JP2011217736A - Recombinant chlamydia and method for producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide recombinant chlamydia having foreign DNA in a genome sequence and a method for producing the recombinant chlamydia.SOLUTION: A combination of 5'→3' exonuclease and single strand DNA annealing protein is used to recombine double strand DNA including, at both ends, regions homologous to a base sequence on chlamydia genome, with chlamydia genome by homologous recombination.

Description

  The present invention relates to a recombinant chlamydia having a foreign DNA in the genome sequence and a method for producing the same.

  Chlamydia infection occurs mainly on the mucosal surface, and there are various groups such as conjunctiva, genital, respiratory and neonatal infections. The infectious agent is Chlamydia which causes obligate intracellular bacterial parasitism of eukaryotic cells. Known bacteria of the genus Chlamydia include Chlamydia trachomatis, Chlamydia psittaci, and Chlamydia pneumoniae. As for the classification of Chlamydia, a new classification was proposed by Evarett et al. In 1999, and C. pneumoniae and C. psittaci were separated from the conventional Chlamydia genus to be Chlamydophila genus (Non-patent Document 1).

  C. trachomatis is a causative bacterium that causes trachoma, sexually transmitted lymphogranulomas, genitourinary infections, inclusion body conjunctivitis, neonatal pneumonia, etc. Chlamydia sshitasi is a causative bacterium such as parrot disease, and C. pneumoniae. Is a causative bacterium such as respiratory infection, atypical pneumonia.

  C. trachomatis, the causative agent of genital chlamydial infection, mainly causes urethritis in men and cervicitis in women. Especially in women, it easily penetrates into the abdominal cavity and causes tubal pregnancy and tubal infertility. Furthermore, when it spreads to the upper abdomen, fulminant perihepatitis (Fitz-Hugh Curtis syndrome) develops and is a clinically problematic disease. C. trachomatis is classified into 15 serovars based on antigenic mutations in the main outer membrane protein (MOMP). This disease includes nongonococcal urethritis, mucopus purulent cervicitis, acute accessory testicularitis, ectopic pregnancy and pelvic inflammatory disease (PID, endometritis, fallopianitis, parauterine histitis and / or Peritonitis). Infection to women causes infertility and is a very serious social health problem. Infants born to Chlamydia-infected mothers have a high risk of developing encapsulated conjunctivitis and pneumonia. In addition, C. trachomatis subtypes A, B, Ba and C cause trachoma, an infection of conjunctival epithelial cells, and chronic and secondary infections induce infiltration of subepithelial lymphocytes to form follicles, Invasion of glandular fibroblasts and blood vessels into the eye leads to blindness. There are approximately 500 million trachoma patients worldwide, with 7-9 million blinded due to the complications. The incidence of trachoma is high at an early age, with 80 million children in need of treatment.

  As a vaccine for prevention of Chlamydia infection, one using a serotype antigen existing in a 40 KDa major outer membrane protein (MOMP) (Patent Literature 1, Patent Literature 2), or Chlamydia glycolipid exoantigen GLXA (Patent Literature 3) The one using is known. Although these are inactivated vaccines, humoral immunity is acquired, but cellular immunity is not acquired, and the development of attenuated vaccines is desired. However, at present, chlamydia genome recombination technology has been established. (Non-Patent Document 2), development of an attenuated vaccine is difficult to achieve.

  Chlamydia is a biologically unusual property of obligate intracellular parasitism, and genome analysis has been carried out vigorously. All nucleotide sequences of C. pneumoniae, C. trachomatis, and C. psittaci were determined by the genome project. (Non-patent literature 3, Non-patent literature 4, Non-patent literature 5), however, there are still many unknown genes. As a tool for elucidating the functions of these genes, gene disruption and Development of an introduction method is required.

  On the other hand, in E. coli, a combination of RecE, which is a 5 ′ → 3 ′ exonuclease derived from Rac prophage, and RecT, which is a single-stranded DNA annealing protein, or Redα, which is a 5 ′ → 3 ′ exonuclease derived from λ phage. In addition, it has been reported that highly efficient homologous recombination was carried out in E. coli by using a combination of single-stranded DNA annealing protein Redβ (Patent Document 4, Non-Patent Document 6, Non-Patent Document 7). ). However, there has been no report of successful recombination of Chlamydia using the RecE / RecT system or Redα / β system.

JP2007-308508 JP 11-123078 Patent No. 2990211 Patent No. 4139561

Karin D. E. Everett, Robin M. Bush and Arthur A. Andersen: Int. J. Syst. Bacteriol. 1999; 49: 415-440 BMC Microbiology 2009, 9: 218 Stephens, RS, Kalman, S., Lammel, C., Fan, J., Marathe, R., Aracind, L., Mitchell, W., Olinger, L., Tatusov, RL, Zhao, Q., Koonin, EV, Davis, RW: Science, 282, 754-759 (1998) Kalman, S., Mitchell, W., Marathe, R., Lammel, C., Fan, J., Hyman, RW, Olinger, L., Davis, RW, Stephenes, R .: Nature Genet., 21, 385 -389 (1999) Read TD, Myers GS, Brunham RC, Nelson WC, Paulsen IT, Heidelberg J, Holtzapple E, Khouri H, Federova NB, Carty HA, Umayam LA, Haft DH, Peterson J, Beanan MJ, White O, Salzberg SL, Hsia RC , McClarty G, Rank RG, Bavoil PM, Fraser CM .: Nucleic Acids Res. Apr 15; 31 (8): 2134-47 (2003) Richard Kolodner, Sharynn D. Hall and Cynthia Luisi-Deluca: Molecular Microbiology 11 (1), 23-30 (1994) Youming Zhang, Joep PP Muyrers, Jeanette Rientjes and A Francis Stewart: BMC Molecular Biology 4 (2003)

  An object of the present invention is to provide a recombinant chlamydia having a foreign DNA in the genome sequence, and a method for producing the same.

  In order to achieve the above object, the inventors have conducted extensive research on a method for homologous recombination of the Chlamydia genome with high efficiency. As a result, the inventors have obtained a plasmid having nucleotide sequences encoding recE gene and recT gene, Chlamydia genome The chlamydial genome was successfully recombined by introducing a double-stranded DNA containing a region homologous to the above base sequence at both ends into chlamydia and culturing it under conditions where the RecE protein and RecT protein are expressed.

That is, the present invention provides the following plasmid for chlamydia genome recombination and a method for homologous recombination of chlamydia genome using the same.

(1) A recombinant chlamydia having a non-chlamydia-derived foreign DNA sequence in the chlamydia genome sequence.
(2) a DNA fragment having a first region and a second region that are homologous to a part of the Chlamydia genome sequence, and a foreign DNA sequence derived from non-Chlamydia between the first region and the second region; A recombinant chlamydia obtained by introducing into chlamydia and allowing homologous recombination between the DNA fragment and the chlamydia genome.
(3) a DNA fragment having a first region and a second region that are homologous to a part of the Chlamydia genome sequence, and a foreign DNA sequence derived from non-Chlamydia between the first region and the second region; A method for producing recombinant chlamydia, which is introduced into chlamydia and allows homologous recombination between the DNA fragment and the chlamydia genome.
(4) The method for producing recombinant chlamydia according to (3), wherein the chlamydia genome is recombined by homologous recombination using a combination of 5 ′ → 3 ′ exonuclease and single-stranded DNA annealing protein.
(5) The method for producing recombinant chlamydia according to (4), wherein the 5 ′ → 3 ′ exonuclease is RecE or Redα, and the single-stranded DNA annealing protein is RecT or Redβ.
(6) A base sequence encoding 5 ′ → 3 ′ exonuclease and a base sequence encoding single-stranded DNA annealing protein are included on the same plasmid or on a separate plasmid downstream of the promoter sequence. A plasmid for recombination of Chlamydia genomes.
(7) The plasmid for chlamydial genome recombination according to (6), wherein the 5 ′ → 3 ′ exonuclease is RecE or Redα, and the single-stranded DNA annealing protein is RecT or Redβ.
(8) a plasmid according to any one of (6) and (7), a DNA fragment comprising a foreign region derived from non-Chlamydia having a first region and a second region that are homologous to a part of the Chlamydia genome sequence; Is introduced into Chlamydia, and the protein encoded by the plasmid is expressed in Chlamydia to homologously recombine the DNA fragment with the Chlamydia genome.

  The present invention provides a plasmid for recombination of Chlamydia genome and a method for homologous recombination of Chlamydia genome using the same. The present invention makes it possible to recombine the Chlamydia genome, which was impossible in the past, and contributes greatly to elucidating the function of unknown proteins found in the Genome Project, and producing Chlamydia vaccines by acquiring disrupted strains of pathogenic genes. It becomes possible.

The map of the plasmid (pCLfrec1) for Chlamydia genome recombination constructed | assembled by this invention is shown. The recE / T gene sequence was cloned downstream of the promoter region, having PL / PR promoter induced by temperature increase and pMB1 ori as the origin of replication. The result of the recombination confirmation of Chlamydia genome is shown. The upper part of the sequence is the base sequence of the insertion site of the wild strain, and the lower part is the base sequence analysis result of the DNA fragment obtained by nested PCR. The result of the recombination confirmation of Chlamydia genome is shown. The upper part of the sequence is the base sequence of the insertion site of the wild strain, and the lower part is the base sequence analysis result of the DNA fragment obtained by nested PCR. It is the result of comparing the mRNA expression level by RT-PCR when a chloramphenicol resistance gene linked to different ompA promoter sites was incorporated into Chlamydia. It is the result of subculturing the lcrE gene-disrupted strain, yscC gene-disrupted strain, and yscN gene-disrupted strain without a selective agent, and comparing the relative abundance. The results show that the lcrE gene-disrupted strain and the yscN gene-disrupted strain can be selected by subculture in the presence of a selective drug.

  Hereinafter, the present invention will be described with reference to embodiments for carrying out the invention. However, the present invention is not limited to the items described in the above items as long as they belong to the concept of the present invention.

(1) Recombinant chlamydia of the present invention The recombinant chlamydia of the present invention is a recombinant chlamydia having a non-chlamydia-derived foreign DNA sequence in the natural chlamydia genome sequence.

  In the present invention, “foreign DNA” refers to DNA that is not derived from the Chlamydia genome, for DNA encoding a gene that is desired to be expressed for substance production and functional modification or functional analysis, or for disrupting Chlamydia gene. Although artificially prepared DNA to introduce | transduce is mentioned, if it is DNA which does not originate in Chlamydia genome, it will not specifically limit.

  Chlamydia having such foreign DNA can be obtained, for example, by the production method of the present invention described below.

(2) Production method of the present invention The production method of the present invention comprises a first region and a second region that are homologous to a part of a Chlamydia genome sequence, and a foreign DNA sequence derived from non-Chlamydia is converted to the first region and the second region. This is a method for producing recombinant chlamydia by introducing a DNA fragment contained between these regions into chlamydia and allowing homologous recombination between the DNA fragment and the chlamydia genome.

  In the present invention, “homologous recombination” means DNA recombination that occurs between homologous regions of a pair of double-stranded DNAs.

  The above-mentioned “homologous region” means a region having the same base sequence as the corresponding region in Chlamydia genomic DNA, or a region having sequence identity such that homologous recombination occurs with the region. That is, in addition to the first region and the second region having the same base sequence as the corresponding region in Chlamydia genomic DNA, one or more bases in the base sequence are substituted and / or missing. It is also possible to include those which have been lost and / or have one or more bases inserted into and / or added to the base sequence, so that the number of these can be homologously recombined. Specifically, it preferably has 90% or more, more preferably 95% or more, more preferably 98% or more, and most preferably 100% identity with the corresponding region in Chlamydia genomic DNA. Here, the identity of the base sequence means that both base sequences are aligned so that both bases match as much as possible (by inserting a gap if necessary), and the number of mismatched bases is calculated as the total number of bases (both sequences). When the total number of bases is different, the number divided by the total number of bases of the shorter sequence) is expressed as a percentage, and can be easily calculated by known software such as BLAST. In particular, the total number of bases to be substituted, deleted, inserted and / or added is preferably less than 10.

  The length of the homologous region is at least 15 nucleotides, more preferably at least 30 nucleotides, and most preferably at least 50 nucleotides in each of the first homologous region and the second homologous region.

  In addition, as the DNA sequence between the first region and the second region, any DNA sequence to be inserted into Chlamydia genomic DNA can be used, but confirm that homologous recombination has occurred. Therefore, it is preferable to use a marker gene such as a chloramphenicol resistance gene or a tetracycline resistance gene.

  The method for introducing the DNA fragment into Chlamydia is not particularly limited, and any method known to those skilled in the art can be appropriately selected. For example, an electroporation method, a method using calcium ions, a lipofection method and the like can be mentioned.

  Homologous recombination can be used in the production method of the present invention as long as it is a protein capable of causing homologous recombination between a DNA fragment introduced into Chlamydia and the Chlamydia genome. For example, 5 ′ → 3 ′. It is extremely preferable from the viewpoint of efficiency to recombine the chlamydia genome by homologous recombination using a combination of exonuclease and single-stranded DNA annealing protein.

  5 '→ 3' exonuclease refers to an enzyme having a function of releasing mononucleotide from a 5 'end toward a 3' end from a single-stranded DNA. In the present invention, any kind of 5 ′ → 3 ′ exonuclease can be used as long as it can be expressed and function in Chlamydia, such as RecE derived from rac prophage or Redα derived from phage λ.

  A single-stranded DNA annealing protein is a protein that binds to a single-stranded DNA having regions that are homologous to each other, and has a function of causing homologous recombination between the homologous regions of these single-stranded DNAs. Point to. In the present invention, any kind of single-stranded DNA annealing protein can be used as long as it can be expressed and function in Chlamydia, such as RecT derived from rac prophage, Redβ derived from phage λ, and Erf derived from phage P22. I can do it.

  RecT and Redβ are known to be functionally similar (Hall et al. J. Bacteriol. 175 (1993) 277-287; Muniappa and Radding, J. Biol. Chem. 261 (1986) 7472-). 7478; Kmiec and Holoman, J. Biol. Chem. 256 (1981) 12636-12539). The Erf protein is also described by Potetete and Fenton (J Mol Biol 163 (1983) 257-275) and references therein, and is known to be functionally similar to Redβ and RedT (Murphy). et al., J Mol Biol 194 (1987) 105-117).

  In E. coli, effective homologous recombination between two DNA molecules occurs frequently by expressing functionally related genes such as recE and recT genes or redα and redβ genes or phage P22 recombination systems. (Kolodner et al., Mol. Microbiol. 11 (1994) 23-30; Fenton, A. C. and Pottete. A. R., Virology 134 (1984) 148-160; Potete. A. R. And Fenton, A.C., Virology 134 (1984) 161-167).

  The present invention is also a functional equivalent of the molecules specifically identified above as RecE, RecT, Redα, Redβ, and Erf and retains the ability to mediate recombination as described herein. Anything can be used. Such functional equivalents include homologues of recombinant elements present in bacteriophage (large DNA phage, T4 phage, T7 phage, small DNA phage, isomeric phage, filament DNA phage, RNA phage, Mu Including, but not limited to, phage, P1 phage, derivative phage and phage-like), and functional homologues of recombination system elements present in the virus (any virus belonging to any of the following groups): Include but are not limited to: plant viruses, insect viruses, yeast viruses, fungal viruses, parasitic microbial viruses, picornaviruses, enteroviruses, polioviruses, coxsackie viruses, echoviruses, rhinoviruses, all livers Virus, Calciviridae, Norwalk virus, Astroviridae, Astrovirus, Togavirus, Alphavirus, Rubella virus, Flaviviridae, Flavivirus, Pestivirus, Coronaviridae, Coronavirus, Lactate dehydrogenase augmentation Viruses and related viruses, Rhabdoviridae, Rhabdovirus, Filoviridae, Marburg virus, Ebola virus, Paramyxoviridae, Parainfluenza virus, mumps virus, measles virus, RS virus, Orthomyxoviridae, Orthomyxovirus, Bunyaviridae, Arenaviridae, Arenavirus, Reoviridae, Reovirus, Rotavirus, Orbivirus, Cortivirus (c ltivirus), retrovirus, human T cell leukocyte virus, human immunodeficiency virus, lentivirus, papoviridae, polyomaviridae, polyomavirus, papillomaviridae, papillomavirus, adenoviridae, adenovirus, parvo Virology, parvovirus, herpesviridae, herpes simplex virus, Epstein-Barr virus, cytomegalovirus, varicella-zoster virus, human herpesvirus, rhesus herpesvirus, B virus, poxviridae, poxvirus, hepadnavirus As well as unclassified factors (e.g., Fields Virology, Third Edition, edi) ed by B. N. Fields, D.D. M.M. Knipe, P.M. M.M. Howley, et al. Lippincott-Raven Publishers, Philadelphia PA USA (1996)).

  Specific examples of the above functional equivalent are RecE protein, RecT protein, Redα protein, Redβ protein or Erf protein, including amino acid substitutions, insertions and / or deletions from the wild type sequence, and Those that can normally exert the function of homologous recombination can be mentioned. Such functional equivalents are preferably at least 20% or more with the wild type sequence, more preferably at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or It has 99% or more amino acid sequence identity.

  That is, the base sequence encoding 5 ′ → 3 ′ exonuclease and the base sequence encoding single-stranded DNA annealing protein that can be used in the present invention include, for example, base sequences encoding RecE and RecT. Includes a sequence of SEQ ID NO: 1 in which the base sequence of the recE gene and the base sequence of the recT gene partially overlap, and includes a base sequence that hybridizes with this under stringent conditions. The stringent condition refers to washing in 0.1 × SSC and 0.5% SDS, preferably at 55 ° C., more preferably at 62 ° C., and even more preferably at 68 ° C. for 30 minutes. The base sequence that hybridizes underneath is a base sequence that is at least 60% or more homologous to SEQ ID NO: 1, preferably a base sequence that is at least 70% or more, more preferably at least 80% or more.

  Examples of the base sequences encoding Redα and Redβ include the sequences of SEQ ID NO: 2 and SEQ ID NO: 3, and also include base sequences that hybridize with this under stringent conditions. The stringent condition refers to washing in 0.1 × SSC and 0.5% SDS, preferably at 55 ° C., more preferably at 62 ° C., and even more preferably at 68 ° C. for 30 minutes. The base sequence that hybridizes underneath is a base sequence that is at least 60% or more homologous to SEQ ID NO: 1, preferably a base sequence that is at least 70% or more, more preferably at least 80% or more.

  As the plasmid for chlamydia genome recombination in the present invention, the following plasmids of the present invention can be used.

(3) Plasmid of the Present Invention The plasmid of the present invention is a plasmid characterized by comprising a base sequence encoding 5 ′ → 3 ′ exonuclease or a base sequence encoding a single-stranded DNA annealing protein downstream of the promoter sequence.

  As described above, the plasmid of the present invention is a plasmid that can be used to prepare the chlamydia of the present invention by the method of the present invention.

  As long as it can be used for such applications, the plasmid of the present invention has either a base sequence encoding 5 ′ → 3 ′ exonuclease or a base sequence encoding single-stranded DNA annealing protein. In view of the above, it is preferable that both are contained in the same plasmid.

  The plasmid of the present invention has a promoter sequence upstream of these gene sequences so as to express a 5 '→ 3' exonuclease and a single-stranded DNA annealing protein. Examples of promoter sequences that can be used in the plasmid of the present invention include lac promoter, tac promoter, T7 promoter, cspA promoter, tal promoter, trp promoter, trc promoter, araBAD promoter, or gal1 promoter. Any kind of promoter that can be induced can be used, but a temperature-sensitive promoter is preferable, and PL and PR promoters of λ phage are exemplified. Such a promoter and a base sequence encoding a 5 ′ → 3 ′ exonuclease and a base sequence encoding a single-stranded DNA annealing protein are appropriately transcribed so as to express the protein. A person skilled in the art can appropriately select a method that is relatively familiar to use.

  Furthermore, the plasmid of the present invention contains a drug resistance gene such as a chloramphenicol resistance gene, a tetracycline resistance gene, or an ampicillin resistance gene as a marker gene for confirming that the introduction of the plasmid of the present invention is appropriate. Those skilled in the art can also arrange them above.

  Specific examples will be described below, but the present invention is not limited to the following examples.

  Primer sequence with restriction enzyme site salI site added to 5 'end and restriction enzyme site XbaI site added to 3' end to amplify recE gene and recT gene using XL-1blue genome derived from E. coli K12 strain as a template ( DNA fragments were amplified by the PCR method using SEQ ID NO: 4 and SEQ ID NO: 5). The obtained DNA fragment was incorporated into a multi-cloning site of pCL476 plasmid having pMB1 ori to construct an expression plasmid. The constructed plasmid is named pCLfrec1 (FIG. 1).

  In order to disrupt the type 3 secretion apparatus component yscC gene on the chlamydia genome by homologous recombination, a partial sequence 50 base of the type 3 secretion apparatus component yscC gene on the chlamydia genome and the promoter sequence of the chlamydia ompA gene (gene encoding MOMP) Using a primer (SEQ ID NOs: 6 and 7) added to the 5 ′ end, PCR was performed using tetracycline or chloramphenicol resistance gene as a template as a drug resistance gene. Similarly, it was carried out for the type 3 secretion apparatus component lcrE gene and yscN gene.

  Two days after infection of Chlamydia to Hela cells, the cell suspension was collected in 250 μl of 2% glycerol using a cell scraper, and Chlamydia was released with an ultrasonic crusher. After removing the nuclei of Hela cells by centrifugation, 10.4 μg of pCLfrec1 and 0.1 μg of the PCR product were introduced by electroporation using a MicroRadr manufactured by BioRad. As for the pulse, an exponential waveform of 1.5 kV was applied once to a 2 mm gap cuvette without a time constant. Immediately after introducing DNA, add 500μl of SPG solution (0.22M sucrose, 3.8mM KH2PO4, 8.6mM NaHPO4, 4.9mM Na glutamate) and incubate at 44 ° C for 20 minutes, then incubate at 37 ° C for 2 hours and again infect Hela cells I let you.

For recombination of the chlamydia genome, a sequence upstream from the recombination position of the yscC gene (SEQ ID NO: 8) and a sequence complementary to the tetracycline or chloramphenicol resistance gene (SEQ ID NO: 9) were used as a forward primer and a reverse primer, respectively. PCR was performed, and the product was further confirmed by performing nested PCR using (SEQ ID NO: 10, SEQ ID NO: 11) and determining the gene sequence (FIG. 2). The same procedure was performed for the lcrE gene and the yscN gene.

Acquisition of Glycogen hydrolase gene (glgX) disruption strain
By using a primer (SEQ ID NOs: 12 and 13) in which a partial sequence 50 base of the Glycogen hydrolase gene is added to the 5 ′ end, PCR using the chloramphenicol resistance gene as a template as a template is performed. Except for the obtained points, the DNA fragment was introduced into chlamydia in the same manner as in Example 1 to perform recombination.

  Confirmation of recombination was performed by performing PCR using a sequence upstream of the glgX gene (SEQ ID NO: 14) and a sequence complementary to the chloramphenicol resistance gene (SEQ ID NO: 9) as a forward primer and a reverse primer, respectively. The product was further confirmed by performing nested PCR using (SEQ ID NO: 15, SEQ ID NO: 11) and determining the gene sequence (FIG. 3).

  A chloramphenicol resistance gene linked to the promoter sequence of the ompA gene encoding Chlamydia major outer membrane protein MOMP (FIG. 4, P1, P2, and P2b) was introduced into Chlamydia, and the amount of mRNA transcription was measured. That is, in the same manner as in Example 1, the chloramphenicol resistance gene having the promoter sequence P1, P2, or P2b shown in FIG. 4 upstream was recombined at the 3 ′ end position of the lcrE gene of Chlamydia genome. Mutant strains were prepared and the mRNA expression level two days after infection of Hela cells was examined by RT-PCR. RNA is prepared from the entire infected cell using Trizol (Invitrogen), cleaved with DNAase I, and then subjected to RT-PCR using primers complementary to the chloramphenicol resistance gene (SEQ ID NOs: 16 and 17). went.

  LcrE gene-disrupted strain (inserted DNA fragment prepared using SEQ ID NOs: 18 and 19), yscC gene-disrupted strain, and yscN gene-disrupted strain (inserted DNA prepared using SEQ ID NOs: 20 and 21) by the method of Example 1 Fragments) were obtained, and the relative abundance by subculture was compared (FIG. 5). The relative abundance was quantified by preparing Real DNA with each of the Hela cells infected with Chlamydia and preparing the DNA as a template. Infected chlamydia is a mixture of genetic variants and wild strains, so the relative abundance of the variants was quantified and compared. That is, the amount of the mutant was determined by using a forward primer (SEQ ID NO: 22, SEQ ID NO: 8, SEQ ID NO: 23) complementary to the 5 ′ adjacent portion of the recombination portion and a reverse primer complementary to the inserted chloramphenicol resistance gene ( Quantification was performed using a TaqMan probe (SEQ ID NO: 24) complementary to the sequence to be amplified. The genome copy number of the entire infected chlamydia was quantified by real time PCR (SEQ ID NO: 25, SEQ ID NO: 26) of the GroEL gene to determine the relative abundance of mutants within the entire chlamydia. The results of FIG. 5 indicate that these type 3 secretion apparatus-deficient strains have low reinfection ability, suggesting the possibility of using these mutant strains as vaccines.

  LcrE gene-disrupted strain (inserted DNA fragment prepared using SEQ ID NOs: 27 and 28) into which tetracycline resistance gene has been incorporated by the method of Example 1, and yscN gene-disrupted strain (inserts prepared using SEQ ID NOs: 29 and 30) DNA fragment) was obtained and cultured for several passages in the presence of 0.1 μg / ml tetracycline to confirm that tetracycline could be selected. (FIG. 6). The gene-disrupted strain was confirmed by nested PCR as in the case of glgX. That is, PCR is performed using a forward primer (SEQ ID NO: 22 or SEQ ID NO: 23) complementary to the 5 ′ adjacent portion of the recombination portion and a reverse primer (SEQ ID NO: 31) complementary to the inserted tetracycline resistance gene, and the product Further, nested PCR was performed using SEQ ID NO: 32 and SEQ ID NO: 33 or SEQ ID NO: 34.

  The recombination of the Chlamydia genome according to the present invention makes it possible to identify Chlamydia pathogenic genes based on elucidation of the functions of proteins of unknown function found in the genome project. Furthermore, it is expected to make significant contributions in the medical field, such as the production of attenuated Chlamydia vaccines by acquiring strains that have disrupted pathogenic genes.

Claims (8)

A recombinant chlamydia having a foreign DNA sequence derived from non-chlamydia in the chlamydia genome sequence. A DNA fragment having a first region and a second region that are homologous to a part of the Chlamydia genome sequence and containing a non-Chlamydia-derived foreign DNA sequence between the first region and the second region in Chlamydia A recombinant chlamydia obtained by introduction and homologous recombination between the DNA fragment and the chlamydia genome. A DNA fragment having a first region and a second region that are homologous to a part of the Chlamydia genome sequence and containing a non-Chlamydia-derived foreign DNA sequence between the first region and the second region in Chlamydia A method for producing recombinant chlamydia, which is introduced and allows homologous recombination between the DNA fragment and the chlamydia genome. The method for producing recombinant chlamydia according to claim 3, wherein the chlamydia genome is recombined by homologous recombination using a combination of 5 '→ 3' exonuclease and single-stranded DNA annealing protein. The method for producing recombinant chlamydia according to claim 4, wherein the 5 '→ 3' exonuclease is RecE or Redα, and the single-stranded DNA annealing protein is RecT or Redβ. A chlamydia comprising a base sequence encoding a 5 ′ → 3 ′ exonuclease and a base sequence encoding a single-stranded DNA annealing protein on the same plasmid or on separate plasmids downstream of the promoter sequence Plasmid for genome recombination. The plasmid for chlamydial genome recombination according to claim 6, wherein the 5 'to 3' exonuclease is RecE or Redα, and the single-stranded DNA annealing protein is RecT or Redβ. A plasmid according to any one of claims 6 and 7, and a DNA fragment comprising a foreign region derived from non-Chlamydia having a first region and a second region that are homologous to a part of the Chlamydia genome sequence. A method for producing a recombinant chlamydia, wherein the DNA fragment is homologously recombined with the chlamydia genome by introducing the gene into chlamydia and expressing the protein encoded by the plasmid in chlamydia.