JP2016135120A - Transgenic cells and vaccine production method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide novel means for producing RD-114 virus-free feline or canine vaccine.SOLUTION: A transgenic cells using Felis-derived cells as an original strain is obtained by artificially knocking out infection env genes of RD-114 virus inherent in the genome of the original strain. By artificially knocking out the infection env gene of RD-114 virus inherent in the genome of the original strain, the Felis-derived cells can be transformed into RD-114 virus-free cells, and further, by applying the transgenic cells to feline or canine vaccine production, RD-114 virus-free feline or canine vaccines can be produced.SELECTED DRAWING: Figure 1

Description

  The present invention is a genetically engineered cell based on a cell derived from a cat genus, wherein the infectious env gene of the RD-114 virus contained in the genome of the original strain is artificially knocked out. Recombinant cells, vaccine production methods using these cells, infectious RD-114 virus expression risk detection method for determining the presence or absence of infectious RD-114 virus expression, genetic recombinant cell production methods, etc. To do.

  As one of vaccine production methods, vaccine production by cell culture has been widely put into practical use. Vaccine production against feline herpes virus, feline calicivirus, feline panleukopenia virus, canine adenovirus, canine distemper virus, canine parvovirus, canine coronavirus, canine parainfluenza virus, etc. In the production of feline, a cell line derived from a cat genus such as CRFK cells (Crandell-Rees feline kidney cells) is used.

  Endogenous retroviruses (ERVs) are retroviruses that are endogenously integrated into a part of the mammalian genome by insertion of genes into the ancestral germ line during retroviral infection in ancient times. It is. About 10% of endogenous retroviruses, which are traces of past retrovirus infections, are present in the mammalian genome. Most of them have lost their ability to replicate and infect as viruses due to accumulation of mutations and deletions of genomic DNA that have occurred over many years, but some endogenous retroviruses retain infectivity. Some cases have been reported.

  The domestic cat has an infectious endogenous retrovirus called the RD-114 virus. Like other retroviruses, the genome of the RD-114 virus in the feline genome contains a gag gene that encodes a structural protein and reverse transcription in a region between two long terminal repeats (LTRs) that have promoter activity. It has a pol gene encoding an enzyme, an env gene encoding an envelope protein, and the like.

  Regarding RD-114 virus, in Non-Patent Document 1, as a knowledge at the time, there were about 20 copies of sequences homologous to RD-114 virus in the genome of domestic cats, and in the coding region of gag gene and pol gene, It was reported that most of the env gene was greatly deleted, while the restriction enzyme sites of the env gene were conserved. In the same document, an infectious RD-114 virus was identified (then named “Sc3c”) and its molecular clone was created (then 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 feline-derived cell lines, produce infectious particles of the RD-114 virus. Was reported. Non-Patent Document 3 reports that infectious particles of infectious RD-114 virus are mixed in several live attenuated vaccines for cats and dogs produced using cat-derived cell lines. It was done.

  On the other hand, although the pathogenicity of the RD-114 virus to cats or dogs is not yet clear, in the aforementioned non-patent document 2, that some cat-derived cell lines are sensitive to the RD-114 virus, Non-Patent Document 4 reported that RD-114 virus can infect and proliferate in canine primary cultured cells and canine-derived cell lines.

In addition, in Non-Patent Document 5, a CRT strain that is an infectious RD-114 virus isolated from CRFK cells was identified, and its molecular clone pCRT1 was prepared, and the gene sequence of pCRT1 was RD-114. It was reported that it was almost identical to that of pSc3c, which was a clone when the virus was first identified, and that there was no substitution 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, and pRD-UCL) of infectious RD-114 virus were compared.
Reeves, RH &O'Brien, SJ Molecular genetic characterization of the RD-114 gene family of inherent 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 inherent 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 principal 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 revealed that infectious RD-114 virus may be mixed in a vaccine produced using a cat-derived cell line.

  Currently, the pathogenicity of the RD-114 virus to cats or dogs is not yet clear, and its effects are unknown. However, several cases of other endogenous retroviruses have been reported, and RD-114 virus cannot completely eliminate the risk of developing in cats and dogs. 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 cats or dogs free of RD-114 virus.

  The inventors have identified (1) six RD-114 virus-related sequences from the genome of the cat, and (2) that the viruses encoded by these sequences are all non-infectious, ( 3) Non-infectious RD-114 virus-related sequence having the coding sequence region of infectious env gene and non-infectious RD-114 virus-related sequence having no coding sequence region of infectious env gene If it is endogenous, infectious RD-114 virus particles are produced by genetic recombination, and (4) infectious RD gene is knocked out by knocking out the infectious env gene of RD-114 virus contained in the genome. -114 virus production can be blocked, (5) Therefore, by producing a vaccine using a recombinant cell in which the infectious env gene of RD-114 virus endogenously contained in the genome is knocked out, RD -114 Made virus-free vaccine I found out that it can be made.

  Therefore, in the present invention, a genetically-recombinant cell having a feline-derived cell as the original strain, and the infectious env gene of the RD-114 virus endogenously contained in the genome of the original strain is artificially knocked out. Provided genetically modified cells and the like.

  As mentioned above, the same sequence as the infectious RD-114 virus has been discovered in the past even though there are multiple sequences in the feline genome that have homology with the genome sequence of the RD-114 virus. The coding region of the infectious RD-114 virus has not been identified. In contrast, the present invention elucidates the production mechanism of infectious RD-114 virus particles by genetic recombination, and the infectious env gene of RD-114 virus is not highly conserved and is infected as a virus. While losing sex, we found that the region where the gene was highly conserved was limited in the genome, and identified the region. In addition, we found that knocking out the infectious env gene can prevent the production of infectious RD-114 virus particles. As described above, the gene to be knocked out could be specified in a limited site, and thus it was possible to realize the production of genetically modified cells.

  In the present invention, by effectively knocking out the infectious env gene of the RD-114 virus inherent in the genome of the original strain, effective reconstitution 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 this genetically modified cell to the production of vaccines for cats or dogs, RD-114 virus-free cats or dogs Vaccines can be manufactured.

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

<Gene-modified cell according to the present invention>
The present invention relates to a genetically modified cell whose original strain is a cell derived from a cat genus, wherein 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. Note that the present invention is not limited to this embodiment.

  As described above, this recombinant cell is knocked out of the infectious env gene of RD-114 virus to prevent the expression of infectious RD-114 virus and is free of RD-114 virus.

  The cell to be the original strain before genetic recombination only needs to have at least the RD-114 virus infectious env gene in its genome, and in principle is a cell derived from a cat genus, It is not limited to it narrowly.

  For example, even if the genome of the original cell 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. A non-infectious RD-114 virus-related sequence having a non-infectious RD-114 virus-related sequence that does not have a coding sequence region of the infectious env gene. This may produce infectious RD-114 virus. Therefore, the present invention encompasses all genetically modified cells based on these strains.

  The original strain may be, for example, a primary cultured cell derived from a cat genus, a passage cell derived from a cat genus, or a cell line derived from a cat genus. 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 feline-derived cell lines, are infected with the RD-114 virus. 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 present 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, for example, the Sc3c strain and the CRT1 strain have been reported as infectious RD-114 viruses. These full-length genome sequences can be obtained from known literature. The full-length genome sequence of CRT1 strain is also registered in GenBank (accession number AB559882.1).

  The Sc3c strain is encoded as a sequence of 8,409 bases, which includes the coding regions of the gag gene (947 to 2,599 positions), the pol gene (2,600 to 6,169 positions), and the env gene (6,204 to 7,901 positions). ing. CRT1 strain is encoded as a sequence of 8,315 bases, including the coding region of gag gene (positions 900-2,552), pol gene (positions 2,553-6,122), and env gene (positions 6,157-7,854) ing. The amino acid sequences encoded by the env genes of the Sc3c strain and the CRT1 strain 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 the Sc3c strain or CRT1 strain, and is infectious RD after expression. -114 A gene having a coding sequence of env gene capable of producing virus particles. In order to retain the ability of the env gene 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 a sequence that is inherent in the genome of a cell derived from a cat genus and has homology with a gene of 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 referred to as “infectious”, and when it is not, it is referred to as “noninfectious”.

<Method for Producing Genetically Modified Cell According to the Present Invention>
The present invention encompasses all methods for producing a recombinant cell, including a procedure for artificially knocking out the infectious env gene of the RD-114 virus endogenously contained in the genome of a cell derived from a cat genus.

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

  By artificially knocking out the infectious env gene of the RD-114 virus endogenously contained in the genome of the cat-derived cell, the cat-derived cell can be made free of RD-114 virus.

<Method for producing vaccine according to the present invention>
The present invention includes all vaccine production methods including a procedure for infecting and amplifying the above-described genetically modified cells with a virus strain for vaccine.

  The virus strains for vaccines include all strains that can be used as vaccines and can grow on feline-derived cell lines during vaccine production. For example, attenuated strains such as feline herpes virus, feline calicivirus, feline panleukopenia virus, canine adenovirus, canine distemper virus, canine parvovirus, canine coronavirus, canine parainfluenza virus are strains used as vaccines Moreover, since a feline genus-derived cell line can be used during vaccine production, it is included in the scope of application.

  For example, in a method including the step of infecting the above-described genetically modified cells with a virus strain for vaccine, the step of culturing and growing the virus strain for vaccine, the step of recovering the virus solution, the step of purifying the recovered virus solution, etc. By producing a vaccine, an RD-114 virus-free vaccine can be produced.

<Infectious RD-114 virus expression risk detection method according to the present invention>
The present invention determines the presence or absence of infectious RD-114 virus infectivity RD-114 by determining the presence or absence of an infectious env gene of RD-114 virus in the genome of a cat-derived cell. Widely includes virus expression risk detection methods.

  As described above, even when all of the RD-114 virus-related sequences present in the genome are non-infectious, in the case of having the coding sequence region of the infectious env gene, infectious RD -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 the cat-derived cell.

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

  For example, after the genomic DNA is extracted, the infectious env gene is cleaved at the restriction enzyme site present on or around the above-mentioned infectious env gene sequence, and the DNA fragment length is confirmed by gel electrophoresis or the like. It may be determined whether or not it exists. In addition, by making a probe of the sequence of the infectious env gene and detecting whether the extracted genomic DNA or fragment thereof hybridizes with the probe, it is determined whether the infectious env gene is present. May be. In addition, the presence or absence of the infectious env gene is determined by specifically amplifying the sequence of the infectious env gene or its surroundings by PCR, etc., and confirming the length of the amplified product by gel electrophoresis or the like. May be performed. In addition, it may be determined whether or not an infectious env gene is present by obtaining a sequence of genomic DNA or a fragment thereof and checking whether or not the sequence of the infectious env gene is contained therein.

  In Example 1, an attempt was made to identify an RD-114 virus-related sequence from the feline genome.

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

  Further, by Southern blot analysis, it was examined whether RD-114 virus-related sequences capable of inducing RD-114 virus exist in the whole feline genome. As a result of digesting genomic DNA extracted from cat-derived cells with restriction enzyme HindIII and hybridizing with gag probe, CRFK cells that are RD-114 virus-producing cells and G355-5 cells that are non-RD-114 virus-producing cells (Feline fetal astrocyte cell line), fcwf-4 cell (Felis catus whole fetus-4 cell line), FeT-J cell (interleukin-2-independent feline T-cell line), etc., 2.6kb fragment was detected . On the other hand, genomic DNA extracted from cat-derived cells was cleaved with restriction enzyme HindIII and hybridized with the env probe. As a result, a 3.2 kb fragment was detected in CRFK cells, which are RD-114 virus producing cells. The band was not detected in 114 virus non-producing cells. In addition, as a result of cleaving with any of the restriction enzymes SacII, EcoRI, SphI and hybridizing 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 gag gene or pol It was found to be lower than the gene copy number. This finding was consistent with the findings of the prior literature.

  Therefore, we performed an inverse nested PCR method targeting the env gene of the RD-114 virus, performed TOPO cloning of the amplified DNA, and examined its sequence. As a result, we found three RD-114 viruses from the genome of CRFK cells. Related sequences were identified. These were named “RDRS A2” and “RDRS C1” based on the name of the chromosome in which the sequence existed in addition to the above “RDRS C2a”.

  Similarly, three RD-114 virus-related sequences were identified from the genome of primary cultured cells of feline PBMCs (peripheral blood mononuclear cells). These were named “RDRS E3” and “RDRS D4” based on the name of the chromosome in which the sequence existed in addition to the above-mentioned “RDRS C2a”.

  Similarly, two RD-114 virus-related sequences were identified from the genome of FER cells, which are cells producing the infectious particles of RD-114 virus described above. These were named “RDRS C2b” based on the name of the chromosome in which the sequence existed in addition to the above “RDRS C2a”.

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

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

  As shown in FIG. 1, of these six RD-114 virus-related sequences, four of 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 sequence in the infectious RD-114 virus clone. In addition, RDRS C1 and RDRS C2b had full-length ORF (open reading frame) 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 full length ORF (open reading frame) in all three genes of gag, pol, and env, and 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 most closely approximated 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 is infectious.

  Compared with the base sequence of pSc3c, a molecular clone of infectious RD-114 virus, RDRS C1 has an adenine (a) at position 491 (within the primer binding site) of pSc3c at position 3,748 (pol) Adenine (a) in the gene) is guanine (g), thymine (t) at position 5,394 (in pol gene) is cytosine (c), and adenine (a) in position 5,719 (in pol gene) is guanine (g ), Respectively.

  Therefore, first, the RDRS C1 DNA sequence was incorporated into the pcDNA3.1 plasmid vector to prepare an RDRS C1 expression vector (pRDRS C1). In addition, the pRDRS C1 was used as a template, and PrimeSTAR GXL Polymerase (TaKaRa) was used to mutate three kinds of a at 3,748 position a to g, 5,394 position t to c, and 5,719 position a to g. A gene 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 as 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 based on the presence or absence of staining by X-gal staining. As a positive control, a virus (Sc3c) prepared by transfecting pSc3c was provided.

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

  In addition, as a sample, a virus was prepared by transfecting a genetically modified plasmid vector in which a at position 3,748a was mutated to g, and a genetically modified plasmid vector in which a at position 5,719 was mutated to g. Was negative, but when the virus was prepared by transfecting a genetically modified plasmid vector in which t at position 5,394 was mutated to c, the result was positive.

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

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

  Therefore, in Example 3, it is assumed that infectious particles of RD-114 virus are produced by genetic recombination in CRFK cells, and infectious RD-114 virus is produced by genetic 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 incorporated into the pcDNA3.1 plasmid vector to create an RDRS A2 expression vector (pRDRS A2), and then cotransfected with HERD293T cells with pRDRS C1 and pRDRS A2. We succeeded in recovering the virus.

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

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

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

  Subsequently, the genome sequence of the recombinant virus obtained by co-transfecting pRDRS C1 and pRDRS A2 was compared with RDRS C1 and RDRS A2. As a result, in the pol gene, position 3,748 was a and position 5,394 was t. , Position 6169 was a, consistent with 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 c at the known Sc3c were mutated to a, and at position 4,357, it was c at RDRS C1, RDRS A2, and the known Sc3c. Mutated to t. At position 491, it was g in RDRS C1 and RDRS A2, but 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 different from the base (gga) at the same site in RDRS A2. The length of LTR is 27 bp shorter than RDRS C1.

  As a result of comparing the genome sequence of the recombinant virus obtained by cotransfecting pRDRS C1 and pRDRS A2 with the known pSc3c, all except for positions 3,224 and 4,357 are identical, and the amino acid sequence is replaced. The mutation was only at position 3,224.

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

  In Example 4, the infectious env gene of the RD-114 virus inherent in the genome of the CRFK cell, which is a feline-derived cell line, was artificially edited by a method using TALEN, whereby RD-114 Viral knockout CRFK cells were generated.

  TALEN is an artificial nuclease in which the DNA binding domain of TAL effector and the DNA cleavage domain of restriction enzyme FokI are linked. The DNA binding domain of TAL effector has a repeat structure of about 17-18 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, each 12th and 13th amino acid residue recognizes one base in the DNA sequence. Therefore, each module is formed so that each 12th and 13th amino acid residue becomes an amino acid that binds to the target base (A, T, G, or C), and they are joined together. Thus, a TAL effector that recognizes a specific base sequence of about 17 to 18 bp in DNA and binds to the recognition sequence can be formed. On the other hand, the DNA cleavage domain of the restriction enzyme FokI exhibits DNA double-strand cleavage activity by dimerization. Therefore, in the target sequence on the DNA base sequence, TALENs that recognize upstream (left) and downstream (right) sequences of the target sequence are formed with an interval of about 14 to 20 bases. Is bound to the upstream side and downstream side of the target sequence, respectively, and the restriction enzyme FokI sites of both TALENs are dimerized, whereby the double-stranded DNA can be cleaved within the specific target sequence. The cleaved DNA duplex is repaired by a mechanism inherent in the cell, and a deletion / mutation is introduced into the target sequence.

  In this example, in order to knock out the infectious env gene of the RD-114 virus endogenous to the genome of CRFK cells by the method using TALEN, the entire genome sequence information (accession number) of the infectious RD-114 virus / CRT1 strain was used. Based on AB559882.1), a set of sequences that recognizes the upstream (left) and downstream (right) sequences of the target sequence at positions 6,938-6,952 in the env gene coding region as the target sequence TALEN (left TALEN and right TALEN, respectively) was designed. Specifically, right TALEN recognizes the upstream side of the target sequence (positions 6,921 to 6,937, SEQ ID NO: 1), and the right TALEN is located downstream of the target sequence (positions 6,953 to 6,969) by known methods. Each TALEN was designed to recognize a sequence from the 5 ′ end to the 3 ′ end, SEQ ID NO: 2). The env gene has an 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 necessary for retroviral infection and enables binding to a specific receptor, 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. The infectious env gene can be reliably, effectively and highly efficiently constructed by knocking out the infectious env gene by editing the genome in the SU region of the infectious env gene (partial deletion of the base sequence, etc.). It may be possible to knock out the gene.

  The base sequence encoding each TALEN designed as described above was incorporated into the plasmid vector pcDNA3.1, and a recombinant plasmid vector expressing left TALEN and a recombinant plasmid vector expressing right TALEN were prepared.

Next, CRFK cells (5 × 10 ⁵ cells) were suspended in 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: 1400 V, pulse width: 20 ms, pulse number: 2).

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

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

  In addition, the PCR product was cloned into pCR BluntII TOPO plasmid vector (Life technologies, Carlsbad, CA, USA) and sequence analysis of the RD-114 virus infectious env locus was performed. Mutation with a shift was observed, the open reading frame of the env gene was destroyed, and the mutation introduction rate in CRFK cells was 20%.

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

  Next, selection of mutation-introduced CRFK cells was performed. First, CRFK cells that had been transfected twice were seeded in a 10 cm dish, and 4 μg / mL Puromycin was added and cultured. At that time, AH927 cells were co-cultured as feeder cells to help the growth of immature colonies. AH927 cells do not have the RD-114 virus-infected env gene locus on their genome. During the culture, the medium supplemented with Puromycin was changed 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 in 96-well plates. Two days later, the cell colony was transferred to a 24-well plate, a part of the cells was collected, the genomic DNA was extracted, PCR was performed with the same primers as above, and the PCR product was analyzed for sequence. As a result, mutation introduction in the targeted env gene was observed 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 Thus, a deletion of 4 bases in one allele and a deletion of 918 bases in the other allele (addition of 64 bases after the env gene and deletion of 982 bases in total) were confirmed. For this clone, in order to increase the purity, the cell cloning process was repeated again, and the resulting cells were designated as RD-114 virus knockout CRFK cells.

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

  The RD-114 virus knockout CRFK cells prepared in Example 4 were cultured for 3 days, and the culture supernatant was collected, sterilized by filtration with a 0.45 μm filter, treated with DNase I (Roche Diagnostics, Indianapolis, IN), and then cultured. The copy number 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 Taqman probe has a sequence of positions 6,938-6,952 of CRT1 (sequence between both TALEN DNA binding sequences; SEQ ID NO: 5) And primers having SEQ ID NO: 6 (sequence of positions 6,857 to 6,876 of CRT1) and SEQ ID NO: 7 (sequence of positions 6,986 to 7,005) were used. As a control, untreated CRFK cells were used to measure the copy number of RD-114 viral RNA in the culture supernatant in the same procedure.

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

Subsequently, the titer of RD-114 virus produced and released from RD-114 virus knockout CRFK cells was evaluated by a focus assay (Sakaguchi et al., J Vet Med Sci., 2008). QN10S cells (1 × 10 ⁴ ) were seeded in 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 transferred to QN10S cells in the presence of 8 μg / mL of polybrene. Vaccinated. Seven days after inoculation, the number of focus was measured. As a control, untreated CRFK cells were subjected to a focus assay in the same procedure using the culture supernatant.

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

  As described above, this example demonstrated that RD-114 virus particle production 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 cell prepared in Example 4 and the untreated CRFK cell as a control were seeded in a 96-well plate (1 × 10 ⁵ / well) and cultured for 2 days, followed by Cell Proliferation Kit Absorbance at a wavelength of 595 nm was measured with Wallac 1420 ARVOsx (PerkinElmer Life Sciences, Boston, Mass.) Using I (MTT) (Roche) according to the attached protocol.

  As a result, the absorbances of RD-114 virus knockout CRFK cells and untreated CRFK cells were 1.077 and 1.105, respectively, which were equivalent. From this, the RD-114 virus knockout CRFK cells produced in Example 4 are at the same level as the untreated CRFK cells in cell viability, and even when RD-114 virus is knocked out using TALEN, It was shown not to affect cell proliferation.

  In Example 7, feline parvovirus growth in RD-114 virus knockout CRFK cells was examined.

  As a test virus strain of feline parvovirus, feline panleukopia virus (FPLV, [feline parvovirus]) V142 strain was used. RD-114 virus knockout CRFK cells prepared in Example 4 and untreated CRFK cells as a control were inoculated with FPLV V142 strain at MOI (multiplicity of infection) = 0.1, and sensitized at 37 ° C. for 1 hour, The plate was 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., respectively.

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

As a result, the titer of parvovirus recovered from RD-114 virus knockout CRFK cells was 6.26 × 10 ² TCID ₅₀ / mL after 2 days of sensitization and 1.57 × 10 ⁵ TCID ₅₀ / mL after 4 days of 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 After 6 days, 6.26 × 10 ⁴ TCID ₅₀ / mL, and after 6 days, 3.03 × 10 ⁶ TCID ₅₀ / mL. The proliferation of feline parvovirus was almost the same in both cells. It was.

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

  Feline calicivirus (FCV) 01-106 was used as a test virus strain for feline calicivirus. The RD-114 virus knockout CRFK cell prepared in Example 4 and the untreated CRFK cell as a control were inoculated with FCV 01-106 strain at MOI = 0.1 or 0.01, and sensitized at 37 ° C. for 1 hour, then PBS Was washed 3 times and cultured with 2 mL of medium. Two 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 in the same procedure as in Example 7 using CRFK cells.

As a result, the titer of virus recovered from RD-114 virus knockout CRFK cells was 1.06 × 10 ⁸ TCID _{50 /} mL inoculated at MOI = 0.1 and 3.99 × 10 ⁷ TCID ₅₀ inoculated at MOI = 0.01. On the other hand, the potency of calicivirus recovered from untreated CRFK cells was 2.95 × 10 ⁸ TCID ₅₀ / mL inoculated at MOI = 0.1 and 4.67 × 10 inoculated at MOI = 0.01. ⁸ TCID ₅₀ / mL. The results indicate that feline calicivirus growth in RD-114 virus knockout CRFK cells was lower than that in untreated CRFK cells, but feline calicivirus also grew sufficiently in RD-114 virus knockout CRFK cells. I understood.

  In Example 9, feline herpesvirus proliferation 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 untreated CRFK cells as a control were inoculated with 50 μL of 1,000-fold or 10,000-fold diluted virus solution for 2 × 10 ⁵ cells, and sensitized for 4 days. Later, the culture supernatant was collected as a virus solution and stored at −80 ° C., respectively.

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

As a result, the virus titer recovered from RD-114 virus knockout CRFK cells was 2.64 × 10 ⁷ TCID ₅₀ / mL at 1,000-fold dilution and 2.73 × 10 ⁸ TCID ₅₀ / mL at 10,000-fold dilution. On the other hand, the titer of herpesvirus recovered from untreated CRFK cells is 8.44 × 10 ⁷ TCID ₅₀ / mL with 1,000-fold dilution, 1.53 × 10 ⁸ TCID ₅₀ / mL with 10,000-fold dilution, In both cells, the feline herpesvirus virus growth was almost equivalent.

  As described above, as shown in Examples 7 to 9, also in RD-114 virus knockout CRFK cells, feline parvovirus, feline calicivirus, feline herpesvirus should proliferate sufficiently as in untreated CRFK cells. It has been shown. These results and the results of Example 5 above show that RD-114 virus knockout CRFK cells are useful as a cell line for production of a live attenuated vaccine against virus infection in dogs and cats, and are infectious to the same vaccine. It shows that contamination of infectious particles of RD-114 virus can be suppressed as much as possible, that is, RD-114 virus-free vaccine can be produced by using RD-114 virus knockout CRFK cells.

The figure which showed typically the genome structure of the full length of six RD-114 virus related sequences identified this time in Example 1. FIG. In Example 3, the graph which shows the titer of the virus obtained by co-transfecting pRDRS C1 and pRDRS A2.

Claims (6)

A genetically modified cell derived from a feline genus cell,
A recombinant cell in which the infectious env gene of the RD-114 virus contained in the genome of the original strain has been artificially knocked out.   A non-infectious RD-114 virus-related sequence having the coding sequence region of the infectious env gene in the genome of the original strain, and a non-infectious RD-114 having no coding sequence region of the infectious env gene 2. The recombinant cell according to claim 1, wherein at least one virus-related sequence is inherent.   3. The genetically modified cell according to claim 1, wherein the original strain is a CRFK cell.   A method for producing a vaccine, comprising a step of infecting and amplifying a virus strain for vaccine in the genetically modified cell according to any one of claims 1 to 3.   Infectious RD-114 virus expression risk detection by judging the presence or absence of infectious RD-114 virus expression by determining the presence or absence of RD-114 virus infectious env gene in the genome of cat-derived cells Method.   A method for producing a recombinant cell, comprising a step of artificially knocking out an infectious env gene of an RD-114 virus endogenous to the genome of a cat-derived cell.

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