JP3677125B2 - Methods for introducing foreign genes into chromosomes - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for introducing a foreign gene into a host chromosome. Specifically, when a foreign gene is introduced into a chromosome using a lysogenic vector, the foreign gene is necessary at the time of introduction. The present invention relates to an introduction method for finally removing an unnecessary gene portion derived from a vector which is unnecessary for the expression and maintenance of.
[0002]
[Prior art]
Introduction of a foreign gene into a host cell using a plasmid is performed in various microorganisms including Escherichia coli. In order to obtain more stable characteristics of foreign genes in host cells, attempts have been made to incorporate foreign genes into host chromosomes using lysogen phages.
[0003]
The present inventors previously described Lactobacillus casei (Lactobacillus casei ; Hereinafter referred to as “L. casei”), and the incorporation mechanism derived from the lysogenic phage φFSW is applied. Proposed vector pMSK742 for Lactobacillus casei and L. A method for incorporating a foreign gene into the chromosome of casei was shown (Japanese Patent Application No. 8-184266).
[0004]
However, this L.P. Not only the casei vector pMSK742 but also a method using a lysogen phage, not only the target foreign gene but also a gene sequence that becomes unnecessary after the introduction is simultaneously introduced. For example, the L. In the casei vector, the site-specific recombinase (int) gene region necessary for the function of the integration mechanism, the drug resistance gene region required for selection of the integrant, and the replication initiation region functioning in E. coli Are integrated on the host chromosome at the same time, but these parts are functionally unnecessary after gene introduction.
[0005]
[Problems to be solved by the invention]
The parts that become functionally unnecessary after the introduction of these genes may become an obstacle from the viewpoint of the use of the subsequent transformant, and finally the unnecessary parts derived from vectors other than foreign genes are removed. Development was an issue.
[0006]
As a result of intensive studies based on such a viewpoint, the present inventor has devised the incorporation of a foreign gene into a vector using a lysogen phage at the time of introduction into a host cell. After confirming that unnecessary genes derived from vectors and the like can be removed, the present invention has been achieved.
[0007]
An object of the present invention is to obtain a method for introducing a foreign gene into a chromosome that can remove an unnecessary gene derived from a vector other than the foreign gene.
[0008]
[Means for Solving the Problems]
In the present invention, a method for incorporating a foreign gene onto a host chromosome using a vector having the characteristics of a lysogen phage,
A first partial sequence in which one end region of a foreign gene intended for introduction is deleted, and a second portion having a region in which the other end region is deleted and overlaps a part of the first partial sequence Obtaining a vector in which the sequence is arranged on both sides of the lysogen phage integration site (attP site),
After integrating this vector onto the host chromosome, select a transformant from which the unnecessary gene portion derived from the vector has been removed by the homologous recombination mechanism of the overlapping region of the first partial sequence and the second partial sequence It is a method to do.
[0009]
The vector utilizing the characteristics of the lysogen phage in the present invention refers to a vector having a function of being integrated on a host chromosome when introduced into a host cell. In other words, any one having an integration site (attP region) that causes recombination with the integration site (attB region) on the chromosome of the host cell may be used. When a foreign gene is integrated into a host chromosome by such a vector, a gene derived from the vector is also incorporated together with the foreign gene.
[0010]
For this reason, the first partial sequence in which one end region of a foreign gene intended to be introduced is deleted, and the first partial sequence in which the other end region is deleted and has a region overlapping with a part of the first partial sequence. A vector in which two partial sequences are arranged on both sides of the integration site (attP site) of the lysogen phage is obtained, and this vector is integrated on the host chromosome to promote homologous recombination.
[0011]
Both the first partial array and the second partial array may have a region where the deletion side arrays overlap. If this overlapping region is short, the frequency of homologous recombination after integration decreases, so that unnecessary genes are not excised by homologous recombination. On the other hand, homologous recombination is expressed even when the overlapping region is lengthened, for example, the first partial sequence and the second partial sequence are made completely the same without excising the terminal region of the foreign gene. However, when the expression of a foreign gene is used as a kind of marker, there is a possibility that a transformant from which an unnecessary gene portion derived from a vector has been removed cannot be selected. Therefore, the lower limit of the overlapping region may be longer than the length at which the homologous recombination mechanism is performed with sufficient frequency. In addition, although the upper limit of the length of the overlapping region is not related to homologous recombination, when the expression of the foreign gene is used as a kind of marker, it is less than the length where the foreign gene is not expressed in the partial sequence with the terminal region excised. If it is.
[0012]
Moreover, about the direction of arrangement | positioning of a 1st partial sequence and a 2nd partial sequence, after homologous recombination is performed by the overlapping area | region and it is performed, it arrange | positions in the direction which becomes an original foreign gene. That is, the first and second partial sequences are oriented in the same direction, and the N and C terminal portions of the foreign gene are on the integration site side on both sides of the integration site (attP site) of the mechanical lysogen phage. Placed in.
[0013]
The term “foreign gene” as used in the present invention does not necessarily mean a gene derived from a bacterium other than the host bacterium to be introduced. It is also included to introduce into the chromosome. Moreover, it may be a gene group as a conjugate of a plurality of protein information (for example, a gene of the whole enzyme composed of a plurality of subunits).
[0014]
In order to select a transformant from which an unnecessary gene portion derived from a vector has been removed, the loss of a drug-resistant trait derived from a vector can be used as a marker. It is simplest to confirm the expression of a foreign gene and use this as a kind of marker. That is, in the method of the present invention, the case where the unnecessary gene derived from the vector is removed is the case where the foreign gene has undergone homologous recombination and has become the original foreign gene. Therefore, selection is made using the expression of the foreign gene as a kind of marker.
[0015]
Specific examples of the present invention include L.P. Casei YIT9018 strain lysogen phage φFSW-derived site-specific recombinase (integrase, int) gene region, integration site into host chromosome (attP region), drug-resistant gene region functioning in lactic acid bacteria and E. coli, E. coli (Escherichia coliL.), which has a replication initiation region that functions in) and a cloning site. Using the vector for casei bacteria, A foreign gene (ABC) consisting of an A region, a B region, and a C region on the chromosome of casei (where the A region and the C region indicate the region containing the cut end of the foreign gene, and the B region Is a method of incorporating an arbitrary overlapping portion between both cleavages in a foreign gene),
(1) The first partial sequence (AB) excluding the C region of the foreign gene (ABC) intended for introduction and the A region of the foreign gene (ABC) were excluded. A step of obtaining a vector in which the second partial sequence (BC) is arranged on both sides of the attP region in the vector;
(2) L. Integrating into the chromosome of casei and obtaining transformants;
(3) After that, a step of selecting a transformant from which an unnecessary gene part including the site-specific recombination enzyme region, the drug resistance region and the replication initiation region has been removed by the homologous recombination mechanism;
The method provided with.
[0016]
The method for introducing a foreign gene into the chromosome of the present invention can be applied to any microorganism as long as there is a combination of a vector and a host bacterium utilizing the characteristics of lysogen phage. Therefore, the present invention relates to L.P. L. using a vector for casei bacteria. Needless to say, the introduction of foreign genes into casei is not limited. Specifically, Lactococcus lactis φLC3 phage, Lactobacillus gasseri φadh phage, Lactobacillus delbruecki subp Bulgaris mv4 phage, Escherichia coli and λ phage, HK22 phage, φ80 phage, P4 phage Application with a vector using a combination with 186 phage, etc. is possible (see L. Dupont et al., J. Bacteriol. 177: 586-595 (1995)).
[0017]
DETAILED DESCRIPTION OF THE INVENTION
As a result of earnest research, in order to obtain a method for introducing a foreign gene into a chromosome capable of removing an unnecessary gene other than a foreign gene derived from a vector or the like utilizing the characteristics of lysogen phage, When a foreign gene intended for introduction is incorporated into a vector, it is not incorporated as it is, but is divided into two partial sequences that overlap with any intermediate sequence of the foreign gene, and this is divided on both sides of the attP sequence. Then, the portion between AB and BC is detached with a considerable probability by a naturally occurring homologous recombination mechanism. As a result, the sequence of ABC is transferred to the chromosome. The present invention was completed by confirming that a host transformant was obtained as an integrated one.
[0018]
That is, when the foreign gene to be introduced is, for example, A-B-C (where A and C indicate a region including a cut end and B indicates an arbitrary overlapping portion of the intermediate sequence portion), A A first partial sequence AB and a second partial sequence BC which are partial sequences of -B-C (AB and BC have overlapping portions in the portion of sequence B); Is placed on both sides of the attP region of the vector and transformed into the host chromosome, which is then sandwiched between AB and BC with considerable probability by a naturally occurring homologous recombination mechanism. The part is detached and as a result the sequence ABC is integrated into the chromosome.
[0019]
First, according to the method of the present invention, for example, L.P. As the vector for casei, those disclosed in the patent application by the present inventor (Japanese Patent Application No. 8-184266) can be used. That is, L. Casei YIT9018 strain lysogen phage φFSW-derived int region and attP region, drug resistance gene region that functions in lactic acid bacteria and E. coli, E. coli (Escherichia coliL.) which has a replication initiation region that functions in) and an appropriate cloning site. It is a vector for casei.
[0020]
Specifically, L.M. A site-specific recombinase (integrase, int) region derived from the casei YIT9018 strain derived from the casei YIT9018 strain, an integration site into the host chromosome (attP region), a drug resistance gene region that functions in lactic acid bacteria and E. coli, and E. coli A replication initiation region that functions in (E. coli) and a cloning site.
[0021]
For more details, see L.L. Site-specific recombinase (integrase, int) gene region derived from the casei YIT9018 strain and the integration site into the host chromosome (attP region), and functions in both Escherichia coli and lactic acid bacteria derived from Enterococcus faecalis The erythromycin (Em) resistance gene and the replication initiation region derived from E. coli have been confirmed.
[0022]
The foreign gene to be introduced is not particularly limited, but in the examples described later, a chloramphenicol (Cm) resistance gene was used. The method of incorporating this foreign gene as two partial sequences on both sides of the attP region of the vector is based on the use of the restriction enzyme site of the vector and after examining the restriction enzyme site etc. of the gene to be introduced. Although it can be arbitrarily designed, the B sequence portion overlapping in the above general formula needs to be long enough to induce the subsequent homologous recombination mechanism (preferably 200 to 1000 bases, more preferably Is about 400 to 600 bases).
[0023]
In the examples described later, once the Cm resistance gene as corresponding to ABC is introduced into both sides of the attP region, and then the excess part is removed, as a result, A- Although the operation was performed so that the partial sequences of B and B-C were introduced as intended, the method is of course not limited to this method.
[0024]
Vectors for gene introduction in which AB and BC are introduced into vector pMSK742 are L.P. Integration into chromosomes by transformation into casei. Using the Em resistance gene of pMSK742 as an index, transformants are selected, and those that have been integrated into the chromosome as intended are obtained. The generated vector still contains the int region derived from pMSK742, the Em resistance gene region, and the like, and the Cm gene intended for introduction is also divided into two and integrated in a complete state. Absent.
[0025]
When the transformant obtained here is subcultured using Em resistance and Cm resistance as indicators, a strain expressing Cm resistance can be obtained with a considerable probability (10 −3). It was Em sensitive (losing Em resistance). In this, homologous recombination occurred between the introduced AB part and BC part, and the part between AB and BC was removed, and this probability is The probability that the Em resistant strain loses Em resistance due to the mutation is sufficiently higher, and the introduction of the foreign gene ABC and the unnecessary portion derived from the vector were removed by the expected mechanism.
Hereinafter, the present invention will be described in more detail with reference to examples.
[0026]
【Example】
1. Materials and methods
(1-1) Strains and plasmids used
Lactobacillus casei YlT9029 (FERM BP-1366, φFSW non-lysogen) was stored at Yakult Central Laboratory. Eschirihia coli JM109 was used as a recombinant plasmid production host. L. has an integrase gene derived from φFSW, attP, plasmid DNA replication initiation region ori177 for Escherichia coli, and an Em resistance gene derived from plasmid pAMβ1 of Enterococcus faecalis. Casei bacteria vector pMSK742 was shown in Japanese Patent Application No. 8-184266. This vector pMSK742 has been deposited with the Biotechnology Research Institute as of Seiko Kenkyu No. 15695 on June 20, 2008. Staphylococcus aureus plasmid pC194 (S. Horinouchi & B. Weisblum. Nucleotide sequence and functional map of pC194, a plasmid that specifies inducible chloramphenicol resistance. J. Bacteriol. 150: 815-825, 1982) It was done.
[0027]
(1-2) Medium
 YlT9029 was grown under aerobic conditions at 37 ° C. using MRS medium (Difco, USA) or ILS medium (still in liquid medium). Since expression of Cm resistance by the Cm resistance gene of plasmid pC194 is inductive, 0.5 μg / ml of Cm (concentration that grows in the same way as Cm-resistant strains) is resistant to Cm-resistant gene-bearing bacteria. Always added, regardless of non-tolerance. When necessary, 20 μg / ml of Em (concentration at which an Em-sensitive strain cannot grow) or 2.5 μg / ml of Cm (concentration at which a Cm-sensitive strain cannot grow) was added. LB medium was used for the growth of E. coli JM109, and 500 μg / ml of Em was added when necessary.
[0028]
(1-3) Preparation and manipulation of DNA and transformation method
Basic DNA manipulation is carried out with reference to the literature (J. Sambrook, EFFritsch & T. Maniatis. Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory Press. 1989), and the characteristics of L. casei YlT9029 and E. coli The conversion method was carried out by the operation described in Japanese Patent Application No. 8-184266. The PCR reaction was repeated 30 times at 98 ° C for 5 seconds 60 ° C for 5 seconds 74 ° C for 30 seconds using MJ Research's DNAEngine PTC-200 using Toyobo's K0D polymerase and its buffer shystem #l. . Primers used were appropriately synthesized with 20-mer or more.
[0029]
(1-4) Determination and analysis of DNA sequence
 The chromosome structure in the YIT9029 transformant was examined using the PCR method. PCR products are produced by Pharmacia Micro Spin^TM Purified by S-400 HR column. Primers of 17 mer or more were appropriately synthesized. The reaction is ABI PRISM^TM Using a 373S DNA Sequencer, Terminator Cycle Sequencing Ready Reaction Kit (PerkinElmer) was used. For analysis of the base sequence, GENETYX ver.9.0 and GENETYX-CD biodatabase software ver.29.0 manufactured by Software Development Co., Ltd. were used.
[0030]
(1-5) Stability test
The YlT9029 derivative, the test bacterium, was cultured overnight in ILS supplemented with 2.5 μg / ml Cm, and the culture broth was diluted 10-5 in ILS supplemented with 0.5 μg / ml Cm and statically cultured at 37 ° C. Then, every 24 hours, ILS containing 0.5 μg / ml Cm^-3The subculture was continued in the same manner after dilution. The viable cell count was measured using an ILS plate (selection plate) to which 2.5 μg / ml Cm was added at each dilution and an ISL (non-selection plate) to which 0.5 μg / ml Cm was added. The number of generations is log₂It was calculated by (number of viable bacteria at each time × dilution rate / number of first-occurring bacteria).
[0031]
2. Overview of foreign gene introduction methods
For example, the transformant obtained by the chromosomal integration vector pMSK742 using the site-specific recombination enzyme integrase for prophage integration possessed by the phage φFSW of YlT9029 reported in Japanese Patent Application No. 8-184266 is used as the target gene. In addition, it has a plasmid DNA replication initiation region (derived from a different species) for propagation in Escherichia coli as a vector plasmid, an Em resistance gene for selection (derived from a different species), and an integrase gene (derived from the same species) that were required for integration. These three regions were necessary for a series of operations up to integration, but were considered unnecessary for stable retention of the target gene in YlT9029 cells. Therefore, an object was to remove the above-described gene region derived from a heterogeneous vector.
[0032]
The first partial sequence (A-B) and the second partial sequence (B-C) obtained by excising each end of the target foreign gene (A-B-C) to be introduced onto the chromosome are located on both sides of the attP region. A vector placed in is prepared and used as a host fungus. After integration into the chromosome of casei, we attempted to construct a plasmid for the purpose of deleting unnecessary gene regions by homologous recombination.
[0033]
FIG. 1 is an explanatory diagram showing an outline of the steps of one embodiment of the method for introducing a foreign gene into a chromosome of the present invention. Details of the plan and procedure will be described with reference to FIGS.
(1) A second partial sequence (BC) lacking the N-terminal part of the target gene (s) on both sides of the site-specific recombination region attP on the phage genome on the vector plasmid as shown in FIG. The first partial sequence (AB) lacking the C-terminal part is arranged in the same direction, and the Em resistance gene (Em), Escherichia coli plasmid DNA are the regions to be removed on the opposite side of attP with respect to both defective genes. A replication initiation region (ori) and an integrase gene (int) are placed.
[0034]
(2) The DNA of this plasmid is introduced into YlT9029 cells, and a strain in which the plasmid is integrated onto the YlT9029 chromosome by site-specific recombination between attP and attB by integrase is selected using Em resistance as an index. As shown in FIG. b, on the chromosome of the transformant, attL to C-terminal deletion target gene (group) (AB) to plasmid DNA replication initiation region for E. coli (ori) to Em resistance gene (Em) to integrase gene ( int) to N-terminal deficient target gene (s) (BC) to attR.
[0035]
(3) Since the B region common to the two target genes (groups) is homologous, a part of the obtained transformant is sandwiched between the two B regions by the homologous recombination mechanism inherent in the cell. May be deleted (Fig. C). In that case, in the DNA introduction part on the deletion chromosome, it is arranged in the order of attL to complete target gene (group) (ABC) to attR, and the phenotype of the deletion is Em sensitive and ABC.⁺ (Fig. D). That is, an unnecessary gene such as a heterologous origin can be removed on this deletion.
[0036]
3. Integration of Cm resistance gene
An integrative plasmid was prepared in which Cm resistance gene (CAT) was used as a model gene (target gene) and expected to be deleted later.
[0037]
YIT9029 Cm resistance gene derived from plasmid pC194 of S. aureus is considered as the target gene to be integrated into the chromosome, taking into account the expression and selectivity in the same strain and Escherichia coli, restriction enzyme cleavage point, gene length, etc. I chose. This structural gene encodes 216 amino acids, and has a restriction enzyme MunI cleavage point at a position corresponding to 9 amino acids from the N-terminus and a restriction enzyme VspI cleavage point at a position corresponding to 42 amino acids from the C-terminal.
[0038]
The target integrative plasmid was prepared according to FIG. First, two plasmids, pMSK755 and pMSK751, were constructed in which the complete Cm gene (CAT) was inserted on each side of attP.
[0039]
Specifically, PCR was used to amplify a fragment containing the Cm-resistant gene of pC194 with both ends at the BstXI breakpoint and a MunI breakpoint immediately inside the BstXI breakpoint on the N-terminal side of the structural gene and cleaved with BstXI And ligated with a SacI partial degradation product of pMSK742 dimer DNA to obtain pMSK753. Similarly, a fragment containing a Cm-resistant gene of pC194 having a NheI cleavage point at the N-terminal end of the structural gene and an XbaI cleavage point at the C-terminal end was amplified and cleaved with NheI and XbaI to completely digest pMSK742 DNA with XbaI To give pMSK751. In addition to the Cm gene (CAT), these two plasmids have an Em resistance gene (Em), an E. coli plasmid DNA replication initiation region (ori), and an integrase gene (int).
[0040]
When YlT9029 was transformed with each plasmid, the frequency was 10^ThreeIn the transformant, it was confirmed that each plasmid was integrated into the chromosome via attP and attB, and that Cm resistance was expressed simultaneously with Em resistance. That is, when Cm at a concentration of 2.5 μ / ml was added to the medium, the Cm-resistant gene-carrying strain could grow and the parent strain could not grow.
[0041]
Next, the Cm resistance genes of the respective plasmids pMSK753 and pMSK751 were deleted. Specifically, pMSK760 and pMSK756 were obtained from the MunI partial degradation product of pMSK753 DNA and the XbaI and VspI degradation products of pMSK751 DNA, respectively. The YIT9029 transformants of these plasmids were sensitive to Cm2.5 μ / ml.
[0042]
Subsequently, both DNAs of pMSK760 and pMSK756 were cleaved with BssHII and ScaI, respectively, and the fragments having the respective defective Cm genes were selected and ligated to obtain the target plasmid pMSK761. The structure of pMSK761 is shown in FIG. The length of the homologous portion common to the two defective genes was 494 bases, and the distance between both defective Cm genes on the side not passing through attP was 3545 bases.
[0043]
4). Acquisition of transformants and selection of Cm expression strains
3. PMSK761 obtained in step 1 was integrated into the YlT9029 chromosome and unnecessary genes were removed. When YIT9029 was transformed with pMSK761, Em resistant transformants could be obtained at the same frequency as pMSK755, and all were sensitive to Cm 2.5 μg / ml.
[0044]
This Em-resistant Cm-sensitive strain was cultured in MRS medium supplemented with 0.5 μg / ml of Cm (non-selective and inductive concentration) for about 40 generations, and then cultured in a medium containing Cm of 2.5 μg / ml. Select 10^-3Resistant strains were isolated at a frequency of All 16 Cm resistant strains examined were Em sensitive.
[0045]
On the other hand, from the transformants of pMSK756 and pMSK760 having only one defective Cm gene, 10^-FiveCm2.5 μg / ml resistant strains were isolated at a frequency of 1 and all 16 strains examined remained Em resistant. This 10^-FiveThe frequency of this is the same as the frequency of isolation of the naturally resistant mutant of Cm2.5 μg / ml in YlT9029.
[0046]
These results show that in some transformants in which the DNA of pMSK761 is integrated into the chromosome, spontaneous homologous recombination in the cell occurs in the homologous region in the two defective Cm resistance genes, and the full-length Cm gene is As a result, resistance was exhibited and the region containing the Em resistance gene was removed.
[0047]
This was supported by analyzing the structure of the obtained Em-sensitive Cm resistant strain chromosome by analyzing the DNA base sequence of the PCR product. That is, i) attL-full length Cm resistance gene-attR were arranged in this order, ii) plasmid DNA replication initiation region for E. coli-Em resistance gene-no integrase gene was found. .
[0048]
4). Stability of chromosomally integrated transformants
Select the stability of the phenotypes of the two Em-sensitive Cm-resistant mutants (integrase gene-deficient) obtained from the pMSK761 transformant, and the proportion of drug-resistant strains after passage in a non-selective medium, It was examined by measuring the number of colonies formed on a non-selected plate. As a result, as shown in FIG. 4, the drug-resistant trait into which any of the integrants was introduced was retained in the YIT9029 transformant at 100% for about 50 generations. In the case of a transformed strain into which a plasmid having no lysogen phage characteristics is introduced, about 5% of strains exhibit drug resistance after about 50 generations of division.
[0049]
Furthermore, when 100 colonies derived from cells after approximately 50 generations of the Em-sensitive Cm-resistant mutant derived from the pMSK761 transformant were examined by individual liquid culture, they all retained drug resistance. From the above results, it is clear that the integration of φFSW into the chromosome by integrase is very effective for stable retention of the target gene. During this period, the presence or absence of the integrase gene did not affect the stability.
[0050]
As described above, in the chromosomal integration vector using intcgrase of the phage φFSW of YIT9029, structural ingenuity was added when inserting the target gene to be introduced onto the chromosome. As a result, the heterogeneous Em resistance gene, the plasmid DNA replication initiation region for E. coli, and the integrase gene derived from the same species could be removed from the final breeding strain by spontaneous homologous recombination in the cell after chromosome integration.
[0051]
In addition, the stability of the phenotype was examined. The trait introduced by site-specific recombination between attP and attB by φFSW integration using this chromosomal integrative vector was transformed into YIT9029 under non-selective conditions. It was quite stable even after cell division in the body for at least about 50 generations. During this period, no difference was found in the stability of the presence or absence of the integrase gene.
[0052]
Based on the above, chromosomal integration using the φFSW integral shown here and subsequent removal of unnecessary genes is stable and safe in the YIT9029 strain, and the final breeding strain is not a recombinant gene depending on the type of target gene. It became clear that engineering breeding was possible.
[0053]
【The invention's effect】
As described above, the present invention has an effect that a method for introducing a foreign gene into a chromosome that can remove an unnecessary gene derived from a vector other than the foreign gene can be obtained.
[Brief description of the drawings]
BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is an explanatory diagram showing an outline of the steps of one embodiment of a method for introducing a foreign gene into a chromosome of the present invention.
FIG. 2 is an explanatory diagram showing a production path of plasmid pMSK761 from plasmid pMSK742.
3 is an explanatory diagram showing the structure of the plasmid pMSK761 obtained in FIG. 2. FIG.
FIG. 4 is a diagram showing the results of a stability test of a chromosomal integration transformant obtained by pMSK761 transformation. The vertical axis represents the ratio of drug resistant strains, and the horizontal axis represents the number of generations.

Claims (1)

Lactobacillus casei YIT9018 strain lysate phage φFSW-derived site-specific recombinase (integrase, int) gene region and integration site into host chromosome (attP region), and a drug that functions in lactic acid bacteria and Escherichia coli A region and B region on the chromosome of Lactobacillus casei using a vector for Lactobacillus casei provided with a resistance gene region, a replication initiation region that functions in Escherichia coli , and a cloning site And the C region, a foreign gene (ABC) (provided that the A region and the C region indicate a region including the cut end of the foreign gene, and the B region is an arbitrary region between both cuts in the foreign gene. Which shows the sequence of the overlapping portion),
1 a first partial sequence (AB) excluding the C region of the foreign gene (ABC) intended for introduction, and a second excluding the A region of the foreign gene (ABC) A step of obtaining a vector in which the partial sequence (BC) is placed on both sides of the attP region in the vector,
2 The process of integrating this into the chromosome of Lactobacillus casei and obtaining a transformant,
And 3) a step of selecting a transformant from which an unnecessary gene portion including the site-specific recombination enzyme region, the drug resistance region and the replication initiation region has been removed by a homologous recombination mechanism. A method for introducing a foreign gene into a chromosome.

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