Method of constructing recombinant strains of microorganisms expressing prokaryotic and eukaryotic genes

 

The invention relates to biotechnology, particularly genetic engineering. Method of constructing recombinant strains of microorganisms provides the expression of the target prokaryotic or eukaryotic gene in any strain of microorganism that supports replication of the vector DNA. Based multicopying plasmids pLF1311 constructed a vector that allows you to easily select the cloned sequence by phenotypic sign bearing his bacteria and Express them. With his help designed probiotic strain Lactobacillus sp. At ONCE (pLF-SL2), which expresses eukaryotic gene somatoliberin. This invention is used to create probiotics and starter cultures drugs on the basis of live cultures of microorganisms. 1 Il.

The invention relates to biotechnology, in particular genetic engineering, and can be used in agriculture, veterinary science, medical, microbiological and food industry to obtain microorganisms with desired properties, including the creation of probiotics and starter cultures drugs on the basis of live cultures of microorganisms.

Currently, use many of sposobnosti closest analogue may be considered a method of obtaining a transformed lactic acid bacteria (international application WO 89/01970; Michiels F., J. Delcour, Mahillon J., Transformed lactic acid bacteria). For the expression of cloned bacterial genes used their natural promoters that are active in the recipient strains of lactic acid bacteria. However, the properties to ensure gene expression in a wide range of cells have not all natural promoters.

Meanwhile, often the challenge is to Express a sequence of the strain of microorganism for which a special expression systems have not been developed, or in various strains of microorganisms that requires the development of a fairly universal way of expression.

The aim of the invention is the creation of DNA molecules for gene cloning, the introduction of cloned genes in lactic acid bacteria and other microorganisms and expression of cloned genes into recipient cells.

The goal is greater flexibility in the expression of the cloned target gene in comparison with analogues is achieved by way of design, which consists in joining one transcription under a universal promoter as replicative vector genes, and subject to the expression of sequences. This method of constructing obespechena.

The invention is implemented in the form of a family of plasmids consisting of a plasmid, a broad host range pLF21, pLF22, pLF-SL1, pLF-SL2. In all of these plasmids include replicon cryptic plasmids pLF 1311, source originating from Lactobacillus fermentum BKM 1311. For their replication is strictly requires the presence in the cells of the gene product gerv, transcription which occurs with identifizierung promoter vector (Aleshin, V. V. et al., FEMS Environ. Lett., 1999, 178, 47-53). The range of bacterial strains hosts, in which replication occurs odnoradiusnyh plasmids family pLF1311, and, accordingly, expression of the gene gerv, was determined (Aleshin, V. V. et al., FEMS Environ. Lett., 1999, 178, 47-53). He was very broad and includes several strains used as probiotics.

The essence of patented genetically engineered solutions illustrated in the drawing, which shows a schematic construction of hybrid plasmids pLF-SL2.

To create the expression vector pLF21 use plasmid pLF20, nucleotide sequence which is fully known. Part plasmids pLF20 is replicative region pLF 1311, including genes Gera and gerv with promoter loci ori+, ori-; cat gene from the plasmid RS (Horinouchi, S., Weisblum C. J. Bacteriol., 1982, 150, 815-825) that directs the synthesis of chlorophenylacetylene pLF20 hydrolyzing restrictase ACC 161 and Bmel42I, this was followed by ligation and transformation in accordance with known methods (Methods of molecular genetics and genetic engineering", Novosibirsk, "Nauka", CO, 1990, S. 7-10, 39-44). After ligation and transformation of selected colonies containing plasmid pLF21, which differs from pLF20 that it does not contain ballast fragment between sites restricts ACC 161 and Bme 142I, within which is located one of the two sites Nae plasmids. In the resulting plasmid pLF21 there are unique sites HindIII and Nae in the spacer between genes Gera and gerv replicative operon that allows you to clone and Express foreign genetic material.

Another significant advantage of the patented method of construction is the increased structural stability of the obtained genetic structures. It is known that the structural stability of recombinant molecules in cells of the microorganism-host poses a serious problem for biotechnology. In the case of structures, obtained a patent pending method, the structural stability of recombinant molecules in cells is provided by the localization of the cloned genes in the intergenic spacer composed rep operon. Any extensive deletions, exciting flankiruyushchei plasmids against whom will thus be a natural selection in living cells without human intervention.

Environmental safety, in particular the prevention of dissemination of recombinant plasmids in nature, is provided by conjugatively and the need for mobilization in specific plasmid-helpers, not capable of replication in gram-positive recipients. Previously it was shown that the origin of the plasmid pLF1311 due to deletion natural mob sites, characteristic for the majority of related plasmids, but not in pLF1311 (Aleshin centuries, Doroshenko Century BC, Cockroaches B. C., Molecular biology, 1998, 32, 415-419).

The invention is illustrated in the examples which characterize the expression of the cloned sequences with promoter replicative genes.

Example 1. Expression-peptide-galactosidase with promoter replicative gene vector plasmids.

To test the expression of foreign DNA from the promoter replicative vector genes between unique sites HindIII and HaeII in the spacer genes Gera and gerv replicative operon was cloned fragment of plasmid pTZ19 (Mead D. A., Szczesna-Skorupa, E., Kemper C. Protein Eng., 1986, 1, 67-74) containing the lacZ gene’. Abrazovanii after hydrolysis with restriction enzyme HindIII and break through completing the 3’protruding end with fragment maple DNA polymerase I of Escherichia coli, connected to the end of the lacZ fragment formed by the hydrolysis of DNA plasmids pTZ19 the BsrBI restriction enzyme in the region of the lacZ operator.

For selection of the desired design ligase mixture were transformed cells of Escherichia coli strain TG1(1 ° C, pro), supE, thi, hsdD5/F’ traD36, proA+B+, ladqand inoculated on selective Cup with LB medium containing chloramphenicol at a concentration of 15 µg/ml of the chromogenic substrate 5-bromo-4-chloro-3-indolyl--D galactopyranoside (X-gal) at a concentration of 50 μg/ml, but not containing inducer of Lac operon isopropyl--D-thiogalactoside (IPTG). As a result, on the second day of incubation at 37oWith colonies were selected, painted in light blue color. They contain plasmid pLF22 and Express working-galactosidase without adding on Wednesday inductor IPTG.

Example 2. Cloning of the synthetic gene somatoliberin part of replicative operon plasmids pLF21.

Cloning of the synthetic gene somatoliberin part of replicative operon plasmids pLF21 carried out in two stages. In the first stage, we solve the problem of amplification of the structural part sinteticheskogo gene somatoliberin and connect it to a regulatory sequence is wyzwania corresponding mRNA bacterial ribosome and initiation of translation. This goal is achieved using the method of amplification of DNA fragments in the in vitro system under the control of thermostable DNA polymerase Taq polymerase chain reaction, PCR). This is done using oligonucleotide primer M1 32 nucleotide residues having the sequence d(5 GAAAGGAAAAHZZZTTT’), in which 19 residues that comprise the 3’ end of the oligonucleotide, complementary to the sequence of the plasmid pMTGRF1 (Brenig Century, G. BREM-Chimica OGGI - Chemistry today, 1993, 11 (1-2), 33-42), carrier synthetic sequence of the structural part of the gene somatoliberin running eukaryotically regulatory elements. This segment M1 has the following sequence: 5’-...ACGTACGTACGTACGT-3’,5’-terminal segment of the Ml primer is not complementary matrix pMTGRF1, this segment contains the sequence 5’-GAITA...3’, corresponding to the consensus SD bacterial genes and canonical spacer elements section between SD and initiation code ATG enriched remnants of adenosine and thymidine. To facilitate the manipulation in the spacer elements between SD sequence and the initiation codon introduced a sequence of recognition of restrictase Apol. Paired primer MP has the structure d(5’-CCATATTCTTGGGAACAA-3’). Amplificatore Techne-3 (England). The composition of the reaction mixture: 10 mm Tris-Hcl (pH of 9.0 at 25°C), 50 mm KS1, 0.1% Triton X-100, 2 mm MgCl2, 0.2 mm of each deoxynucleotidase: DSTF, dATP, dTTP, dCTP 5 PM oligonucleotides MI and MII, 1 ng DNA pMTGRFl and 1 unit of Taq-polymers production Silex (Moscow), the volume of the reaction mixture of 50 μl under a layer of mineral oil. At the end of the process amplificatory DNA fragment from clone by known methods in the vector plasmid pBluescriptKS(+) (Stratagene), pre-linearizing the endonuclease Eco. Bacteria E. coli TG1 containing the plasmid with the gene of somatoliberin form on the indicator Petri dishes in LB medium with ampicillin, chromogenic substrate X-gal and inductor IPTG at a concentration of 50 μg/ml, colonies are white or light blue depending on the orientation of the insert, in contrast to colonies formed by cells with the vector plasmid pBluescriptKS(+), painted in intense blue color. Among light blue colonies are selected colony containing plasmid pSLD2 with tandem duplication sequence somatoliberin in the same orientation as that of lacZvector. Plasmid pSLD2 occurs through transcription somatoliberin+lacZmRNA with bacterial lactose prom is Yong somatoliberin is also under the promoter of bacteriophage T3 and can be expressed in systems in vitro or in vivo in various modifications.

Sequence somatoliberin comprising plasmids pSLD2 not contain transcription terminators, as well as a fragment of the lacZ. Moreover, the asymmetric location of the sites HindIII and Nae in pSLD2 provides cloning of the target fragment in the desired orientation. In the second phase, DNA plasmids pSLD2 and pLF21 in the amount of 0.5 μg sequentially hydrolyzing restrictase Nae and HindIII, purified from the reaction products, are ligated and the ligase mixture was used to transform E. coli TG1. Trasferimenti plated on selective Petri dishes with LB medium with the addition of chloramphenicol with the concentration of 15 μg/ml of the chromogenic substrate X-gal at a concentration of 50 μg/ml After incubation cups at 37°C for 20 h them transferred to a refrigerator with a temperature of 12°C and incubated for 5 days. After incubation select light blue colony phenotype CmrApsthe cells which contain the plasmid pLF-SL2. The sequence of the structural gene region of somatoliberin and lacZplaced inside rep operon vector and transcribed independently from IPT.

Plasmid pLF-SL2 administered Lactobacillus ps. RA processing cell suspension in a solution of DNA pLF-SL2 in a concentration of 0.1 µg in 50 µl of cell suspension using UIN M. C., Livshits C. A., Biol. membrane, 1994, 11, 117-139). To a suspension of cells in an ice bath, add a single rectangular pulse of positive polarity duration of 2.6 MS and the electric field strength of 17 kV/see Immediately after application of the pulse, the sample is diluted chilled nutrient medium and incubated in ice bath for 10 min, then 1 h at 37°C, and then plated on selective medium MRS.

Lactobacillus ps. At ONCE (pLF-SL2) deposited in Russian national collection of industrial microorganisms under registration number In-7495.

The resulting strain is characterized by the following cultural-morphological features. Gram-negative short rods, located disordered clusters or short chains of 3-5 cells. On agar medium MPC-1 and Cattails form a whitish, convex, opaque colonies.

The strain has the following physiological and biochemical properties. The optimum temperature for growth is 37°C, tolerance to the presence of oxygen. Breaks down glucose without the formation of gas. Sprayway sucrose, maltose, lactose, galactose, cellobiose, melezitose, mannose, sorbitol, mannitol, salicin. Not ferments tagatose and rhamnose.

Under the strain of Lactobacil the relevant areas of somatoliberin have bacterial broadcast signal. Transcribe cloned genes somatoliberin comes with a universal replicative promoter, it is constitutive and regulated mainly through chopinot plasmids in the cell.

Claims

1. Method of constructing recombinant strains of microorganisms expressing prokaryotic or eukaryotic gene, including the definition of the functional areas in the composition vector of the molecule, the integration coding region of the gene to be expressed, coupled with regulatory elements, recognizable in the cell host, and the introduction of the DNA into the cells of the microorganism, characterized in that the coding region of the gene is integrated into the structure of replicative operon vector molecules in the direction of transcription.

2. The method according to p. 1, characterized in that the replicative operon vector molecules pre-enter the area with the unique restriction sites.

3. The method according to p. 1, characterized in that the recipient use the microorganism strain of probiotic.

4. The method according to p. 1, characterized in that as a regulatory element use synthetic oligonucleotide sequence corresponding to ri is Iberia.

 

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