JP2002281966A - Transformed lactobacillus obtained by allergen gene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a transformed lactobacillus producing extraneous allergen protein. SOLUTION: This transformed lactobacillus is obtained by transforming lactobacillus with a recombinant vector obtained by inserting a polynucleotide encoding the allergen protein under a control of a promoter acting in the lactobacillus, and is characterized by producing the extraneous allergen protein.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The invention of this application relates to a lactic acid bacterium transformed with an allergen gene. More specifically, the present application relates to a new transformed lactic acid bacterium that produces a specific allergen protein and can be used as a lactic acid bacterium beverage for desensitization therapy of allergic patients, for example.

[0002]

2. Description of the Related Art Allergic diseases (type I allergy)
A disease that develops as a result of an immune response (reaction involving an IgE antibody) to an allergen (antigen), and diseases such as atopic bronchial asthma, hay fever, some urticaria, and anaphylactic shock are known. . This allergic disease is caused by sensitization of the body to allergens
When an E antibody is produced and the allergen that has re-entered mast cells or basophils to which the IgE antibody binds via the IgE receptor acts, cross-linking occurs between IgE receptors on the cell membrane, and various influxes such as influx of Ca ions occur. After the reaction, histamine,
Symptoms develop due to release of chemical mediators such as leukotriene, eosinophil chemotactic factor, platelet activating factor.

[0003] Therefore, in the treatment of allergic diseases, drugs that block the above-mentioned reaction pathway (for example, antihistamines and the like) are used, but they are merely coping treatments for temporary relief of symptoms. I cannot expect a radical cure. For this reason, "allergen removal therapy" has long been used for treating allergic diseases. In other words, allergic diseases are caused by allergens entering the body where specific IgE antibodies are already present, and are caused by various tests (interviews, skin tests, RAST, histamine release test, provocation test, etc.) Improvement of living environment and improvement of eating habits for identifying allergens and avoiding allergens as much as possible have been attempted.

[0004] However, in a modern era in which lifestyles and eating habits are diversified, it is practically impossible to completely avoid the cause allergen. Therefore, "hypersensitization therapy" has attracted attention as a treatment for suppressing the onset of allergic symptoms even when the cause allergen has entered the living body. This is a treatment method in which a small amount of the allergen is injected, the amount is gradually increased, and allergic symptoms do not occur even when exposed to a large amount of the allergen. Its history is old and since it was used to treat hay fever in 1911,
It has been adopted for the treatment of various allergic diseases, and its efficacy has been confirmed.In recent years, with the diversification of allergic symptoms and the severity of the symptoms, various allergen proteins have been produced in large quantities and at low cost by DNA recombination technology Due to the availability, it is becoming widely used clinically.

[0005] However, in the case of desensitization therapy, it is necessary to repeat many allergen injections before the effect is obtained, and the burden on the patient is not insignificant.

On the other hand, lactic acid bacteria have been used for a long time in food production because they suppress the growth of putrefactive bacteria and have a good taste of lactic acid. For example, fermented milk, cheese, alcohol, miso,
It is known that it plays an important role in the production of fermented foods such as soy sauce and pickles. In addition, lactic acid bacteria living in the human intestinal tract have numerous effects on the human body, including intestinal action, serum cholesterol lowering action, immunity enhancing action, antitumor action, antimutant action, and improved absorption of proteins, fats, calcium, and iron. It is becoming clear in recent years that it provides utility. The presence of lactic acid bacteria has been reviewed as probiotics (environmental microorganisms that have beneficial effects on the host).

[0007]

The object of the present invention is to provide a transformed lactic acid bacterium in which an allergen gene has been introduced into a lactic acid bacterium which has been used safely in food production and has many effects on the human body. It is an issue. That is, such a transformed lactic acid bacterium is used not only for producing an allergen protein, but also for ingesting a fermented food produced using the lactic acid bacterium, for example, to enable simple and effective desensitization therapy.

[0008]

SUMMARY OF THE INVENTION The present invention relates to an invention for solving the above-mentioned problems, which is to transform a recombinant vector into which a polynucleotide encoding an allergen protein is inserted under the control of a promoter sequence acting on lactic acid bacteria. And a transformed lactic acid bacterium, which produces the allergen protein.

Hereinafter, embodiments of the present invention will be described in detail.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The transformed lactic acid bacterium of the present invention can be produced by transforming a lactic acid bacterium with a recombinant vector into which a polynucleotide encoding an allergen protein has been inserted under the control of a promoter sequence acting on the lactic acid bacterium. it can.

The vector is not particularly limited, and a known expression vector having an origin capable of replicating in lactic acid bacteria, a ribosome binding site, a DNA cloning site, a terminator and the like can be used, and a plasmid vector derived from lactic acid bacteria is preferably used. I do. Examples of plasmid vectors derived from lactic acid bacteria include plasmid pRN14 developed by the inventors of the present application. This pRN14
Plasmid pLTK2 of lactic acid bacteria Lactobacillusplantarum L137
(About 2.3 kb) and plasmid vector pBluescrip for E. coli
This is a shuttle vector of Escherichia coli and lactic acid bacteria, in which tIISK + (about 3.0 kb) and an erythromycin resistance gene (about 1.5 kb) acting on Gram-positive bacteria are linked (Journal of Japan).
Bioscience and Bioengineering 89 (1): 62-67, 200
0).

Other vectors that can be used for lactic acid bacteria include general pGK13 (Applied and Envir
onmental Microbiology 48: 726-731, 1084) and pSA3
(Applied and Environmental Microbiology 49: 115-11
9, 1985), and these vectors can also be used in the present invention.

A vector for transformation is constructed by inserting a polynucleotide encoding an allergen protein under the control of a promoter sequence acting on lactic acid bacteria. The polynucleotide may be inserted into a vector having a promoter acting on lactic acid bacteria, or may be inserted into the vector after the promoter and the polynucleotide are ligated in advance.

The promoter linked to the polynucleotide encoding the allergen protein may be any promoter as long as it acts on lactic acid bacteria. Preferably, the promoter sequence of any genomic gene of lactic acid bacteria is used. . Lactic acid bacteria genes are, for example, DDBJ / EMBL / GenBank
The database contains more than 1000 entries.Based on these sequence information, a probe is produced, a gene is isolated by screening a lactic acid bacterium genomic library, and its promoter region is excised to control its expression in lactic acid bacteria. Can be obtained. For example, L. cloned by the inventors of this application.
Acetyl coenzyme Acarboxylase from plantarum L137
(Acc) Operon (GenBank accession No. AB025973)
(Acc promoter: base sequence positions 664-960 of AB025973) can be used.

As the polynucleotide encoding the allergen protein, a genomic gene, mRNA, cDNA or the like of the protein acting as an allergen can be used. For example,
The DDBJ / EMBL / GenBank database contains more than 1000 allergen genes and cDNAs from various animals and plants, and by screening the genomic and cDNA libraries of each animal and plant based on these sequence information. And a target polynucleotide can be prepared. For example, Dermatophagoides farinae major allergen Derf1 (Immunology 44 (2): 239-255, 1)
981) and Dermatophagoides farinae allergen Derf7 (Clinical an
d Experimental Allergy 25 (10): 1000-1006, 1995). When producing a fermented food using the transformed lactic acid bacterium of the present invention,
It is preferable to express a safe allergen derived from food microorganisms. For example, a mold-derived alkaline serine protease (Int. Arch. Allergy Immunol. 116 (1): 29-35, 1)
998) and xylosidase (J. Allergy Clin. Immunol. 1
02 (2): 256-264, 1998), cDNA encoding wheat allergen (eg, GenBank accession No. U91981)
And cDNAs encoding milk allergens (eg, GenBa
nk accession No. AJ404653) can also be used.

The lactic acid bacteria transformed by the above-mentioned recombinant vector are lactic acid bacteria (genus lactobacillus or Streptoco
ccus) or microorganisms that inhabit the human intestinal tract and have beneficial effects on digestion and absorption (eg, La
bacteria belonging to the genus ctococcus, the genus Lactobacterium, the genus Bifdobacterium, and the like. Such a lactic acid bacterium can be obtained by isolating it from a commercially available product or a dairy beverage, and culturing and growing it.

In order to introduce a recombinant vector into lactic acid bacteria,
It can be performed by a known method such as a calcium treatment method, a protoplast method, and electroporation.

Hereinafter, the invention of this application will be described in more detail and specifically with reference to examples, but the invention of this application is not limited by the following examples.

[0019]

[Examples] (1) Transformation efficiency of the plasmid vector pRN14 L. plantarum and the shuttle vector pR of E. coli
N14 (FIG. 1) was introduced into bacteria of the genus Lactobacillus by electroporation, and the transformation efficiency was examined. Electroporation conditions are: DNA 0.1 μg; electric capacity 25
μF; electric field strength 10.0 kV / cm; resistance 400 Ω; ssDNA 0.05%. In addition, L. plantarum L137 used in this test was
It was isolated from the Philippine fermented food Burong Isda. Also
L. casei K95-5 was isolated from a commercially available lactic acid beverage.

The results are as shown in Table 1, and L. pla
The transformation efficiency of ntarum L137 was 1.8 × 10 ⁴ per 1 μg of DNA, and the transformation efficiency of L. casei K95-5 was 1.8 × 10 ³ per 1 μg of DNA.

[0021]

[Table 1]

(2) Confirmation of Transformation with Recombinant Vector Acc promoter derived from L. plantarum L137 (Genebank a
ccession No. AB025973, a polynucleotide comprising the nucleotide sequence at positions 664-960), and a cDNA of choA, a cholesterol oxidase gene derived from actinomycetes (J. Bacteriol. 171: 596-6).
01, 1989; J. Bacteriol. 172: 3644-3653, 1990; GenB
Ank accession No. M31939) was inserted into the shuttle vector pRN14 to construct a recombinant vector pRNa-fchoA whose restriction enzyme map is shown in FIG. That is, as shown in FIG. 3, the acc promoter region was cloned into a pT7Blue vector (Novagen) to construct pT7blue-Pacc, and the SacI-PstI fragment of
Plasmid pRNPacc was constructed by inserting into the acI-PstI site. And choA's c on NdeI-EcoRV site of this pRNPacc
The DNA fragment was inserted and the recombinant vector pRNa-
fchoA was constructed. In addition, the choA gene and acc
Vector pR for inserting a promoter and expressing choA directly
Na-dchoA was also constructed.

Lactic acid bacteria are prepared by electroporation.
L. plantarum NCL21 and L. casei K95-5, and E. coli E.
coli JM109 was transformed. Erythromycin or ampicillin resistant transformants obtained at 30 ° C or 37 ° C
The cells were centrifuged, collected, centrifuged, and the obtained cells were treated with a lytic enzyme to disrupt the cells. In addition, the presence of the recombinant vector was confirmed from the same transformant. L.
plantarum NCL21 is a strain in which 9 plasmids out of 15 plasmids of L. plantarum L137 have been eliminated by Novobiocin.

Cholesterol oxidase activity in the obtained transformant was measured by a colorimetric assay using a chromogen. The results are as shown in Table 2. Enzyme activity was observed in Escherichia coli and lactic acid bacteria, and it was confirmed that the transgene was expressed. However, when choA was directly expressed, the specific activity was reduced to about 1/10 compared to that obtained by fusion expression with the acc promoter. It was confirmed that it could be used as a promoter for expression.

[0025]

[Table 2]

(3) Preparation of Lactic Acid Bacteria Transformed by Mite Allergen Gene Plasmid pBS-Derf1 containing cDNA of major allergen gene Derf1 of Dermatofagoides farinae
(Immunology 44 (2): 239-255, 1981) as a template,
The insert region of this plasmid was amplified by PCR. XbaI and BamHI recognition sequences were added to the PCR primers. The obtained PCR product was subcloned into a pGEM-T vector (Promega) to construct pGEM-Derf1 (see FIG. 4).

Next, as shown in FIG. 5, pGEM-Derf1
Was cut out with XbaI-PstI and pRNPacc (FIG. 3)
Into the SpeI-PstI site of the Derf1 expression vector pRN
a-Derf1 was constructed.

The expression vector pRNa-Derf1 was ligated to L. plantarum NCL21 and L. plantarum by electroporation.
After introduction into L. casei K95-5, the expression of Derf1 protein in erythromycin-resistant transformants was examined by dot western blotting using an anti-Derf1 mouse monoclonal antibody.

The results are as shown in FIG. 6, and compared to the control (lactic acid bacteria into which pRN14 was introduced), pRNa-De
A strong signal was detected in lactic acid bacteria transformed with rf1.

[0030]

As described in detail above, the present application provides a transformed lactic acid bacterium that produces an exogenous allergen protein. From this transformed bacterium, a purified allergen protein that can be used for desensitization therapy or the like can be produced in large quantities at low cost. In addition, by ingesting fermented foods produced using the transformed lactic acid bacteria, it becomes possible to perform desensitization therapy without resorting to regular hospital visits and injections.

[Brief description of the drawings]

FIG. 1 is a restriction map of a lactic acid bacterium-E. Coli shuttle vector pRN14 that can be used in the present invention. pLTK2
Indicates a plasmid sequence of lactic acid bacteria L. plantarum, and Em indicates an erythromycin resistance gene sequence.

FIG. 2 is a restriction map of an actinomyces choA gene expression vector pRNa-fchoA.

FIG. 3 is a restriction map of a plasmid vector showing a process for producing a vector pRNPacc having an acc promoter.

FIG. 4. Derf, a major allergen gene of Dermatophagoides farinae
1 is a restriction map of a plasmid, showing a process for producing a cloning vector pGEM-Derf1 having a 1.

FIG. 5 is a restriction map showing a process for producing a recombinant vector pRNa-Derf1 having an acc promoter and a Derf1 gene.

FIG. 6: Der in lactic acid bacteria transformed by Derf1 gene
It is the result of dot western blotting which examined f1 expression. 1 is transformation with pRN14 L. plantarum NCL2
1 and 2 were transformed with pRNa-Derf1 L. plantarum NCL2
1 and 3 are transformed with pRN14 L. casei K95-5, 4 is pRNa
Transformation with -Derf1 L.. Casei K95-5.

 ────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Masaji Kawamoto 8-6-3-203 Saijo Chuo, Higashihiroshima City, Hiroshima Prefecture F term (reference) 4B018 LB08 LE05 MD86 ME07 MF13 4B024 AA01 BA31 CA04 DA05 EA04 FA15 GA14 HA01 4B064 AG31 CA02 CA19 CC24 DA01 4B065 AA30X AA99Y AB01 AC14 BA02 BA03 CA24 CA44

Claims (1)

[Claims] 1. A lactic acid bacterium transformed by a recombinant vector into which a polynucleotide encoding an allergen protein has been inserted under the control of a promoter sequence acting on the lactic acid bacterium, wherein the lactic acid bacterium produces the allergen protein. Converted lactic acid bacteria.