Recombinant plasmid dna rdti 23, a method of obtaining a recombinant plasmid dna rdti 23 and the bacterial strain escherichia coli containing a recombinant plasmid dna rdti 23 - producer fused protein til 2

 

(57) Abstract:

Usage: biotechnology, biomedical research T cells. The inventive preparation of recombinant plasmid DNA RDI that determines the synthesis of fused protein of diphtheria toxin-interleukin 2 (DIL) and the bacterial strain Escherichia coli containing this plasmid DNA. Recombinant plasmid DNA GTI has a size of 4.7 T. p. O., gene DTIL is under control of the inducible promoter lactose operon of plasmid V18 that allows you to increase the level of expression of the protein in dozens of times. To obtain plasmids RTI connect the reading frame of the gene of diphtheria toxin (DT) of carinifera and natural gene of Mature interleukin 2 people when using the Eco RI site of the gene DT, embed Bam HI linker, treatment with restriction enzyme Bam HI and subsequent ligation of a DNA fragment into a vector plasmid. The strain E. Li grows well on ordinary nutrient media, resistant to ampicillin and producing a fused protein TIL after induction with isopropyl-b-D-thiogalactoside. 3 S. p. f-crystals, 5 Il.

The invention relates to genetic engineering and relates to a method of obtaining a recombinant protein of diphtheria toxin in the basis diphtheria toxin gene of carinifera genes and polypeptide hormones, such as melanocytestimulating hormone (MSH) or interleukin 2 [1]

Designing genes fused proteins described in this patent only for DT-AMSC and DT-b using gene Assembly DF of individual nucleotide sequences encoding the a and b fragments of diphtheria toxin.

The known method of constructing gene fused protein TIL consisting of cloning the synthetic gene IL2 received on the published sequence IL2 [2] in single Paradise the website of the gene DT.

However, low expression of the fused protein under the control of the native promoter of diphtheria toxin and a high degree of proteolytic degradation are not effectively produce the polypeptide in cells of E. coli.

The task of the invention the receiving slit protein DTIL with antigenic determinants of diphtheria toxin and interleukin 2.

The difference of the present invention is that gene fused protein is constructed on the basis of the diphtheria toxin gene of carinifera w and the natural gene of Mature interleukin 2 people. Connection reading frames made using Eco RI site of the gene DT, embed, You HI linker, processing alnoe difference created plasmids is what gene TIL in rdti is under control of the inducible promoter lactose operon of plasmid pV18 that allows you to increase the level of expression of this protein in ten times compared with previously described [3] ADP-ribosoma activity obtained chimeric toxin corresponds to the activity of the source of diphtheria toxin.

Plasmid RTI encoding a protein of diphtheria toxin-interleukin 2 has a molecular weight of 3.2 d ($4.7 T. p. O.) and consists of the following elements:

fragment Sma I SalGI DNA plasmids VC18, the size of which 2,6 T. p. O.

fragment Eco 24.I SalGI encoding the synthesis of the fused protein of diphtheria toxin-interleukin 2.

The fragments contain the following genes:

gene bla encodes the synthesis of b-lactamase,

gene fused protein DTIL.

Method of constructing plasmids rdti that encodes the synthesis of the fused protein DIL, is that the fragment (2,3, etc., O.) DNA plasmids RDT after complete hydrolysis of restrictase RI and Bam HI and fitting ends to blunt clone in plasmid Rea by blunt RI site; the resulting plasmid rtil restriction enzyme Eco.I and obtuse gap embed You HI linker, after processing which Andonites recombinant plasmid, and gene fused protein TIL distinguish with complete hydrolysis by restrictase ESO.I and SalGI, the protruding 3' ends are removed by treatment T4 polymerase, then the fragment clone by SmaI SalGI sites in the plasmid pVC18.

For solving the problem using the bacterial strain Escherichia coli JM109 (RTI) ACM CR D producer fused protein DTIL.

Cultural-morphological features of the proposed strain

Cells are straight, rod-shaped forms 1,2 1,6 2,0 6,0, mm, gram-negative, risperadone.

Cultural characteristics

Cells grow well on simple nutrient media. When growth on meat-peptone agar, L-agar colonies are smooth, round, shiny, yellowish-white, smooth edge, opaque.

When culture growth in meat-peptone broth and LB-medium formed flat intensive haze.

Physiological and biochemical signs

Cells grow in the range of 4 to 45oC at the optimum pH of 7.2 to 7.4.

As the carbon source used by many carbohydrates, including d-glucose, d - fructose, arabinose, trehalose. Cells do not metabolize acetate, adunit galactose.

The source of nitrogen is predominantly organic in form of peptone and amino acids.

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Antibiotic resistance

Are resistant to ampicillin (up to 800 µg/ml), due to the presence of plasmids RTI.

Example 1.

The method of obtaining the plasmid RDI.

The starting plasmid for the construction of plasmid RTI are plasmid RDT (Fig. 1) cloned in her genome of diphtheria toxin from the SOG. diphteriae (w + tox) and plasmid Rea (Fig. 2) carrying the gene of Mature interleukin 2 people, kindly provided by the E. I. Gren (Institute of organic synthesis, Academy of Sciences of Latvia).

DNA plasmids RDT and RAA allocate according to the method of [4] the Concentration of plasmid DNA determined spectrophotometrically (extinction coefficient e2602600.02 OTP. units/μg. The obtained preparations of plasmids RDT and RAO used to construct plasmids GTI. The design is carried out in several stages in the following sequence (Fig. 3): 6 μg of plasmid DNA RDT incubated with the restriction enzyme RI (15 units) in buffer 3 (0.5 M Tris-HCl, pH 8.0, 10 mm MgCl2, 100 mm NaCl). The reaction is performed at 37oC for one hour. The weight restriction is determined by electrophoresis with 0.8% agarose gel. Obtained after the first restriction analysis of plasmid DNA RDT periostat ethanol and PfP agarose gel. Double restriction enzymes EcoRI and Bam HI from plasmids RDT visiplate fragment of 2.3 kV, carrying diphtheria toxin gene with its own promoter and the sequence encoding the signal peptide. Fragment allocate preparative electrophoresis with 0.8% agarose gel. After completion of electrophoresis the gel is stained in 0.05% solution of ethidium bromide and visualize DNA fragments by irradiating ultraviolet light irradiator DESAGA, Germany) with a wavelength of 340 nm. The band corresponding to the DNA fragment size of 2.3 kV, carefully cut out with a scalpel, placed in a dialysis bag with 0.4 ml of buffer for electrophoresis (10 mm Tris-acetate, pH 8.0, 1 mm EDTA) and subjected to electrophoresis for 1.5 hours, in which DNA moves from the gel in buffer solution. DNA in buffer solution cleanse twice the processing of buffered phenol and ether. Then periostat ethanol, after which the protruding 5' ends of the DNA fragment to complete a blunt fragment maple DNA polymerase I in the presence of four deoxyribonucleotides.

4 kg of plasmid DNA RAA incubated with the restriction enzyme Eco RI (10 units) in buffer 3 for 1 2 hours. Check the completeness enzyme electrophoresis DNA RAA to blunt.

The connection of the DNA fragment of the plasmid RDT size of 2.3 kV and vector DNA (RAA) is carried out in the buffer for ligation (10 mm Tris-Hcl, pH to 7.6, 10 mm MgCl2, 10 mm DTT, 0,0025 mm ATP) in the presence of RNA ligase (20 E. A.) and DNA ligase (10 E. A. ). For ligation mix 3 µg DNA fragment of the plasmid DDT with 1 μg of plasmid DNA RAA. The ligation reaction is carried out at 4oC for 16 hours. Quality ligating checked by electrophoresis with 0.8% agarose gel. The obtained DNA preparation transform cells of bacteria E. Li K12 strain jM109 with the use of calcium chloride by the method of [4] From the obtained ampicillin-resistant(Apr) clones of E. coli produce plasmid DNA by the method Birnbaum [5] and compare the mobility of the selected plasmids 0.8% agarose gel with mobility plasmids RAA. Reduced mobility indicates the presence of insert DNA fragment from a plasmid rd. Plasmid DNA Arrclones of E. coli with reduced mobility is isolated and subjected to restriction analysis. Restriction analysis of plasmids rtil shown in Fig. 3, suggests that the genes of diphtheria toxin and interleukin 2 are tandem and direction of transcription of both genes is the same.

the bottom of the transcriptional unit with the simultaneous deletion of the gene sequence DT, coding receptornegative domain of diphtheria toxin. The literature suggests that receptornegative activity has a C-terminal sequence consisting of 50 A. about. located distal to the second disulfide loop DT. This sequence is encoded, since 1640 nucleotides (counted from 5' Hind III site of the gene DT), and so forth. The design is based on the fact that in the area of 1660 nucleotide gene DT plasmid rtil is a single website to restrictase ESO.I, which generates blunt ends. For this purpose, 5 μg of plasmid DNA Gil treated with restriction enzyme Eco.I (15 E. A.) buffer (10 mm Tris-Hcl, pH of 7.8, 150 mm NaCl, 5 mm MgCl2, 100 μg/ml bovine serum albumin) at 37oC for two hours. The weight restriction is checked by electrophoresis with 0.8% agarose. Fully gidralizovanny DNA plasmids rtil periostat ethanol. Next 0,002 O. E. You HI linker CGGAATCG (NGOs "Enzyme", , Vilnius) built using the enzyme DNA ligase(10 E. A.) and RNA ligase (20 E. A.) in buffer 2 at 4oC for 16 hours. DNA plasmids after ligation reactions periostat ethanol, dissolved in buffer 3, add a restriction enzyme You HI (15 E. A.) and incubated 2 hours at 37oC. After checking the completeness of R is described. The selected DNA fragment is treated with DNA ligase (10 E. A.) in the buffer for ligation at 4oC for 16 hours. Quality ligating checked by electrophoresis with 0.8% agarose gel. Received ligate transform cells of E. Li strain NV using l2methods [4] Transformants are selected on L agar medium containing ampicillin (30 μg/ml). From Aprclones on the results of immunoblotting using antilittering and antiinterleukin antibodies selected those that synthesize protein diphtheria toxin-interleukin 2 (Fig. 4). The molecular weight of this polypeptide containing antigenic determinants as DT and IL2, 70 KD, which coincides well with the results of the calculation based on the nucleotide sequence. Thus was selected plasmid SF39, the nucleotide sequence of which was tested in the area of "joint" genes DT and IL2 by the method of chemical degradation of Maxima-Gilbert. The nucleotide sequence is shown below:

DT.GTG CAC CGG ATC CCA ATC.IL2

Given the data on the primary structure of DT and IL2, and the results of sequencing pSF39, we can conclude that the protein consists of 650 A. with. of which the first 25 N-concebir gene DT, which in E. Li directs constitutive synthesis of DT with average efficiency. To increase the level of synthesis TIL and regulation of this process gene fused protein was placed under the control of the inducible lac promoter plasmids pV18. For this purpose, 5 μg plasmid incubated with the restriction enzyme Eco24.I (15 E. A. ) in buffer (10 mm Tris-Hcl, pH 8.0, 10 mm MgCl2, 1 mm DDT, 100 μg/ml bovine serum albumin) at 37oC for two hours. The weight restriction is checked by electrophoresis. DNA restrictional plasmids SF39 periostat ethanol and remove the protruding 3' Oh ends polymerase of phage T4 (10 E. A.) in the buffer (0,033 M Tris-acetate, pH 7,9; of 0.066 M potassium acetate, 0.01 M magnesium acetate, 0.5 mm DTT, 0,01 mg/ml albumin) at 37oC for 10 minutes. After the resultant deposition rates of ethanol DNA plasmids psF39 treated with restriction enzyme salGI. Check the completeness of restriction and produce preparative electrophoresis, the DNA fragment size of 2.1 kV, carrying the gene fused protein DTIL.

Plasmid DNA vector pVC18 (5 μg) treated consistently in a nuclease enzyme SmaI (15 units) in buffer (20 mm Tris-Hcl, pH 7.4, 5 mm gCl2; 50 mm KCl) at 37oC for 2 hours, and after checking the completeness of the restriction and the resultant deposition rates of ethanol by restriction enzyme Sa. The connection of fragments of DNA plasmids psF39 and vC18 spend legirovaniem the ligase of phage T4 according to the described method. Received medication transform cells jM109 E. Li. From Apr-clones are selected those that contain plasmid VC18 co insertion of a fragment from pSF39 by reduced mobility of plasmid DNA, as described previously. Recombinant plasmids containing the insert is subjected to restriction analysis, and then conduct sequencing of the 5'- and 3'-ends of the gene fused protein by the method of Sanger. As the results of these experiments, the selected recombinant plasmid GTI fully satisfies the map shown in Fig. 5, and has the following structure early gene:

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Thus, plasmid RTI gene fused protein DTIL is under control of the inducible lac promoter. Expression of protein DTIL in the cells of jM109 E. Li (RTI) is achieved only after induction with isopropyl--D-thiogalactoside (IPTG).

Example 2.

Induction of the synthesis of the fused protein in the cells of jM109 E. Li (RTI).

Cells of E. coli jM109 (RTI) grown in 5 ml LB-medium with ampicillin (30 μg/ml) at 37oC for 16 hours. Next night culture was diluted in 15 times Wednesday LB c ampicillin, podras the Noy concentration of 0.001 M and continue to grow up in the same mode [6] the cells are harvested by centrifugation (3000 g, 10 min) and are lysed by freezing and thawing in the presence of lysozyme 2 mg/ml PBRT (10 mm For Na phosphate buffer, pH of 7.2 to 7.4, 0.15 M NaCl, 0.05% tween-20). After the second freeze thawing, centrifuged (10000 g, 15 min), select the supernatant. The presence of a fused protein in the supernatant determined by enzyme immunoassay using equine antibodies to diphtheria toxin and monoclonal antibodies to interleukin 2, as described earlier.

Literature

1. Williams D. P. Parker K. P. Bacha Bishai W. M. Borowski Genbauffe F. Strom, T. B. Murphy J. R. reagent grade toxin receptor binding domain substitution with interleukin-2: genetic construction and properties of a diphteria toxin-related interleukin-2 fusion protein // Protein engineering. - 1987. Vol.1. P. 493-498.

2. Bishai W. R. Rappuoli R. Murphy J. R. High-level expression of a proteolytically sensitive reagent grade toxin fragment in Escherichia coli // J. of Bacteriology. 1987. Vol. 169. P. 5140-5151.

3. Shemyakin I. Anisimov, C. A. Mitrofanova, N. Construction and expression of hybrid proteins diphtheria toxin-interleukin 2 // Molecular biology. 1992, T. 26, S. 1088-1098.

4. Maniatis T. Fritsch E., Sambrook Century Methods of genetic engineering: molecular cloning. TRANS. with ang. M. Mir, 1984.

5. Birnboim, H. C. and Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA // NAR. 1978. Vol.7. P. 1513-1516.

6. Kapralek F. Jecmen P. Sedlacek J. M. Fabry Zadrazil S. Fermentation conditions for high-level expression DNA rdti 23 size of 4.7 T. p. O. and mol.m. 3.1 MDA, which determines the synthesis of fused protein of diphtheria toxin-interleukin 2 (DTIL) containing Sma SalG I I - fragment of plasmid pUC18 size of 2.6 T. p. O. Eco 24.1 SalG I fragment of plasmid pSF39 size of 2.1 T. p. O. corresponding gene fused protein DIL; genetic marker: bla gene, which provides resistance to ampicillin.2 2. A method of obtaining a recombinant plasmid DNA rdti 23, which determines the synthesis of fused protein of diphtheria toxin-interleukin 2 (TIL), namely, that plasmid RDT completely hydrolyzing restrictase EcoRI and BamHI, isolated DNA fragment size 2,3, etc., of O. the corresponding gene of diphtheria toxin (DT) with its own promoter and the sequence of the signal peptide, the 5'ends of the fragment indicated complete to blunt using DNA polymerase I maple, connect it with vector DNA RAA by EcoRI site to obtain plasmid rtil, this plasmid digested with the restriction enzyme Eco 72.1 and obtuse gap embed BamHI-linker, and then the plasmid rtil cleaved by endonucleases BamHI and produce a DNA fragment with a size of 7.2 T. p. O. realize it ligation obtained by ligate transform cells of E. coli bacteria and selected plasmid psF39 containing gene fused belkaid 3'-OH ends and then by restriction enzyme SalGI isolated DNA fragment size of 2.1 T. p. O. the corresponding gene fused protein DTIL, embed it on SmaI SalGI-site in the plasmid pUC18 and get the target plasmid, rdti 23, which determines the synthesis of fused protein DTIL under control of the inducible promoter.2 3. The strain of bacteria Esherichia coli ACM CR-D containing recombinant plasmid DNA rdti 23, producing fused protein DIL.

 

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