RU2077585C1 - RECOMBINANT PLASMID DNA "pДТИ-23", METHOD OF PREPARING RECOMBINANT PLASMID DNA "pДТИ-23" AND STRAIN OF BACTERIUM ESCHERICHIA COLI CONTAINING RECOMBINANT PLASMID DNA "pДТИ-23" - A PRODUCER OF THE FUSED PROTEIN DTI-2 - Google Patents

Abstract

FIELD: biotechnology. SUBSTANCE: invention relates to preparing the recombinant plasmid DNA "pДТИ-23" determining synthesis of the fused protein diphtherin toxin/interleukin-2 (DTI-2) and strain of bacterium Escherichia coli containing the indicated plasmid DNA. Size of recombinant plasmid DNA "pДТИ-23" is 4.7 thousand base pairs. Gene diphtheric toxin (DT) containing in this plasmid is under control of inducible lactose operon promoter from plasmid pV18. Method of preparing plasmid "pДТИ-23" involves linkage of frame reading of gene diphtheric toxin from corynephage ω and natural gene of the human mature interleukin-2 using Eco RI-site of gene DT, insertion of Bam HI-linker, treatment with restriction endonuclease Bam HI followed by ligation of DNA-fragment in vector plasmid. Strain E. coli grows well on the simple nutrient media, shows resistance to ampicillin and produces the fused protein DTI-2 after induction with isopropyl-b-D-thiogalactoside. Recombinant plasmid can be used in medicine and biological investigations of T-cells. EFFECT: improved method of plasmid preparing. 3 cl, 5 dwg

Description

 The invention relates to genetic engineering and relates to a method for producing a recombinant protein diphtheria toxin interleukin 2 (DTIL2) in bacteria cells of E. coli.

A known method of creating redirected toxins based on the diphtheria toxin gene of corynephage β and polypeptide hormone genes such as melanocytostimulating hormone (MSH) or interleukin 2 [1]
The design of fusion protein genes is described in this patent only for DT-aMSH and DT-bMSH using the assembly of the DT gene from individual nucleotide sequences encoding A and B fragments of diphtheria toxin.

 A known method of constructing the gene fused protein DTIL2, consisting in cloning a synthetic gene IL2 obtained from the published sequence of IL2 [2] in a single Paradise site of the DT gene.

 However, the low expression of the fusion protein gene under the control of its own promoter of diphtheria toxin and the high degree of its proteolytic degradation do not allow efficient production of this polypeptide in E. coli cells.

 The objective of the invention is the obtaining of a fused protein DTIL2 having antigenic determinants of diphtheria toxin and interleukin 2.

 The difference of the invention is that the fusion protein gene is constructed on the basis of the diphtheria toxin gene from corynephage w and the natural human mature interleukin 2 gene. The reading frames were connected using the Eco RI site of the DT gene, embedding the Bam HI linker, Bam HI restriction endonuclease treatment and subsequent ligation of the DNA fragment (Fig. 3) in a vector plasmid. An additional difference of the created plasmid is that the DTIL2 gene in rDTI23 is controlled by the inducible promoter of the lactose operon from the plasmid pVC18, which makes it possible to increase the expression level of this protein tens of times in comparison with the previously described [3] ADP-ribosalizing activity of the obtained chimeric toxin corresponds to activity of the original diphtheria toxin.

The plasmid rDTI23 encoding a diphtheria toxin-interleukin 2 fusion protein has a molecular weight of 3.2 Md (size 4.7 kb) and consists of the following elements:
a Sma I SalGI fragment of plasmid pVC18 DNA, the size of which is 2.6 kbp

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

Fragments contain the following genes:
bla gene encoding b-lactase synthesis,
DTIL2 fusion protein gene.

 A method of constructing the rDTI23 plasmid encoding the synthesis of the DTIL2 fusion protein is that a fragment (2.3 kbp) of the rDT555 plasmid DNA, after complete hydrolysis with restriction enzymes EcoRI and Bam HI and completion of blunt ends, is cloned into blunt plasmid pAA1213 EcoRI website; the obtained plasmid rDTIL34 is restricted by the Eco72.I enzyme and a Ham HI linker is inserted into the obtuse gap, after which a 7.3 kV DNA fragment is isolated by restriction endonuclease, ligated and the bacteria are transformed with the selected recombinant plasmid, and the DTIL2 fusion gene is isolated using a complete digestion with restriction enzymes Eco24.I and SalGI, protruding 3 'ends are removed by treatment with T4 polymerase, then the fragment is cloned at SmaI SalGI sites in plasmid pVC18.

 To solve the problem, a bacterial strain Escherichia coli JM109 (rDTI23) VKM CR 341D producer of the fused protein DTIL2 is used.

Cultural and morphological features of the proposed strain
Cells are straight, rod-shaped 1.2 1.6 • 2.0 6.0, microns, gram-negative, non-spore-bearing.

Cultural signs
Cells grow well on simple nutrient media. When growing on meat-peptone agar, L-agar, the colonies are smooth, round, shiny, yellowish-white, the edge is even, opaque.

 With the growth of the culture in the meat-peptone broth and LB-medium uniform intense turbidity is formed.

Physiological and biochemical characteristics
Cells grow in the range from 4 to 45 ^o C at an optimum pH of 7.2 to 7.4.

 Many carbohydrates are used as a carbon source, including d-glucose, d-fructose, arabinose, trehalose. Cells do not absorb acetate, adanite, galactose.

 The source of nitrogen is primarily an organic form in the form of peptone and amino acids.

 Cells do not dilute gelatin, indole does not form.

 Urease activity is not detected.

Antibiotic resistance
Resistant to ampicillin (up to 800 μg / ml) due to the presence of the plasmid rDTI23.

 Example 1

 The method of obtaining plasmids rDI23.

 The initial plasmids for constructing the rDTI23 plasmid are the rDT555 plasmid (Fig. 1) with the diphtheria toxin gene from Cl cloned in it. diphteriae (w + tox) and plasmid pAA1213 (Fig. 2), carrying the mature human interleukin 2 gene, kindly introduced by E. Ya. Grenom (Institute of Organic Synthesis, Academy of Sciences of Latvia).

DNA of plasmids rDT555 and pAA1213 was isolated according to the method [4]. The concentration of plasmid DNA was determined spectrophotometrically (extinction coefficient e _{260 260} 0.02 unit / μg. The resulting preparations of plasmids rDT555 and pAA1213 were used to construct plasmids rDTI23. Construction was carried out in several stages in of the following sequence (Fig. 3): 6 μg rDT555 plasmid DNA was incubated with EcoRI restriction endonuclease (15 units) in buffer 3 (0.5 M Tris-HCl, pH 8.0, 10 mM MgCl ₂ , 100 mM NaCl). The reaction is carried out at 37 ^o C for one hour.The completeness of the restriction is determined by electrophoresis on a 0.8% agarose gel The rDT555 plasmid DNA obtained after the first restriction was reprecipitated with ethanol and Bam HI restriction (12 units) was carried out in buffer 3, after which the restriction was again checked by electrophoresis on a 0.8% agarose gel. As a result of double restriction by EcoRI and Bam HI enzymes, a 2.3 kb fragment that carries the diphtheria toxin gene with its own promoter and a signal peptide coding sequence is cleaved from the plasmid rDT555. The fragment is isolated by preparative electrophoresis in 0.8% agarose gel. After electrophoresis, the gel is stained in a 0.05% solution of ethidium bromide and visualized DNA fragments by irradiating with ultraviolet light (irradiator DESAGA, Germany) with a wavelength of 340 nm. A strip corresponding to a 2.3 kV DNA fragment is neatly cut with a scalpel, placed in a dialysis bag with 0.4 ml of electrophoresis buffer (10 mM Tris-acetate, pH 8.0, 1 mM EDTA) and subjected to electrophoresis for 1 , 5 hours, as a result of which the DNA moves from the gel to the buffer solution. DNA in the buffer solution is purified by double treatment with buffered phenol and ether. Then, ethanol is reprecipitated, after which the protruding 5 'ends of the DNA fragment are extruded to the blunt Klenov fragment of DNA polymerase I in the presence of four deoxyribonucleotide triphosphates.

 4 kg of plasmid DNA pAA1213 incubated with restriction enzyme Eco RI (10 units) in buffer 3 for 1 2 hours. Check the completeness of restriction by electrophoresis in 0.8% agarose gel. Plasmid DNA is reprecipitated with alcohol and the protruding 5 'ends of the linear form of pAA1213 DNA are completed to blunt.

The connection of the DNA fragment of plasmid rDT555 with a size of 2.3 kV and vector DNA (pAA1213) is carried out in a ligation buffer (10 mm Tris-Hcl, pH 7.6, 10 mm MgCl ₂ , 10 mm DTT, 0.0025 mm ATP) in the presence of RNA ligases (20 ea) and DNA ligases (10 ea). For ligation, 3 μg of dDT555 plasmid DNA fragment is mixed with 1 μg of pAA1213 plasmid DNA. The ligation reaction is carried out at 4 ^o C for 16 hours. The quality of the ligation is checked by electrophoresis in 0.8% agarose gel. The obtained DNA preparation was used to transform bacteria cells of E. coli K12 strain jM109 using calcium chloride according to the procedure [4]. From the obtained ampicillin-resistant (Ap ^r ) E. coli clones, plasmid DNA was isolated according to the Birmboyme method [5] and the mobility of the isolated plasmids was compared to 0 , 8% agarose gel with plasmid mobility pAA1213. Reduced mobility indicates the presence of an insert of a DNA fragment from plasmid rDT555. The plasmid DNA of Ar ^r clones of E. coli with reduced mobility is isolated and subjected to restriction analysis. The restriction analysis of the rDTIL34 plasmid shown in FIG. 3, indicates that the genes for diphtheria toxin and interleukin 2 are located in tandem and the direction of transcription of both genes coincides.

The plasmid rDTIL34 served as the basis for combining the coding sequences of both genes (DT and IL2) into one transcriptional unit with the simultaneous deletion of the DT gene sequence encoding the diphtheria toxin receptor-binding domain. The literature data indicate that the C-terminal sequence consisting of 50 a.a. has a receptor-binding activity. located distal to the second disulfide loop DT. This sequence is encoded starting at 1640 nucleotides (counting from the 5 'Hind III site of the DT gene), and beyond. The design is based on the fact that in the region of 1660 nucleotides of the DT gene in plasmid rDTIL34 there is a single site for the restriction enzyme Eco72.I, which generates blunt ends. For this, 5 μg of rDIL34 plasmid DNA is treated with the restriction endonuclease Eco72.I (15 ea) in buffer (10 mM Tris-Hcl, pH 7.8, 150 mM NaCl, 5 mM MgCl ₂ , 100 μg / ml albumin) at 37 ^o C for two hours. The completeness of restriction is checked by electrophoresis in 0.8% agarose. The fully hydrolyzed DNA of rDTIL34 plasmid was reprecipitated with ethanol. Further, 0.002 p.u. Your HI linker CGGAATCG (NPO Enzyme, Vilnius) was inserted using DNA ligase (10 ea) and RNA ligase (20 ea) enzymes in buffer 2 at 4 ^o C for 16 hours . After ligation reactions, plasmid DNA was reprecipitated with ethanol, dissolved in buffer 3, Bam HI restriction endonuclease (15 ea) was added and incubated for 2 hours at 37 ^° C. After checking the completeness of cleavage in 0.8% agarose gel, preparative electrophoresis was isolated a 7.2 kb DNA fragment, as already described. The isolated DNA fragment is treated with DNA ligase (10 EA) in a ligation buffer at 4 ^° C. for 16 hours. The quality of the ligation is checked by electrophoresis in 0.8% agarose gel. The resulting ligates transform E. coli cells of strain HB101 using CaCl ₂ methodology [4]. Transformants were selected on agarized L medium containing ampicillin (30 μg / ml). From Ap ^r clones, according to the results of immunoblotting using antidiphtheria and anti-interleukin antibodies, those are selected that synthesize the diphtheria toxin-interleukin 2 fusion protein (Fig. 4). The molecular weight of this polypeptide containing antigenic determinants of both DT and IL2 is 70 kD, which is in good agreement with the counting results based on the nucleotide sequence. Thus, plasmid pSF39 was selected, the nucleotide sequence of which was verified in the region of the “junction” of the DT and IL2 genes by the Maxam-Gilbert method of chemical degradation. The nucleotide sequence is shown below:
DT.GTG CAC CGG ATC CCA ATC.IL2
Considering the data on the primary structure of DT and IL2, as well as the results of pSF39 sequencing, we can conclude that the fusion protein consists of 650 a.s. of which the first 25 N-terminal A.O. constitute the signal peptide DT. The DTIL2 fusion protein gene is located in the pSF39 plasmid under the control of the DT gene promoter, which in E. coli directs the constitutive synthesis of DT with moderate efficiency. To increase the level of DTIL2 synthesis and regulate this process, the fusion protein gene was placed under the control of the inducible lac promoter of plasmid pVC18. To do this, 5 μg of the plasmid is incubated with the restriction enzyme Eco24.I (15 ea) in buffer (10 mM Tris-Hcl, pH 8.0, 10 mM MgCl ₂ , 1 mM DDT, 100 μg / ml albumin) at 37 ^o C for two hours. The completeness of restriction is checked by electrophoresis. The DNA of the restricted plasmid pSF39 was reprecipitated with ethanol and the protruding 3 'OH ends of the T4 phage polymerase (10 ea) were removed in buffer (0.033 M tris acetate, pH 7.9; 0.066 M potassium acetate, 0.01 M magnesium acetate, 0 , 5 mm DTT, 0.01 μg / ml albumin) at 37 ^o C for 10 minutes. After ethanol reprecipitation, the plasmid psF39 DNA was treated with the restriction endonuclease salGI. The completeness of restriction is checked and a 2.1 kV DNA fragment carrying the DTIL2 fusion protein gene is isolated by preparative electrophoresis.

Таким образом, в плазмиде рДТИ23 ген слитого белка ДТИЛ2 находится под контролем индуцибельного lac-промотора. Экспрессия белка ДТИЛ2 в клетках Е. соli jM109 (рДТИ23) достигается только после индукции изопропил-β-D-тиогалактозидом (ИПТГ).The plasmid DNA of pVC18 vector (5 μg) was sequentially treated with SmaI restriction nuclease (15 units) in buffer (20 mM Tris-Hcl, pH 7.4, 5 mM MgCl ₂ ; 50 mM KCl) at 37 ^° C for 2 hours, and after checking the completeness of restriction and reprecipitation with ethanol by the restriction enzyme SalGI (15 units) in buffer 3 at 37 ^° C for 2 hours. A large pVC18 DNA fragment is isolated by preparative electrophoresis. The connection of DNA fragments of the plasmid psF39 and pvC18 is carried out by ligation with T4 phage ligase according to the described procedure. The resulting preparation transforms E. coli cells jM109. From Ap ^r clones, those are selected that contain the plasmid pVC18 with the insertion of a fragment from pSF39 by the reduced mobility of plasmid DNA, as described previously. Recombinant plasmids containing the insert are subjected to restriction analysis, and then the 5 ′ and 3 ′ ends of the fusion protein gene are sequenced by the Sanger method. As the results of these experiments show, the selected recombinant plasmid rDTI23 fully complies with the map shown in FIG. 5, and has the following gene start structure:

Thus, in the plasmid rDTI23, the gene for the fused protein DTIL2 is under the control of an inducible lac promoter. The expression of DTIL2 protein in E. coli cells jM109 (rDTI23) is achieved only after induction with isopropyl-β-D-thiogalactoside (IPTG).

 Example 2

 Induction of fusion protein synthesis in E. coli cells jM109 (rDTI23).

Bacterial cells of E. coli jM109 (rDTI23) were grown in 5 ml of LB medium with ampicillin (30 μg / ml) at 37 ^° C for 16 hours. Next, the night culture was diluted 15 times with LB medium with ampicillin, grown at 37 ^o C with intense swing (180 rpm) for two hours, after which IPTG was added to a final concentration of 0.001 M and continued to grow under the same regimen [6] The cells are then harvested by centrifugation (3000 g, 10 min) and lysed by freezing-thawing in the presence of lysozyme 2 mg / ml PBS (10 mM Na phosphate buffer, pH 7.2, 7.4, 0.15 M NaCl, 0.05 % Tween 20). After the second freezing, thawing is centrifuged (10,000 g, 15 min), and the supernatant is taken. The presence of a fusion protein in the supernatant is determined by enzyme immunoassay using equine antibodies to diphtheria toxin and monoclonal antibodies to interleukin 2, as described previously.

Literature
1. Williams DP Parker K. Bacha P. Bishai W. Borowski M. Genbauffe F. Strom TB Murphy JR Diphtheria 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 diphtheria toxin fragment in Escherichia coli // J. of Bacteriology. 1987. Vol. 169. P. 5140-5151.

 3. Shemyakin I.G. Anisimova V.A. Mitrofanova G.N. Design and expression of hybrid proteins diphtheria toxin-interleukin 2 // Molecular Biology. 1992, vol. 26, pp. 1088-1098.

 4. Matiatis T. Fritsch E. Sambrook V. Methods of genetic engineering: molecular cloning. Per. with eng. M. World, 1984.

 5. Birnboim H.C. 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. Fabry M. Zadrazil S. Fermentation conditions for high-level expression or the tac-promoter-controlled calf prochymosin cDNA in Escherichia coli HB101 // Biotechnology and Bioengineering. 1991. Vol. 37. P.71-79.

Claims (1)

1 1. Recombinant plasmid DNA rDTI 23 4.7 kbp and mol.m. 3.1 MDa, which determines the synthesis of a diphtheria toxin-interleukin 2 human fusion protein (DTIL2), containing Sma I SalG I - a 2.6 kb fragment of pUC18 plasmid. Eco 24.1 SalG I - a fragment of plasmid pSF39 with a size of 2.1 kb corresponding to the DTIL2 fusion protein gene; genetic marker: bla gene, which provides resistance to ampicillin. 2 2. A method for producing recombinant plasmid DNA rDTI 23, which determines the synthesis of a fusion protein of human diphtheria toxin-interleukin 2 (DTIL2), which consists in the fact that plasmid rDT555 is completely hydrolyzed by restriction enzymes EcoRI and BamHI a 2.3 kb DNA fragment is isolated. corresponding to the diphtheria toxin (DT) gene with its own promoter and signal peptide sequence, the 5'-ends of the indicated fragment are blunted using Klenov’s DNA polymerase I, connected to the pAA1213 vector DNA via the EcoRI site to obtain the rDTIL34 plasmid, this plasmid is cleaved by restriction enzyme Eco 72.1 and a BamHI linker is inserted into the blunt rupture, then the plasmid rDTIL34 is digested with BamHI endonuclease and a 7.2 kb DNA fragment is isolated. ligation is carried out, the resulting ligates transform Escherichia coli bacterial cells and select the plasmid psF39 containing the DTIL2 fusion protein gene under the control of the DT gene promoter, then digest the plasmid pSF39 with Eco 24.1 restriction enzyme to remove protruding 3'-OH ends and SalGI DNA fragment is isolated 2.1 kbp corresponding to the DTIL2 fusion protein gene, insert it at the SmaI SalGI site into pUC18 plasmid and obtain the target plasmid rDTI 23, which determines the synthesis of the DTIL2 fusion protein under the control of an inducible promoter.2 3. Esherichia coli bacterial strain VKM CR-341D containing recombinant rTI plasmid 23, - producer of the fused protein DTIL2.

Images