠心で精製し、12.5%SDS-PAGEで蛋白質を分離したところ、ここに含まれる蛋白質は、センダイウイルスの蛋白質と同じ大きさのものであった。 Table 1 shows the Sendai virus cDNA used as a template introduced into LLC-MK2 cells, the amounts of pGEM-L, pGEM-P / C and pGEM-NP, which are factors necessary for RNA replication, incubation time, The number of cells inoculated, HAU, and PFU are shown.

A sample showing both HAU and PFU was made into a sediment by ultracentrifugation, resuspended, purified by 20% -60% sucrose density gradient centrifugation, and separated by 12.5% SDS-PAGE. The protein contained was the same size as the Sendai virus protein.

  From this result, it was shown that Sendai virus can be reconstituted by introducing cDNA into cells. In addition, it was shown that when the cDNA that transcribes the (+) strand was introduced into the cell, the virus particles were reconstituted more efficiently than when the cDNA that transcribes the (−) strand was introduced. Furthermore, it was shown that when the cDNA was introduced in a circular form, the virus particles were reconstituted more efficiently than when the cDNA was introduced in a linear form.

[Example 3] Examination of RNA replication factors required for Sendai virus reconstitution
An experiment was conducted to determine whether all three plasmids expressing L, P / C and NP were necessary. The method is the same as in Example 2, but in Example 2, three genes, pGEM-L, pGEM-P / C, and pGEM-NP, were introduced into the cell together with the cDNA. In this experiment, pGEM- Any two or only one of L, pGEM-P / C, and pGEM-NP were introduced into the cells together with the cDNA.

表２から、どの組合わせの２者を導入した場合もウイルスの生産が認められなかった。この結果、この３種の蛋白質すべてが、再構成には必須であることが確認された。 Table 2 shows Sendai virus cDNA as a template introduced into LLC-MK2 cells, the amounts of pGEM-L, pGEM-P / C and pGEM-NP, which are cDNAs for factors necessary for RNA replication, incubation time, and inoculation of chicken eggs Cell number, HAU and PFU were shown respectively.

From Table 2, no virus production was observed when any combination of the two was introduced. As a result, it was confirmed that all of these three proteins are essential for reconstitution.

[Example 4] Sendai virus reconstitution experiment from in vitro transcribed RNA In Example 2, it was shown that Sendai virus was reconstituted from cDNA, but products obtained by further transcribed cDNA in vitro, namely vRNA and cRNA. But we examined whether we could do the same.

この結果より、どちらのセンスのRNAを細胞内に導入しても、ウイルスを再構 成することができた。 Sendai virus transcription unit pUC18 / T7 (-) HVJRz.DNA and pUC18 / T7 (+) HVJRz.DNA were linearized with the restriction enzyme MluI, and then used as a template, purified T7 polymerase (EPICENTRE TECHNOLOGIES: Ampliscribe T7 In vitro RNA synthesis was performed using Transcription Kit). The method of in vitro RNA synthesis followed the kit protocol. The RNA product obtained here was used in place of the cDNA of Example 2 and the same experiment was performed, and virus production was evaluated by the HA test.
The results are shown in Table 3.

From these results, it was possible to reconstitute the virus regardless of which sense RNA was introduced into the cell.

[Example 5] Examination of expression in host of foreign gene inserted in Sendai virus vector
(1) Preparation of Sendai virus vector “pSeVgp120” inserted with foreign gene (HIV-1 gp120 gene) 3 ′) (SEQ ID NO: 2) was used to amplify the HIV-1 gp120 gene on “pNI432” by a standard PCR method. TA cloning was performed, digested with NotI, and inserted into “pSeV18 ⁺ ” digested with NotI. Next, this is transformed into E. Coli, the DNA of each colony of E. Coli is extracted by the “Miniprep” method, electrophoresed after DraIII digestion, and the expected size by insertion of the migrated DNA A clone that was confirmed to contain the DNA fragment was selected to obtain a positive clone (hereinafter, this positive clone is referred to as “clone 9”). After confirming the target base sequence, the DNA was purified by cesium chloride density gradient centrifugation. The pSeV18 ⁺ inserted with gp120 thus obtained is referred to as “pSeVgp120”.

(2) Reconstitution of Sendai virus (SeVgp120) carrying pSeVgp120 and analysis of gp120 expression
In addition to pGEM NP, P, and L in LLCMK2 cells, pSeVgp120 was further introduced, and the urine fluid of the hen's eggs was collected in the same manner as in Example 2 to measure HAU and study gp120 expression (ELISA) ) The measurement of HAU was performed in the same manner as in Example 2.

  Moreover, ELISA was performed as follows. 100 μl of a sample was added to a 96-well plate covered with a monoclonal antibody against HIV-1 and reacted at 37 ° C. for 60 minutes. After washing with PBS, 100 μl of HRP-conjugated anti-HIV-1 antibody was added and reacted at 37 ° C. for 60 minutes. This was washed with PBS, tetramethylbenzidine was added, and the expression level of gp120 was measured by detecting the amount of the reaction product converted by HRP activity under an acidic condition at an absorbance of 450 nm. The results are shown on the left of Table 4.

表４から明らかなように、CV-1細胞で特に高濃度のgp120が生産され（表中央）、また再度発育鶏卵に接種した尿しょう液からも高濃度のgp120が検出された（表右）。なお、表４左と表４中央には３クローンの結果を示してある。 The obtained virus solution was infected with CV-1 cells, and the same examination was performed. CV-1 cells were grown at 5x10 ⁵ cells per plate, the medium was discarded, washed with PBS (-), virus solution was added at a multiplicity of infection of 10, and the cells were infected at room temperature for 1 hour. . The virus solution was discarded, washed with PBS (-), plainMEM medium (MEM medium supplemented with antibiotics AraC, Rif and trypsin) was added, and the mixture was reacted at 37 ° C for 48 hours. After the reaction, the medium was collected, and HAU was measured (the same method as in Example 2) and gp120 expression was examined (ELISA). The results are shown in the center of Table 4. The culture supernatant of CV-1 cells was again inoculated into the embryonated chicken eggs, and the results of HAU measurement and gp120 expression examination (ELISA) of the virus solution thus obtained are shown on the right side of Table 4.

As is clear from Table 4, a particularly high concentration of gp120 was produced in CV-1 cells (the center of the table), and a high concentration of gp120 was also detected from the urinary fluid inoculated into the hen's eggs again (the front right). . In addition, the results of 3 clones are shown in Table 4 left and Table 4 center.

  Furthermore, the expression of gp120 was analyzed by Western blotting. CV-1 cells infected with SeVgp120 were centrifuged at 20,000 rpm for 1 hour to precipitate the virus, and the supernatant was TCA (10% (v / v), 15 minutes on ice) or 70% ethanol (15% on ice). -20 ° C), centrifuged at 15,000 rpm for 15 minutes, the precipitated protein was mixed with "SDS-PAGE Sample buffer" (Daiichi Kagaku), reacted at 90 ° C for 3 minutes, and SDS on a 10% acrylamide gel -Polyacrylamide gel electrophoresis (SDS-PAGE) was performed. After electrophoresis, the protein was transferred to a PVDF membrane (Daiichi Kagaku) and allowed to react with the monoclonal antibody 902 at room temperature for 1 hour. Subsequently, it was washed with T-TBS, anti-mIgG (Amersham) was reacted at room temperature for 1 hour, and washed with T-TBS. Further, HRP-binding protein A (Amersham) was reacted at room temperature for 1 hour and washed with T-TBS. 4-Chloro-1-naphthol (4CNPlus) (Daiichi Kagaku) was added to this to detect gp120. As a result, a band was detected at the expected molecular weight of gp120.

Furthermore, the relationship between the time after infection of SeVgp120 into CV-1 cells, the value of HAU, and the expression level of gp120 was analyzed. CV-1 cells are seeded on a 10cm plate to become 5x10 ⁶ cells, infected with SeVgp120 at a multiplicity of infection of 10, and then 1 ml of the medium is collected at 30, 43, 53, and 70 hours. Were mixed with HAU, and gp120 expression (ELISA) and Western blotting were performed. The result is shown in FIG. As is apparent from FIG. 3, the production of gp120 showed a tendency to increase with the increase of Sendai virus HAtiter.

なお、表左は種々の型の細胞へのSeVgp120の感染後の時間を示す。この結果、検討を行ったすべての細胞でSeVgp120の増殖及びgp120の発現が検出された。 [Example 6] Proliferation of SeVgp120 and expression of gp120 in various types of cells Measurement of HAU and examination of gp120 expression in the same manner as in Example 5 except that various types of cells were used (ELISA) Went. The results are shown in Table 5.

The table on the left shows the time after infection of various types of cells with SeVgp120. As a result, proliferation of SeVgp120 and expression of gp120 were detected in all the cells examined.

[Example 7] Examination of expression in host of luciferase gene inserted into Sendai virus vector Primer (5'-AAGCGGCCGCCAAAGTTCACGATGGAAGAC-3 '(30mer)) (SEQ ID NO :) for isolating the luciferase gene for vector insertion : 3) and a primer (5'-TGCGGCCGCGATGAACTTTCACCCTAAGTTTTTCTTACTACGGATTATTACAATTTGGACTTTCCGCCC-3 '(69mer)) (SEQ ID NO: 4), using "pHvluciRT4" as a template and adding a NotI site at both ends by standard PCR method Was isolated. Next, this was inserted into pSeV18 ⁺ digested with NotI to obtain a Sendai virus vector into which the luciferase gene was inserted. Next, the cells were introduced into LLCMK2 cells and inoculated into growing chicken eggs. The chorioallantoic membrane of the developing egg was cut out, washed twice with cold PBS (−), added with 25 μl of “lysis buffer” (Picagene WACO), stirred well, and then centrifuged at 15000 rpm for 2 minutes. 5 μl of the supernatant was collected, 50 μl of substrate (IATRON) was added, put into a 96-well plate, and the fluorescence intensity was measured with a luminometer (Luminous CT-9000D, DIA-IATRON). The activity was expressed in cps (counts per second). As a result, particularly high luciferase activity was detected in CV-1 cells 24 hours after infection (Table 6). In addition, Sendai virus into which no luciferase gene was introduced was used as a control (indicated by “SeV” in the table). The table also shows the detection results of 2 clones.

  According to the present invention, a system for reconstructing virus particles more efficiently than Sendai virus cDNA has been established, enabling genetic manipulation in Sendai virus, including a desired foreign gene, or a genome in which a desired gene has been deleted or inactivated. It has become possible to obtain a recombinant Sendai virus that retains the ability to transmit and has a transmission power.

FIG. 1 shows the structure of pUC18 / T7 (+) HVJRz.DNA. FIG. 2 shows the structure of pUC18 / T7 (−) HVJRz.DNA. FIG. 3 shows the relationship between the time after infection of CV-1 cells with SeVgp120, the value of HAU, and the expression level of gp120.

Claims (13)

  A recombinant Sendai virus containing a desired foreign gene or retaining a genome in which the desired gene is deleted or inactivated, and has a transmission ability.   The recombinant Sendai virus according to claim 1, wherein one or more functional protein genes are modified.   3. The recombinant Sendai virus according to claim 1 or 2, which has a foreign gene that can be expressed in a host.   An RNA comprising RNA contained in the recombinant Sendai virus according to any one of claims 1 to 3.   RNA containing cRNA of RNA contained in the recombinant Sendai virus according to any one of claims 1 to 3.   (a) DNA containing a template cDNA capable of transcribing the RNA according to claim 4 or 5, and (b) transcription of the RNA according to claim 4 or 5 in a test tube or a cell using the DNA as a template. A kit containing possible units.   (a) a host expressing NP protein, P / C protein and L protein of Sendai virus (each protein may be a protein having the same activity); and (b) the RNA according to claim 4 or 5. Including kit.   Including introducing the RNA according to claim 4 or 5 into a host expressing a Sendai virus NP protein, P / C protein and L protein (each protein may be a protein having equivalent activity). The manufacturing method of the recombinant Sendai virus in any one of the ranges 1-3.   (a) a host that expresses Sendai virus NP protein, P / C protein and L protein, (b) DNA containing a template cDNA capable of transcribing RNA or cRNA according to any one of claims 4 or 5, c) A kit comprising three members capable of transcribing the RNA according to claim 4 or 5 in a test tube or a cell using the DNA as a template.   A host containing a Sendai virus NP protein, P / C protein and L protein, a DNA containing a template cDNA capable of transcribing the RNA according to claim 4 or 5, and using the DNA as a template in a test tube or in a cell A method for producing a recombinant Sendai virus according to any one of claims 1 to 3, comprising introducing a unit capable of transcribing the RNA according to claim 4 or 5.   A method for producing a foreign protein, comprising the step of infecting a host with the recombinant Sendai virus according to claim 3 and recovering the expressed foreign protein.   A culture solution or chorioallantoic fluid containing an expressed foreign protein, which can be obtained by introducing the recombinant Sendai virus according to claim 3 into a host and recovering the culture broth or chorioallantoic fluid.   A protein for expressing a protein encoded by the exogenous gene incorporated in a Sendai virus vector, comprising the exogenous gene arranged downstream of the promoter in such a direction that the antisense RNA of the encoded protein is transcribed and the promoter. DNA.

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