JP6775728B2 - Genetically modified cells and vaccine production method - Google Patents

Description

The present invention is a recombinant cell based on a cell derived from the genus Cat, and a gene in which the infectious env gene of the RD-114 virus contained in the genome of the original strain is artificially knocked out. Related to recombinant cells, vaccine production methods using those cells, infectious RD-114 virus expression risk detection method for determining the presence or absence of infectious RD-114 virus expression, gene recombinant cell production method, etc. To do.

Vaccine production by cell culture has been widely put into practical use as one of the vaccine production methods. In the production of vaccines against infectious diseases of dogs and cats, vaccines against feline herpesvirus, feline calicivirus, feline panleukopenia virus, canine adenovirus, canine distemper virus, canine parvovirus, canine coronavirus, canine parainfluenza virus, etc. CRFK cells (Crandell-Rees feline kidney cells) and other strains derived from the genus Cat are used for the production of dogs.

Endogenous retrovirus (ERV) is a retrovirus that is endogenously integrated into a part of the mammalian genome by inserting a gene into the germline of an ancestor during a retrovirus infection in ancient times. Is. It is estimated that about 10% of endogenous retroviruses, which are traces of past retrovirus infections, are present in the mammalian genome. Most of them have lost their replication ability and infectivity as viruses due to the accumulation of mutations and deletions in genomic DNA that have occurred over many years, but some endogenous retroviruses retain their infectivity. It is said that it has occurred, and some cases have been reported.

Domestic cats carry an infectious endogenous retrovirus called the RD-114 virus. Like other retroviruses, the genome of the RD-114 virus, which is inherent in the genome of cats, has a gag gene encoding a structural protein, reverse transcription, in a region sandwiched between two LTRs (long terminal repeats) with promoter activity. It has a pol gene that encodes an enzyme, an env gene that encodes an envelope protein, and the like.

Regarding the RD-114 virus, as a finding at that time, in Non-Patent Document 1, about 20 copies of sequences having homology with the RD-114 virus exist in the genome of the domestic cat, and among them in the coding region of the gag gene and the pol gene. It was reported that many of the env genes were significantly deleted, whereas the restriction enzyme sites of the virus were preserved. In the same document, the infectious RD-114 virus was identified (later named "Sc3c") and its molecular clone was generated (later named "pSc3c").

In Non-Patent Document 2, CRFK cells, MCC cells (feline large granular lymphoma cells), and FER cells (feline embryonic fibroblasts cells), which are cells derived from the genus Felis, produce infectious particles of RD-114 virus. Was reported. Non-Patent Document 3 reports that infectious particles of the infectious RD-114 virus are contaminated in several live attenuated vaccines for cats and dogs produced using cat-derived strained cells. Was done.

On the other hand, the pathogenicity of the RD-114 virus to cats or dogs is not yet clear, but in Non-Patent Document 2 mentioned above, it is stated that some cat-derived strained cells are susceptible to the RD-114 virus. In Non-Patent Document 4, it was reported that the RD-114 virus can infect and proliferate in primary canine cultured cells and canine-derived strained cells, respectively.

In addition, in Non-Patent Document 5, a CRT strain, which is an infectious RD-114 virus isolated from CRFK cells, is identified, a molecular clone thereof, pCRT1, is produced, and the gene sequence of the pCRT1 is RD-114. It was reported that it was almost identical to that of the clone pSc3c when the virus was first identified, and that there were no substitutions in the amino acid sequence corresponding to the coding region of the env gene. In Non-Patent Document 6, the sequences of three molecular clones (pSc3c, pCRT1, pRD-UCL) of the infectious RD-114 virus were compared.
Reeves, RH &O'Brien, SJ Molecular genetic characterization of the RD-114 gene family of endogenous feline retroviral sequences. J Virol 52, 164-171 (1984). Okada, M., Yoshikawa, R., Shojima, T., Baba, K. & Miyazawa, T. Susceptibility and production of a feline endogenous retrovirus (RD-114 virus) in various feline cell lines. Virus Res 155, 268- 273 (2011). Miyazawa, T., Yoshikawa R., Golder M., Okada M., Stewart H., and Palmarini M. Isolation of an infectious endogenous retrovirus in a proportion of live attenuated vaccines for pets. J. Virol. 84: 3690-3694 (2010). Yoshikawa R., Yasuda J., Kobayashi T., Miyazawa T. Canine ASCT1 and ASCT2 are functional receptors for RD-114 virus in dogs J. Gen. Virol. 93, 603-607 (2012) Yoshikawa, R., Sato, E., Igarashi, T. & Miyazawa, T. characterization of RD-114 virus isolated from a commercial canine vaccine manufactured using CRFK cells. J Clin Microbiol 48, 3366-3369 (2010). Shimode, S. et al. Sequence comparison of three infectious molecular clones of RD-114 virus. Virus Genes 45, 393-397 (2012).

As described above, it has been clarified that the infectious RD-114 virus may be contaminated in the vaccine produced using the cat-derived strained cells or the like.

At present, the pathogenicity of the RD-114 virus to cats or dogs is not yet clear, and its effects are unknown. However, some cases of developing it with other endogenous retroviruses have been reported, and the risk of developing RD-114 virus in cats and dogs cannot be completely ruled out. Therefore, it is desirable to produce an RD-114 virus-free vaccine that reliably eliminates the possibility of RD-114 virus contamination in the vaccine.

Therefore, an object of the present invention is to provide a novel means for producing a vaccine for RD-114 virus-free cats or dogs.

We have identified six RD-114 virus-related sequences from the cat's genome, and (2) all viruses encoded by those sequences are non-infectious ((1) 3) A non-infectious RD-114 virus-related sequence having an infectious env gene coding sequence region and a non-infectious RD-114 virus-related sequence having no infectious env gene coding sequence region are present in the genome. If endogenous, infectious RD-114 virus particles are produced by gene recombination, and (4) infectious RD by knocking out the infectious env gene of RD-114 virus that is endogenous to the genome. -The production of viral particles can be blocked, (5) therefore, by producing a vaccine using recombinant cells in which the infectious env gene of the RD-114 virus, which is endogenous to the genome, has been knocked out, RD -114 Newly found that virus-free vaccines can be produced.

Therefore, in the present invention, the infectious env gene of the RD-114 virus, which is a recombinant cell based on a cell derived from the genus Cat and is inherent in the genome of the original strain, is artificially knocked out. Provided are genetically modified cells and the like.

As described above, although it is said that there are multiple sequences in the cat's genome that are homologous to the genome sequence of the RD-114 virus, the same sequence as the infectious RD-114 virus has been found in the past. The code region of the infectious RD-114 virus was not identified. On the other hand, the present invention clarifies the production mechanism of infectious RD-114 virus particles by gene recombination, and infectious env gene of RD-114 virus is infected as a virus unless it is highly conserved. While losing sex, we found that the region where the gene was highly conserved was limited in the genome and identified that region. We also found that knocking out the infectious env gene can block the production of infectious RD-114 virus particles. In this way, by identifying the gene to be knocked out in a limited site, it was possible to realize the production of recombinant cells.

In the present invention, by artificially knocking out the infectious env gene of the RD-114 virus inherent in the genome of the original strain, effective rearrangement of infectious virus particles can be prevented even if the virus is expressed. .. Therefore, cells derived from the genus Cat can be made RD-114 virus-free, and further, by applying the recombinant cells to vaccine production for cats or dogs, RD-114 virus-free cats or dogs can be made. Vaccines for cats can be produced.

INDUSTRIAL APPLICABILITY According to the present invention, a vaccine for RD-114 virus-free cats or dogs can be produced.

<Genetically modified cell according to the present invention>
The present invention is a genetically modified cell based on a cell derived from the genus Felis, in which the infectious env gene of the RD-114 virus contained in the genome of the original strain is artificially knocked out. Includes all recombinant cells. The present invention is not narrowly limited to this embodiment.

As described above, this recombinant cell is RD-114 virus-free because the expression of the infectious RD-114 virus is blocked by knocking out the infectious env gene of the RD-114 virus.

The cell that is the original strain before gene recombination may be at least one that contains the infectious env gene of RD-114 virus in the genome, and is basically a cell derived from the genus Felis. It is not narrowly limited to that.

For example, even if the genome of the original strain does not contain the same sequence as the coding sequence of the infectious RD-114 virus, the genome of the original strain contains the coding sequence region of the infectious env gene. If at least one non-infectious RD-114 virus-related sequence having the above and at least one non-infectious RD-114 virus-related sequence having no coding sequence region of the infectious env gene are present, the gene set The replacement may produce the infectious RD-114 virus. Therefore, the present invention also includes all genetically modified cells based on these strains.

The original strain may be, for example, any of a primary cultured cell derived from Felis, a passage cell derived from Felis, and a strained cell derived from Felis. As described above, for example, CRFK cells (Crandell-Rees feline kidney cells), MCC cells (feline large granular lymphoma cells), and FER cells (feline embryonic fibroblasts cells), which are strains derived from the genus Felis, are infected with the RD-114 virus. It produces sex particles and the present invention can be applied.

For example, when the original strain is a CRFK cell, there are three RD-114 virus-related sequences in the genome of the CRFK cell, as described later. Among them, the RD-114 virus-related sequence existing on chromosome C1 is non-infectious, but the coding sequence region of the env gene has 100% homology with the sequence of the molecular clone of the infectious RD-114 virus. ..

As described above, Sc3c strain and CRT1 strain have been reported as infectious RD-114 viruses, for example. These full-length genome sequences are available from publicly known literature. The full-length genome sequence of the CRT1 strain is also registered in GenBank (accession number AB559882.1).

The Sc3c strain is encoded as a sequence of 8,409 bases, which contains the coding regions of the gag gene (positions 947 to 2,599), the pol gene (positions 2,600 to 6,169), and the env gene (positions 6,204 to 7,901). ing. The CRT1 strain is encoded as a sequence of 8,315 bases, which contains the coding regions of the gag gene (positions 900 to 2,552), the pol gene (positions 2,553 to 6,122), and the env gene (positions 6,157 to 7,854). ing. The amino acid sequences encoded by the env genes of the Sc3c and CRT1 strains show 100% homology.

The "RD-114 virus infectious env gene" to be knocked out is endogenous to the genome of cells derived from the genus Cat, shows high homology with the env gene of Sc3c strain or CRT1 strain, and is infectious RD after expression. -114 A gene having the coding sequence of the env gene capable of producing virus particles. In addition, in order for the env gene to retain the ability to produce infectious RD-114 virus particles, in principle, 100% or close homology with the env gene of the Sc3c strain or CRT1 strain is required.

In the present invention, the RD-114 virus-related sequence is, in principle, a sequence that is endogenous to the genome of a cell derived from Felis and has homology with the gene of the RD-114 virus (for example, Sc3c strain, CRT1 strain, etc.). is there. When the RD-114 virus or RD-114-like virus expressed from this sequence is infectious, it is defined as "infectious", and when it is not, it is defined as "non-infectious".

<Method for producing genetically modified cells according to the present invention>
The present invention includes all methods for producing recombinant cells, including a procedure for artificially knocking out the infectious env gene of RD-114 virus contained in the genome of a cell derived from Felis.

The method of knocking out the infectious env gene of the RD-114 virus may be any method of knocking out a specific position of the genome in the cell, and a known method can be widely used. For example, a method using an artificial nuclease such as TALEN (Transcription Activator-Like Effector Nucleases) or zinc finger nuclease, or a method applying a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / CAS system can be adopted.

By artificially knocking out the infectious env gene of the RD-114 virus that is inherent in the genome of Felis-derived cells, the Felis-derived cells can be made RD-114 virus-free.

<Vaccine production method according to the present invention>
The present invention includes all vaccine production methods including a procedure for infecting and amplifying a vaccine virus strain in the above-mentioned recombinant cells.

The vaccine virus strain includes all strains that can be used as a vaccine and that can be propagated in Felis-derived strain cells or the like during vaccine production. For example, attenuated strains such as cat herpesvirus, cat calicivirus, cat panleukopenia virus, canine adenovirus, canine distemper virus, canine parvovirus, canine coronavirus, and canine parainfluenza virus are strains used as vaccines. In addition, since cells derived from the genus Cat can be used in the production of the vaccine, it is included in the applicable range.

For example, a method including a step of infecting the above-mentioned recombinant cells with a virus strain for vaccine, a step of culturing and propagating the virus strain for vaccine, a step of collecting a virus solution, a step of purifying the collected virus solution, and the like. By producing the vaccine, the RD-114 virus-free vaccine can be produced.

<Method for detecting infectious RD-114 virus expression risk according to the present invention>
The present invention determines the presence or absence of the infectious RD-114 virus infectious RD-114 by determining the presence or absence of the infectious env gene of the RD-114 virus in the genome of cells derived from the genus Cat. Widely includes methods for detecting the risk of virus expression.

As described above, even if all the RD-114 virus-related sequences existing in the genome are non-infectious, if they have the coding sequence region of the infectious env gene, they are infectious RD by gene recombination. -114 Viral particles may be produced.

On the other hand, the presence or absence of the infectious RD-114 virus can be determined by determining the presence or absence of the infectious env gene of the RD-114 virus in the genome of a cell derived from Felis.

Whether or not the infectious env gene of the RD-114 virus is present in the genome of a cell derived from the genus Felis can be determined by a known method.

For example, after extracting the genomic DNA, the infectious env gene can be obtained by cleaving at the restriction enzyme site existing on or around the above-mentioned infectious env gene sequence and confirming the DNA fragment length by gel electrophoresis or the like. It may be determined whether or not it exists. In addition, by creating a probe for the sequence of the infectious env gene and detecting whether the extracted genomic DNA or a fragment thereof hybridizes with the probe, it is determined whether or not the infectious env gene exists. You may. In addition, it is determined whether or not the infectious env gene exists by specifically amplifying the sequence of the infectious env gene or its surroundings by a PCR method or the like and confirming the length of the amplified product by gel electrophoresis or the like. May be done. In addition, it may be determined whether or not the infectious env gene is present by acquiring the sequence of the genomic DNA or a fragment thereof and collating whether or not the sequence of the infectious env gene is contained therein.

In Example 1, we attempted to identify the RD-114 virus-related sequence in the genome of a cat.

First, the SSEARCH program was used to search for the RD-114 provirus coding region within the domestic cat genome (Felis_catus_6.2) based on the homology with the complete genome sequence of the CRT strain of the RD-114 virus. did. As a result, the coding region of the provirus having the same sequence as the three clones of the infectious RD-114 virus (pSc3c, pCRT1, pRD-UCL) was not identified, but an incomplete RD- on chromosome C2. A 114 virus-related sequence (RDRS; RD-114 virus-related sequence) was discovered and designated as "RDRS C2a".

In addition, Southern blot analysis was performed to determine whether RD-114 virus-related sequences capable of inducing RD-114 virus were present in the entire cat genome. Genomic DNA extracted from cat-derived cells was cleaved with the limiting enzyme Hind III and hybridized with a gag probe. As a result, CRFK cells, which are RD-114 virus-producing cells, and G355-5 cells, which are RD-114 virus-non-producing cells. A 2.6 kb fragment was detected in (feline fetal astrocyte cell line), fcf-4 cells (Felis catus whole fetus-4 cell line), FeT-J cells (interleukin-2-independent feline T-cell line), etc. .. On the other hand, as a result of cutting genomic DNA extracted from cat-derived cells with the restriction enzyme HindIII and hybridizing with the env probe, a 3.2 kb fragment was detected in CRFK cells, which are RD-114 virus-producing cells, but RD- 114 The band was not detected in non-virus producing cells. In addition, as a result of cleavage with any of the restriction enzymes SacII, EcoRI, and SphI and hybridization with any of the gag probe, pol probe, and env probe, the copy number of the env gene in the RD-114-related sequence is the gag gene or pol. It was found to be lower than the copy number of the gene. This finding was consistent with that of the prior literature.

Therefore, an inverse nested PCR method targeting the env gene of the RD-114 virus was performed, TOPO cloning of the amplified DNA was performed, and then the sequence was examined. As a result, three RD-114 viruses were found in the genome of CRFK cells. A related sequence was identified. In addition to the above-mentioned "RDRS C2a", they were designated as "RDRS A2" and "RDRS C1", respectively, based on the name of the chromosome on which the sequence existed.

Similarly, three RD-114 virus-related sequences were identified from the genomes of primary cultured cells of PBMCs (peripheral blood mononuclear cells) in cats. In addition to the above-mentioned "RDRS C2a", they were designated as "RDRS E3" and "RDRS D4", respectively, based on the name of the chromosome on which the sequence existed.

Similarly, two RD-114 virus-related sequences were identified from the genome of FER cells, which are cells producing the above-mentioned infectious particles of RD-114 virus. In addition to the above-mentioned "RDRS C2a", they were designated as "RDRS C2b" based on the name of the chromosome on which the sequence existed.

As described above, a total of 6 RD-114 virus-related sequences were identified from the genome of cats. None of them had the same sequence as the above three infectious RD-114 virus molecular clones.

FIG. 1 is a diagram schematically showing the full-length genomic structure of the six RD-114 virus-related sequences identified this time. In the figure, Δ is the deletion of nucleic acid, filled Δ is the insertion of nucleic acid, “gag”, “pol” and “env” are the ORF (open reading frame) of each gene, and “LTR” is the long terminal. The repeat region and the numerical value represent the homology with the infectious RD-114 virus clone.

As shown in Figure 1, of these six RD-114 virus-related sequences, four of the RDRS A2, RDRS C1, RDRS D4, and RDRS C2b have the full-length ORF of the env gene, of which RDRS C1, The two env gene coding sequences of RDRS C2b showed 100% homology with the env gene coding sequences in the infectious RD-114 virus clone. In addition, RDRS C1 and RDRS C2b had full-length ORFs (open reading frames) in all three genes, gag, pol, and env.

As a result of Example 1, as shown in FIG. 1, two of RDRS C1 and RDRS C2b have a full-length ORF (open reading frame) in all three genes of gag, pol, and env, and the env gene code. The sequence showed 100% homology with the env gene coding sequence in the infectious RD-114 virus clone. In particular, RDRS C1 was the closest approximation to the coding sequence in the infectious RD-114 virus clone.

Therefore, in Example 2, it was examined whether the RD-114 virus encoded by RDRS C1 was infectious.

RDRS C1 has adenine (a) at position 491 (inside the primer binding site) of pSc3c at position guanine (g) and position 3,748 (pol) compared to the nucleotide sequence of pSc3c, which is a molecular clone of the infectious RD-114 virus. Adenine (a) at position 5 (in the gene) is guanine (g), thymine (t) at position 5,394 (in the pol gene) is cytosine (c), and adenine (a) at position 5,719 (in the pol gene) is guanine (g). ), Each was mutated.

Therefore, first, the DNA sequence of RDRS C1 was incorporated into the pcDNA3.1 plasmid vector to prepare an expression vector of RDRS C1 (pRDRS C1). In addition, using the pRDRS C1 as a template, PrimeSTAR GXL Polymerase (manufactured by TaKaRa) mutated a at position 3,748 to g, t at position 5,394 to c, and a at position 5,719 to g, respectively. A recombinant plasmid vector was prepared.

HEK293T cells were transfected with pRDRS C1 or each recombinant plasmid vector, and after 48 hours, the supernatant was collected and prepared to prepare a sample virus. The virus was applied to target cells (TE671 cells) and a LacZ marker rescue assay was performed, and the infectivity of each virus was examined by the presence or absence of stainability by X-gal staining. A virus (Sc3c) prepared by transfecting pSc3c was used as a positive control.

As a result, the result was negative when the virus prepared by transfecting pRDRS C1 was used as a sample, and it was found that the RD-114 virus encoded by RDRS C1 was not infectious.

In addition, the results when a virus prepared by transfecting a recombinant plasmid vector in which a at position 3,748 was mutated to g and a recombinant plasmid vector in which a at position 5,719 was mutated to g were used as samples. Was negative, whereas the result was positive when a virus prepared by transfecting a recombinant plasmid vector in which t at position 5,394 was mutated to c was used as a sample.

This result suggests that when timine (t) at position 5,394 of the nucleotide sequence of pSc3c, which is a molecular clone of infectious RD-114 virus, is mutated to cytosine (c), it becomes non-infectious and RDRS A mutation in cytosine (c) at position 5,394 in C1 suggests that the RD-114 virus encoded by RDRS C1 is the definitive cause of non-infectiousness.

As mentioned above, CRFK cells have been shown to produce infectious particles of the RD-114 virus. However, in Example 1, it was found that CRFK cells had three RD-114 virus-related sequences of RDRS C2a, RDRS A2, and RDRS C1, but from Example 1 and Example 2, all of these sequences were CRFK. It was found that they did not match the coding sequences of CRT1, which is a cell-derived infectious RD-114 virus, and that those sequences were non-infectious.

Therefore, in Example 3, it is assumed that infectious particles of RD-114 virus are produced by gene recombination in CRFK cells, and infectious RD-114 virus is produced by gene recombination between RDRS A2 and RDRS C1. It was examined whether it was produced.

Similar to RDRS C1, the DNA sequence of RDRS A2 was integrated into the pcDNA3.1 plasmid vector to prepare an expression vector of RDRS A2 (pRDRS A2), and then cotransfected with pRDRS C1 and pRDRS A2 into HEK293T cells. Succeeded in recovering a certain virus.

The virus was applied to target cells (TE671 cells) and a LacZ marker rescue assay was performed to measure the virus titer. The Titor was measured by triplicate.

The result is shown in figure 2. FIG. 2 is a graph showing a virus titer obtained by cotransfecting pRDRS C1 and pRDRS A2. The horizontal axis in the figure represents the number of days (dpi) from the date of virus infection to the measurement date, and the vertical axis represents the virus titer (unit: log ₁₀ ffu / mL). The "A2" fold line is the virus titer obtained by transfecting pRDRS A2 alone, and the "C1" fold line is the virus titer obtained by transfecting pRDRS C1 alone, "A2 + C1". The fold line represents the viral titer obtained by cotransfecting pRDRS C1 and pRDRS A2, and "RD-114" represents the viral titer obtained by transfecting pCRT1 as a positive control.

As shown in FIG. 2, the virus obtained by transfecting pRDRS C1 or pRDRS A2 alone had a low titer and was non-infectious, whereas it was obtained by cotransfecting pRDRS C1 and pRDRS A2. Titor recovered to almost the same level as the known infectious RD-114 virus, CRT1.

Subsequently, as a result of comparing the genomic sequences of the recombinant virus obtained by cotransfecting pRDRS C1 and pRDRS A2 with RDRS C1 and RDRS A2, the 3,748th position was a and the 5,394th position was t in the pol gene. , 6169 was a, which matched the sequence of RDRS A2. On the other hand, in the env gene, position 7,049 was c, which matched the sequence of RDRS C1.

In addition, at position 3,224 in the pol gene, RDRS C1, RDRS A2 and known Sc3c were c, whereas they were mutated to a, and at position 4,357, RDRS C1, RDRS A2 and known Sc3c were c. Was mutated to t. At position 491, RDRS C1 and RDRS A2 were g, whereas they were mutated to a, which was consistent with Sc3c. The base at positions 5,072 to 5,073 was ga, which was consistent with RDRS C1 and Sc3c, and was different from the base (gga) at the same site in RDRS A2. The length of the LTR is 27 bp shorter than that of the RDRS C1.

As a result of comparing the genomic sequences of the recombinant virus obtained by cotransfecting pRDRS C1 and pRDRS A2 with the known pSc3c, all but positions 3,224 and 4,357 are consistent, and the amino acid sequences are replaced. The mutation was only at position 3,224.

As described above, it was clarified that the infectious RD-114 virus was produced by the gene recombination of RDRS C1 and RDRS A2 in this example.

In Example 4, the infectious env gene of the RD-114 virus contained in the genome of CRFK cells, which is a cell derived from the genus Felis, was artificially edited by a method using TALEN to RD-114. Virus knockout CRFK cells were generated.

TALEN is an artificial nuclease that links the DNA-binding domain of the TAL effector with the DNA-cleaving domain of the restriction enzyme FokI. The DNA-binding domain of TAL effector has a repeat structure of about 17 to 18 times in which the amino acid sequence is highly conserved, and each repeating unit (module) is composed of about 34 amino acids. Of the 34 amino acids in each module, the 12th and 13th amino acid residues of each recognize one base in the DNA sequence. Therefore, each module is formed so that the 12th and 13th amino acid residues of each are amino acids that bind to the target base (A, T, G, or C), and these are joined together. This makes it possible to recognize a specific base sequence of about 17 to 18 bp in DNA and form a TAL effector that binds to the recognition sequence. On the other hand, the DNA cleavage domain of the restriction enzyme FokI exhibits DNA double-strand cleavage activity by dimerizing. Therefore, in the target sequence on the base sequence of DNA, TALENs that recognize the upstream (left) and downstream (right) sequences of the target sequence are formed with an interval of about 14 to 20 bases, and each TALEN is formed. Is bound to the upstream side and the downstream side of the target sequence, respectively, and the FokI sites of both TALEN restriction enzymes are dimerized, whereby double-stranded DNA can be cleaved within a specific target sequence. The cleaved DNA double strand is repaired by a mechanism inside the cell, and at that time, a defect or mutation is introduced into the target sequence.

In this example, since the infectious env gene of RD-114 virus contained in the genome of CRFK cells is knocked out by the method using TALEN, the whole genome sequence information (accession number) of the infectious RD-114 virus / CRT1 strain is used. Based on AB559882.1), a set that recognizes the upstream (left) and downstream (right) sequences of the target sequence by targeting positions 6,938 to 6,952 in the env gene coding region in the sequence. Designed TALEN (left TALEN and right TALEN, respectively). Specifically, right TALEN is an antisense strand at positions 6,953 to 6,969 so that left TALEN recognizes the upstream side of the target sequence (positions 6,921 to 6,937, SEQ ID NO: 1) by a known method. TALEN was designed so as to recognize the sequence from the 5'end to the 3'end on the side, SEQ ID NO: 2). The env gene has a SU region (positions 6,157 to 7,278 in the CRT1 strain) and a TM region (positions 7,279 to 7,854). The SU region is a site required for retroviral infection and enables binding to specific receptors, and the interaction between the SU region and the receptor induces structural changes in the TM region and induces fusion with the host cell membrane. (Coffin et al., Retroviruses, 1997, Zhao et al., J. Virol., 1998). Based on this finding, in this example, a target sequence (positions 6,938 to 6,952) was set in the SU region of the infectious env gene. By editing the genome in the SU region of the infectious env gene (partial deletion of the base sequence, etc.) and knocking out the infectious env gene, the infectious env gene can be reliably, effectively and highly efficiently used. There is a possibility that the gene can be knocked out.

The nucleotide sequence encoding each TALEN designed as described above was incorporated into the plasmid vector pcDNA3.1 to prepare a recombinant plasmid vector expressing left TALEN and a recombinant plasmid vector expressing right TALEN, respectively.

Next, CRFK cells (cell number 5x10 ⁵ ) were suspended in a 100 μL R buffer (Neon Transfection system, invitrogen), and 1.8 μg of each TALEN plasmid vector and 0.36 μg of puromycin resistance gene expression vector were electroporated. (Electroporation conditions: pulse voltage: 1400V, pulse width: 20ms, pulse number: 2).

In addition, as a control, a GFP expression vector was transfected for evaluation of transfection efficiency, and a pcDNA3.1 vector was transfected as a negative control (mock) by the same method. As a result, one day after transfection, GFP-positive cells accounted for 90% or more, confirming high transfection efficiency.

Two days after transfection, TALEN-induced mutations were detected by the Surveyor (Cel-I) nuclease assay. Genomic DNA was extracted using the QIAamp DNA Blood Mini kit (QIAGEN, Valencia, CA), and primers of SEQ ID NOs: 3 and 4 (CRT1, 6,897-, respectively) using PrimeSTAR GXL polymerase (TaKaRa, Shiga, Japan). PCR was performed at positions 6,914 and 7,111 to 7,130). The PCR amplification product was heat-denatured, cleaved by Surveyor nuclease (Transgenomic, Omaha, NE, USA), and TALEN-induced mutations were detected by agarose gel electrophoresis. As a result, high mutagenesis efficiency could be confirmed. An agarose gel electrophoresis image was analyzed by ImageJ software (National Institute of Health, Bethesda, MD, USA, Hansen et al., J. Vis. Exp., 2012) and found to be nonhomologous with a probability of 29.32%. It was speculated that end joining, NHEJ) was occurring.

In addition, the PCR product was cloned into a pCR BluntII TOPO plasmid vector (Life technologies, Carlsbad, CA, USA), and sequence analysis of the infectious env gene locus of the RD-114 virus was performed. As a result, 2 out of 10 clones were frameshifted. A shift-related mutation was observed, the open reading frame of the env gene was disrupted, and the mutation transfer rate in CRFK cells was 20%.

After culturing the transfected CRFK cells with the addition of 4 μg / mL Puromycin (InvivoGen, San Diego, CA, USA), the same procedure and conditions as above were applied to the cells in order to increase the mutagenesis efficiency. -Transfection was performed again with the reagent.

Next, a selection of mutagenized CRFK cells was performed. First, CRFK cells subjected to two transfections were seeded on a 10 cm dish, and 4 μg / mL Puromycin was added and cultured. At that time, AH927 cells were co-cultured as feeder cells in order to support the growth of immature colonies. In addition, AH927 cells do not carry the locus of the RD-114 virus-infected env gene on their genome. During the culture, the medium supplemented with puromycin was replaced every 3 days. One month after the start of co-culture with AH927 cells, 31 single cell-derived clones were picked up and separated and cultured on a 96-well plate. Two days later, the cell colonies were transferred to a 24-well plate, some cells were collected, their genomic DNA was extracted, PCR was performed with the same primers as above, and the sequence analysis of the PCR products was performed. As a result, mutations were introduced in the targeted env gene in one of the 31 clones. In this clone, the env gene in the sequence of RDRS A2 in the genome of CRFK cells, the insertion of 4 bases in one allele and the deletion of 5 bases in the other allele, the env gene in the sequence of RDRS C1 A deletion of 4 bases in one allele and a deletion of 918 bases in the other allele (a total of 982 bases were deleted in succession by adding 64 bases after the env gene) were confirmed. For this clone, in order to increase the purity, the cell cloning process was repeated again, and the obtained cells were designated as RD-114 virus knockout CRFK cells.

In Example 5, it was evaluated whether the production of RD-114 virus particles was suppressed in the RD-114 virus knockout CRFK cells.

The RD-114 virus knockout CRFK cells prepared in Example 4 were cultured for 3 days, the culture supernatant was collected, filtered and sterilized with a 0.45 μm filter, treated with DNase I (Roche Diagnostics, Indianapolis, IN), and then cultured. The number of copies of RD-114 viral RNA in the supernatant was measured by Real-Time RT-PCR. In Real-Time RT-PCR, TaKaRa Ex Taq HS DNA polymerase (TaKaRa) is used, and the Taqman probe has the sequence of CRT1 at positions 6,938 to 6,952 (the sequence between the DNA binding sequences of both TALENs; SEQ ID NO: 5). The primers used were SEQ ID NO: 6 (sequence at positions 6,857 to 6,876 of CRT1) and SEQ ID NO: 7 (sequence at positions 6,986 to 7,005). As a control, untreated CRFK cells were used, and the copy number of RD-114 viral RNA in the culture supernatant was measured by the same procedure.

As a result, RD-114 virus particles of 4.41 × 10 ⁶ copies / mL or more were detected at the RNA level in untreated CRFK cells (control), whereas RD-114 in RD-114 virus knockout CRFK cells. Virus particle production was below the detection limit.

Subsequently, the titers of the RD-114 virus produced and released from the RD-114 virus knockout CRFK cells were evaluated by a focus assay (Sakaguchi et al., J Vet Med Sci., 2008). QN10S cells (1 × 10 ⁴ ) were seeded on a 24-well plate, and one day later, the culture supernatant of RD-114 virus knockout CRFK cells (filtered with a 0.45 μm filter) was applied to QN10S cells in the presence of 8 μg / mL of polybrene. Inoculated. Seven days after inoculation, the number of focal points was measured. As a control, untreated CRFK cells were also subjected to a focus assay in the same procedure using the culture supernatant.

As a result, in the focus assay, the titer of the culture supernatant of untreated CRFK cells was 3.25 × 10 ³ ffu / mL, whereas in the RD-114 virus knockout CRFK cells, the infectious RD- The production of 114 virus particles was strongly suppressed to levels below the detection limit.

As described above, it was demonstrated from this example that the production of RD-114 virus particles was suppressed in the RD-114 virus knockout CRFK cells prepared in Example 4.

In Example 6, the viability of RD-114 virus knockout CRFK cells was examined.

The RD-114 virus knockout CRFK cells prepared in Example 4 and the untreated CRFK cells as controls were seeded on 96-well plates (1 × 10 ⁵ / well), cultured for 2 days, and then the Cell Proliferation Kit. Absorbance at a wavelength of 595 nm was measured with Wallac 1420 ARVOsx (Perkin Elmer Life Sciences, Boston, MA) using I (MTT) (Roche) according to the attached protocol.

As a result, the absorbances of the RD-114 virus knockout CRFK cells and the untreated CRFK cells were 1.077 and 1.105, respectively, which were equivalent. From this, the RD-114 virus knockout CRFK cells prepared in Example 4 have the same level of cell viability as the untreated CRFK cells, and even if the RD-114 virus is knocked out using TALEN, It was shown to have no effect on cell proliferation.

In Example 7, the proliferation of cat parvovirus in RD-114 virus knockout CRFK cells was examined.

As a test virus strain of cat parvovirus, a feline panleukopenia virus (FPLV, [feline parvovirus]) V142 strain was used. The RD-114 virus knockout CRFK cells prepared in Example 4 and the untreated CRFK cells as controls were inoculated with the FPLV V142 strain at MOI (multiplicity of infection) = 0.1, and after sensitization at 37 ° C. for 1 hour, The cells were washed 3 times with PBS and cultured with 2 mL of medium. Then, 2, 4 and 6 days after the sensitization, the culture supernatant was collected as a virus solution and stored at -80 ° C.

The titers of the recovered parvovirus were measured using FL74 cells (Ikeda et al., J Vet Med Sci., 1998, Sassa et al., J Vet Med Sci., 2006). FL74 cells were seeded on a 96-well plate, the collected virus solution was thawed, and 100 μL of each well was inoculated. One week later, the presence or absence of CPE was determined, and TCID ₅₀ / mL was calculated by the Reed & Muench formula.

As a result, the titers of parvovirus recovered from RD-114 virus knockout CRFK cells were 6.26 × 10 ² TCID ₅₀ / mL 2 days after sensitization and 1.57 × 10 ⁵ TCID ₅₀ / mL 4 days after sensitization. , the 6 days was 1.78 × 10 ⁷ TCID ₅₀ / mL in later ones, whereas the titer of parvovirus recovered from CRFK cells _{^{untreated, 3.16 × 10 2 TCID 50 /}} mL in those after sensitization 2 days , 6.26 × 10 ⁴ TCID ₅₀ / mL after 4 days, 3.03 × 10 ⁶ TCID ₅₀ / mL after 6 days, and the proliferation of cat parvovirus was almost the same in both cells. It was.

In Example 8, the proliferation of feline calicivirus in RD-114 virus knockout CRFK cells was examined.

As a test virus strain of feline calicivirus, feline calicivirus (FCV) 01-106 strain was used. The RD-114 virus knockout CRFK cells prepared in Example 4 and the untreated CRFK cells as controls were inoculated with the FCV 01-106 strain at MOI = 0.1 or 0.01, sensitized at 37 ° C. for 1 hour, and then PBS. The cells were washed 3 times with and cultured in 2 mL of medium. Then, 2 days after the sensitization, the culture supernatant was collected as a virus solution and stored at -80 ° C.

The titer of the recovered feline calicivirus was measured using CRFK cells in the same procedure as in Example 7.

As a result, the titers of the virus recovered from the RD-114 virus knockout CRFK cells were 1.06 × 10 ⁸ TCID _{50 /} mL inoculated with MOI = 0.1 and 3.99 × 10 ⁷ TCID ₅₀ inoculated with MOI = 0.01. On the other hand, the titers of calicivirus recovered from untreated CRFK cells were 2.95 × 10 ⁸ TCID ₅₀ / mL inoculated with MOI = 0.1 and 4.67 × 10 inoculated with MOI = 0.01. ⁸ TCID was ₅₀ / mL. From this result, the proliferation of feline calicivirus in RD-114 virus knockout CRFK cells was lower than that in untreated CRFK cells, but feline calicivirus was sufficiently propagated in RD-114 virus knockout CRFK cells. I found out.

In Example 9, the proliferation of feline herpesviruses in RD-114 virus knockout CRFK cells was examined.

As a test virus strain of feline herpesvirus, feline viral rhinotracheitis virus (FVRV, [feline herpes virus type 1; FHV-1) 00-015 strain was used. RD-114 virus knockout CRFK cells prepared in Example 4, and, in CRFK cells untreated as a control, with respect to a cell count of 2 × 10 ⁵ were inoculated with 1,000-fold or 10,000-fold diluted virus solution 50 [mu] L, sensitization 4 days Later, the culture supernatant was collected as a virus solution and stored at -80 ° C.

The titer of the recovered herpesvirus was measured using CRFK cells in the same procedure as in Example 7.

As a result, the titers of the virus recovered from the RD-114 virus knockout CRFK cells were 2.64 × 10 ⁷ TCID ₅₀ / mL for the 1,000-fold dilution and 2.73 × 10 ⁸ TCID ₅₀ / mL for the 10,000-fold dilution. On the other hand, the titers of herpesvirus recovered from untreated CRFK cells were 8.44 × 10 ⁷ TCID ₅₀ / mL for 1,000-fold dilution and 1.53 × 10 ⁸ TCID ₅₀ / mL for 10,000-fold dilution. In both cells, the proliferation of the feline herpesvirus virus was about the same.

As described above, as shown in Examples 7 to 9, in the RD-114 virus knockout CRFK cells, the cat parvovirus, the feline calicivirus, and the feline herpesvirus are sufficiently proliferated as in the untreated CRFK cells. It has been shown. Based on these results and the results of Example 5 above, the RD-114 virus knockout CRFK cells are useful as strain cells for producing a live attenuated vaccine against viral infections in dogs and cats, and are infectious to the vaccine. It is shown that the contamination of infectious particles of RD-114 virus can be suppressed as much as possible, that is, the RD-114 virus-free vaccine can be produced by using RD-114 virus knockout CRFK cells.

The figure which schematically showed the full-length genomic structure of the 6 RD-114 virus-related sequences identified this time in Example 1. The graph which shows the titer of the virus obtained by cotransfecting pRDRS C1 and pRDRS A2 in Example 3. FIG.

Claims (5)

It is a genetically modified cell based on a cell derived from the genus Felis.
A non-infectious RD-114 virus-related sequence having an infectious env gene coding sequence region in the genome of the original strain, and a non-infectious RD-114 virus having no infectious env gene coding sequence region. At least one related sequence is inherent and
A recombinant cell in which the infectious env gene in a non-infectious RD-114 virus-related sequence having a coding sequence region of the infectious env gene is artificially knocked out. The genetically modified cell according to claim 1, wherein the original strain is a CRFK cell. A vaccine production method comprising a procedure for infecting and amplifying a virus strain for vaccine in the recombinant cell according to claim 1 or 2 . A non-infectious RD-114 virus-related sequence having an infectious env gene coding sequence region in the genome of a cell derived from the genus Cat , and a non-infectious RD-114 having no infectious env gene coding sequence region. 114 virus-related sequences are inherent at least one and
The presence or absence of the infectious RD-114 virus can be determined by determining the presence or absence of the infectious env gene in the non-infectious RD-114 virus-related sequence having the coding sequence region of the infectious env gene. Infectious RD-114 virus expression risk detection method to be determined. A non-infectious RD-114 virus-related sequence having an infectious env gene coding sequence region in the genome of a cell derived from the genus Cat , and a non-infectious RD-114 having no infectious env gene coding sequence region. 114 virus-related sequences are inherent at least one and
A method for producing a recombinant cell, which comprises a procedure for artificially knocking out the infectious env gene in a non-infectious RD-114 virus-related sequence having a coding sequence region of the infectious env gene.

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