KR100676296B1 - A vector for a microorganism of Lactobacillus a host cell containing the same and method for integrating foreign polynucleotide into the chromosome of a microorganism of Lactobacillus - Google Patents

Abstract

The present invention provides a vector for Lactobacillus comprising Lactobacillus-derived 16S rDNA, a host cell comprising the same, and a method of inserting a foreign polynucleotide into the chromosome of Lactobacillus.

Lactobacillus, 16S rDNA

Description

Vector for a microorganism of Lactobacillus, a host cell containing the same and method for integrating foreign polynucleotide into the chromosome of a microorganism of Lactobacillus}

1 is a view showing a restriction map of the pCJ100 vector according to an embodiment of the present invention.

Figure 2 is an electrophoresis picture showing the results of PCR using genomic DNA of the Lactobacillus paracasei strain transformed with the pCJ100 vector as a template, oligonucleotides having the nucleotide sequence of SEQ ID NO: 6 and 7 as a primer.

The present invention relates to a vector for Lactobacillus microorganisms, a host cell comprising the same, and a method for inserting a foreign polynucleotide into the chromosome of Lactobacillus microorganisms.

Microorganisms of the genus Lactobacilli are probiotics commonly found in the human stomach and have functions to promote human health. Recently, many studies have been conducted to use Lactobacillus as an enzyme or vaccine carrier, but there is no established integration system such as Escherichia coli.

In the past, there have been studies to insert foreign polynucleotides into chromosomes in Lactobacillus microorganisms. Serror P, Appl Environ Microbiol, 2002 Jan; 68: 46-52, discloses a method using phage integrative elements. That is, a method of inserting a foreign polynucleotide into a chromosome of a Lactobacillus microorganism using a vector having an integrase gene of bacteriophage and an attP site is disclosed. Also known are site-specific chromosomal insertion methods that do not use phage. For example, Russell WM, Appl Environ Microbiol, 2001 Sep; 67: 4361-4 discloses a method of inactivating the lacL gene using two plasmids. In addition, a method of inserting an extraneous polynucleotide into a chromosome of a Lactobacillus microorganism using the extracellular endoglucanase A ( celA ) gene derived from Clostridium thermocellum strain as a selection marker has recently been disclosed . (Jang SJ, FEMS Microbiol Lett. 2003 Jul 29; 224: 191-5).

However, according to the above-described prior art, since a specific gene or a specific selection marker is used, the range of applicable strains is not wide and the selection process is complicated. Therefore, a new vector for Lactobacillus was required even in the prior art.

The present inventors earnestly studied to solve the problems of the prior art as described above, and confirmed that the vector containing 16S rDNA derived from the microorganism of the genus Lactobacillus was easily inserted into the chromosome to complete the present invention.

An object of the present invention is to provide a vector that can be inserted into the chromosome of the genus Lactobacillus.

Another object of the present invention is to provide a host cell comprising the vector.

Still another object of the present invention is to provide a method for inserting a foreign polynucleotide into a chromosome of a genus Lactobacillus using the vector of the present invention.

The present invention provides a vector for the genus Lactobacillus comprising the polynucleotide of SEQ ID NO: 1.

In the vector of the present invention, the polynucleotide of SEQ ID NO: 1 encodes a 16S rRNA of Lactobacillus paracasei , a strain of the genus Lactobacillus .

One embodiment of the invention is a vector further comprising a polynucleotide encoding an activity for imparting resistance to antibiotics that are lethal to Lactobacillus genus bacteria. The polynucleotide of SEQ ID NO: 1 and the polynucleotide encoding the activity conferring resistance to the antibiotic are operably linked to appropriate regulatory sequences.

In one embodiment of the invention, the vector of the present invention is a vector comprising a polynucleotide of SEQ ID NO: 2. The polynucleotide of SEQ ID NO: 2 encodes a resistance protein for erythromycin. The polynucleotides of SEQ ID NOs: 1 and 2 are operably linked to appropriate regulatory sequences. Preferably, the vector of the present invention is a pCJ100 vector having a nucleotide sequence of SEQ ID NO: 3.

In the present invention, "vector" is used in the meaning commonly known in the art. A "vector" is a foreign chromosomal element that usually contains genes that are not part of the cell's central metabolism, usually circular double-stranded DNA. The element may be a self-replicating sequence, genome insertion sequence, phage or nucleotide sequence, linear or circular, single or double stranded DNA or RNA. Generally, such vectors include sequences that direct the transcription and translation of appropriate genes, selectable markers, and sequences that allow self-replicating or chromosomal insertion. Suitable vectors include the 5 'region of the gene containing transcription initiation regulation and the 3' of the DNA fragment that controls transcription termination. The "suitable regulatory sequence" refers to a sequence that functions to control the transcription and translation of the polynucleotide. Such regulatory sequences include, for example, ribosomal binding sequences (RBS), promoters and terminators. The promoter may be any sequence as long as it can direct the initiation of transcription of the gene of the present invention. For example, CYC1, HIS3, GAL1, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3 , LEU2, ENO, TPI (useful for expression in Saccharomyces), lac, trp, λP _L , λP _R , T7, tac and trc (useful for expression in E. coli ) can be used. In addition, the terminator region may be derived from various genes of a preferred host cell, and may also be optionally not required.

The invention also provides host cells comprising the vectors of the invention. Such host cells include, for example, genus Escherichia and microorganisms of the genus Lactobacillus. Preferably, the host cell is Escherichia coli or Lactobacillus paracasei .

The vector of the present invention contains a polynucleotide encoding Lactobacillus-derived 16S rDNA and, when introduced into a microorganism of the genus Lactobacillus, has the property of being inserted into the chromosome. Therefore, the vector of the present invention can be usefully used for stably inserting the vector itself or foreign genes contained therein into the chromosomes of Lactobacillus microorganisms.

Accordingly, the present invention provides a method for inserting a foreign polynucleotide into a chromosome of a Lactobacillus microorganism, comprising introducing a vector of the present invention into a Lactobacillus microorganism.

As a method for introducing a vector of the present invention into the genus Lactobacillus microorganism, any vector introduction method known in the art may be used. For example, electroporation and particle bombardment may be used, but is not limited to these examples. The microorganism may include any of the microorganisms of the genus Lactobacillus, but is preferably Lactobacillus paracasei .

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example

Example 1 16S rDNA erm ^R  Gene cloning

In this example, the Lactobacillus paracasei 16S rRNA gene (hereinafter, also referred to as 16S rDNA) sequence of all species belonging to Lactobacillus by nucleotide search from the National Institute of Biological Information (NCBI) ( www.ncbi.nlm.nih.gov ) We confirm that we have about 99% homology to. From this, when preparing a vector comprising the 16S rRNA gene sequence, it is assumed that foreign polynucleotides can be easily inserted into a wide range of Lactobacillus microorganisms, and from there, oligonucleotides having the nucleotide sequences of SEQ ID NOs: 4 and 5 Primer was prepared.

Next, genomic DNA was extracted from the Lactobacillus paracasei strain, which was subjected to PCR using an oligonucleotide having a nucleotide sequence of SEQ ID NOs: 4 and 5 as a template, as a primer, and 16S rDNA (about 829 bp). Was amplified.

In addition, PCR using a oligonucleotide having the nucleotide sequences of SEQ ID NOs: 6 and 7 as a template using genomic DNA of Streptococcus pyogenes containing an erythromycin resistant gene (erm ^R ) as a template performed by the erythromycin resistance gene was amplified ^{(erythromycin resistant gene, erm R)} ( approximately 1362 bp). The primers having the nucleotide sequences of SEQ ID NOs: 6 and 7 contain the recognition site of restriction enzyme Hind III.

Amplified 16S rDNA and erm ^R By cloning the gene in each of pCR2.1-TOPO vector using the TOPO TA Cloning Kit (Invitrogen, USA), was prepared in the pCR2.1-TOPO-16S rDNA and the pCR2.1-TOPO- ^R erm.

Example 2 : Preparation of new Lactobacillus chromosome integration vectors

The pCR2.1 vector was autoligated by treatment with the restriction enzyme Dra I to remove the amp ^R gene from the vector. The 16S rDNA amplification product obtained in Example 1 was cloned into a pCR2.1-TOPO-16S rDNA vector treated with EcoR I to cut out the 16S rDNA gene and purified. In addition, pCR2.1 vector, in which amp ^R was removed, was treated with EcoR I and then treated with CIP (calf intestinal phosphatase) at a volume of 30 μl.

Into 100 ng of 16S rDNA gene prepared above and 10 ng of pCR2.1 (Δamp ^R ) , 1 μl of T4 DNA ligase and 1 μl of ligase buffer were added and adjusted to a total volume of 10 μl. . After the reaction, E. coli DH5α was transformed to isolate and purify pCR2.1 ( Δamp ^R ) -16S rDNA containing the 16S rDNA gene.

PCR2.1-TOPO- erm ^R cloned with the erm ^R amplification product obtained in Example 1 was treated with the vector Hind III to cut out the erm ^R gene, and was isolated and purified. In addition, pCR2.1 ( Δamp ^R ) -16S rDNA vector containing the 16S rDNA gene was treated with Hind III, and then treated with CIP (calf intestinal phosphatase) at a volume of 30 μl.

As described above, 1 μl of T4 DNA ligase and 1 μl of ligase buffer were added to 100 ng of the Hind III and CIP treated erm ^R gene and 10 ng of pCR2.1 ( Δamp ^R ) -16S rDNA and adjusted to a total volume of 10 μl. Then, the reaction was carried out at 16 ° C. for 6 hours. After the reaction, E. coli DH5α was transformed to isolate and purify pCR2.1 ( Δamp ^R ) -16S rDNA- erm ^R containing the erm ^R gene.

1 is a diagram showing a restriction map of pCR2.1 ( Δamp ^R ) -16S rDNA- erm ^R prepared in the present example. The vector has a size of about 5405 bp, and has a erythromycin resistance gene and 16S rDNA, as well as a kanamycin resistance gene, which can be used to select a transformed host cell.

PCR2.1 (Δamp ^R ) -16S rDNAerm ^R (hereinafter also referred to as pCJ100 vector) prepared in this example was used as a vector for inserting foreign polynucleotides into the Lactobacillus chromosome.

Example 3 : pCR2.1 (△ amp ^R ) -16S rDNA- erm ^R Transformation into Lactobacillus

Lactobacillus paracasei strain was inoculated in MRS medium (Difco, cat # 288130) (25 mL culture in 250 mL flask) and incubated at 37 ° C., 200 rpm for 12 hours, followed by 100 mL MRS medium (1% glycine, 250 mL flask) was inoculated to OD ₆₀₀ = 0.25 and incubated until OD ₆₀₀ = 0.6. Thereafter, centrifugation was performed at 1500 g for 5 minutes, followed by washing twice with 100 mL SM (326 g sucrose and 0.71 g MgCl ₂ 6H ₂ O to 1 L, filter sterilized). After resuspending in 1 mL SM buffer and 100 ㎕ were dispensed.

The insertion vector for production in Example 2 on the comb peteon bit cell made as ^{above, pCR2.1 (amp R) -16S rDNA-} erm R 2 mm electroporation cuvette (electroporation cuvet) being 1 μg were stored on ice after addition of And electroporated to 1.5 kV, 25 μF, 800 Ω and immediately added 1 mL of MRSSM (MRS + 0.5 M sucrose + 0.1 M MgCl ₂ ). After reacting for 6 minutes in a 46 ℃ water bath and incubated for 3 hours at 37 ℃ and plated on MRS plate containing 5 μg of erythromycin. The resulting colonies were once again confirmed on MRS plates containing 20 μg of erythromycin.

Example 4  : Confirmation of Vector Insertion in Lactobacillus

In this example, genomic DNA was extracted from the transformed Lactobacillus paracasei strain obtained in Example 3 and the Lactobacillus paracasei strain before transformation. PCR was carried out using oligonucleotides having the nucleotide sequences of SEQ ID NOs: 6 and 7 as primers as the template of each genomic DNA to finally confirm whether the vector was inserted into the chromosome. That is, each PCR product was subjected to electrophoresis, and from there it was confirmed whether there was a band corresponding to the size of the pCJ100 vector, thereby confirming whether the vector was inserted into the chromosome.

Figure 2 is an electrophoresis picture showing the results of PCR using genomic DNA of the Lactobacillus paracasei strain transformed with the pCJ100 vector as a template, oligonucleotides having the nucleotide sequence of SEQ ID NO: 6 and 7 as a primer. In Figure 2, lane 1: genomic DNA of Lactobacillus paracasei, lanes 2-6: genomic DNA of Lactobacillus paracasei, transformed to show resistance to erythromycin, lane 7: PCR results of pCJ100 vector as a template Indicates. As shown in Figure 2, it can be seen that the strain of lane 4 retains the pCJ100 vector inserted into the chromosome.

From the results of the present invention as described above, it was confirmed that by using a vector containing 16s rDNA of Lactobacillus paracasei, the vector itself or foreign polynucleotides can be easily inserted into the chromosome of the microorganism of the genus Lactobacillus. .

According to the vector of the present invention, foreign polynucleotides or genes can be efficiently inserted into chromosomes of microorganisms, in particular, Lactobacillus microorganisms. Therefore, the vector of the present invention is useful as a carrier when the foreign polynucleotide or gene is to be expressed in a microorganism of the genus Lactobacillus.

According to the host cell of the present invention, foreign polynucleotides or genes carried by the vector of the present invention can be stably expressed.

According to the method of inserting the foreign polynucleotide of the present invention into the chromosome of the Lactobacillus microorganism, the foreign polynucleotide can be efficiently inserted into the chromosome.

Attach sequence list electronic file  

Claims (8)

A recombinant vector for the genus Lactobacillus comprising a polynucleotide of SEQ ID NO: 1 operably linked to one or more regulatory sequences selected from the group consisting of a self-replicating sequence, a ribosomal binding sequence, and a promoter. The recombinant vector of claim 1, wherein the recombinant vector further comprises a polynucleotide of SEQ ID NO: 2 operably linked to one or more regulatory sequences selected from the group consisting of a self-replicating sequence, a ribosomal binding sequence, and a promoter. delete The recombinant vector of claim 1, wherein the recombinant vector is a pCJ100 vector having a nucleotide sequence of SEQ ID NO. A host cell comprising the recombinant vector according to any one of claims 1, 2 or 4. The host cell of claim 5, wherein the host cell is Escherichia coli or Lactobacillus paracasei . Inserting a foreign vector into the chromosome of the genus Lactobacillus, comprising introducing a recombinant vector according to any one of claims 1, 2 or 4 comprising the foreign polynucleotide into a genus Lactobacillus microorganism. How to let. The method of claim 7, wherein the microorganism is Lactobacillus paracasei .

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