RU2313576C1 - RECOMBINANT PLASMID EXPRESSING VIBRIO CHOLERAE CLONED Ace GENE AND ESCHERICHIA COLI STRAIN - PRODUCER OF VIBRIO CHOLERAE ACCESSORY CHOLERA ENTEROTOXIN - Google Patents

Abstract

FIELD: biotechnology, genetic engineering, medicinal microbiology.

SUBSTANCE: invention proposes recombinant plasmid pAce90 expressing Vibrio cholerae cloned ace gene (accessory cholera enterotoxin) and E. coli strain M15[pREP4]pAce90 (KM 194) as a producer of accessory cholera enterotoxin carrying indicated plasmid. Plasmid is prepared by incorporation of Vibrio cholerae ace gene by sites of Bam HI - Hind III in polylinker of vector plasmid pQE30 in orientation providing the transcription direction under control of T5 promoter. The strain E. coli M15[pREP4]pAce90 (KM 194) is a producer of recombinant protein ace wherein this strain is prepared by transformation of the strain E. coli M15[pREP4] with recombinant plasmid pAce90. Production of ace protein occurs by induction with isopropyl-β-O-thiogalactoside with the yield 7-10% of cellular proteins. Using plasmid pAce90 and the strain E. coli (KM 194) provides accelerating the process for preparing Vibrio cholerae ace protein.

EFFECT: improved preparing method of protein ace, valuable properties of plasmid and strain.

3 cl, 2 dwg, 3 ex

Description

The invention relates to biotechnology, genetic engineering, medical microbiology and can be used to study the properties and biological activity of accessory cholera enterotoxin (ACE) Vibrio cholerae.

Asa's studies require its preparations, the most promising way to obtain which is to use producers - recombinant strains of Escherichia coli containing the cloned ace gene.

Known cholerogenic strain Vibrio cholerae E8946 (see article Trucksis M., Conn TL, Wasserman SS, Sears CL Vibrio cholerae ACE stimulates Ca ⁺ dependent Cl ^- / HCO ₃ ^- secretion in T84 cells in vitro: American Journal of Cell Physiology, 279, 2000, C567-C577), from the culture fluid of which Ace was isolated, which is a heat-labile protein with a molecular mass (MM) of 9 kDa, forming dimers with 18 kDa MM and with the ability to cause an increase in the permeability of intestinal tissue culture in Ussing chambers.

A disadvantage of the known producer is that in addition to Ace, it forms cholera toxin and many biologically active substances, which greatly complicates the selection of fractions containing Ace by biological activity, as well as the need to comply with the regime of work with causative agents of especially dangerous infections, which is possible only in specialized laboratories institutions of the antiplague system.

A non-choleric recombinant strain of Vibrio cholerae CVD110 (pCVD630) is known (see Trucksis M., Galen GE, Michalski J. et al. Accessory cholera enterotoxin (Ace), the third toxin of a V. cholerae virulence cassette: Proc. Natl. Sci. USA, 90, 1993, 5267-5271), the culture fluid of which contained Ace and caused fluid accumulation in isolated loops of the intestine of the rabbit and an increase in short circuit current (I _sc ) in the intestinal tissue when examined in Ussing chambers.

However, the use of this producer is also associated with the need to achieve a high degree of Ace purification, which requires a multi-stage process using expensive equipment.

The method was chosen for the prototype (see article Trucksis M., Conn TL, Fasano A., Kaper JB Production of Vibrio cholerae accessory cholera enterotoxin (Ace) in the yeast Pichia pastoris: Infection and Immunity, 65, 1997, 4984-4988), consisting in the cloning and expression of the ace gene of cholera vibrio in the chromosomal DNA of the yeast Pichia pastoris.

The disadvantage of the prototype is the long period of its cultivation (36-48 hours) and the need to use an extremely toxic methyl alcohol as an inductor, which narrows the possibilities of its technological use.

The technical task of the invention is the creation of a recombinant plasmid and strain of Escherichia coli expressing the cloned gene of accessory cholera enerotoxin (ace) Vibrio cholerae under the control of the T5 promoter, which provides accelerated toxin production.

The problem is solved by creating:

- a new recombinant plasmid pACe90 expressing the cloned ACE gene of cholera vibrio in E. coli strains;

- strain Escherichia coli M15 [pREP4] pAce90 - producer of Ace cholerae vibrios by transforming E. coli strain M15 [pREP4] with the recombinant plasmid pACE90.

Figure 1 shows the scheme of the vector plasmid pQE30 (QIAGEN), which is the starting one for obtaining the recombinant plasmid pACe90.

Figure 2 presents the design scheme of the recombinant plasmids pACe90.

The plasmid pQE30 (see Fig. 1) carries the gene for resistance to ampicillin (bla) and contains the promoter operator region, including the T5 promoter and two tandem lac operators, providing maximum repression of Ace synthesis in the presence of glucose, as well as a synthetic binding site ribosomal RNA, start codon, sequence of triplets encoding the synthesis of hexahistidine (6-His), polylinker (MCS) and two transcription terminators (t0 phage lambda phage and T1 from the rrnB operon of E. coli). The expression of cloned genes occurs upon induction with isopropyl-β-D-thiogalactoside (IPTG) and begins with a plasmid start codon, and a hybrid protein is formed with the hexahistidine block in front of the first amino acid.

The plasmid pACe90 is a genetically engineered variant obtained by inserting the V. aceolerae O1 gene elto biovar into the pQE30 vector (see FIG. 2).

Being transformed into strains of E. coli Jm103 or M15 [pREP4], the recombinant plasmid expresses the cloned gene under the control of the T5 promoter. Gene expression is suppressed in the presence of glucose and IPTG is induced.

The strain E.coli M15 [pREP4] pAce90 is a genetically engineered variant obtained by transformation of the recombinant plasmid pACE90 into the strain E.coli M15 [pREP4] and is a producer of Ace V. cholerae. The strain is deposited in the State collection of pathogenic bacteria "Microbe" under the number KM 194.

The resulting producer strain is characterized by the following features:

Cultural and morphological properties

In liquid nutrient media (Hottinger broth, meat-peptone broth) forms a uniform turbidity, on dense - round, convex, smooth, white translucent colonies with a smooth edge, a doughy consistency.

Physiological and biochemical properties

The strain decomposes glucose, arabinose and mannitol with the formation of acid and gas, does not decompose sucrose, and forms lactose-negative colonies on the Endo medium. Auxotroph.

Antibiotic resistance

The strain is resistant to 25-30 μg / ml kanamycin, which is a consequence of the presence of the plasmid pREP4 in the initial strain, and to 50-100 μg / ml ampicillin due to the expression of the bla gene contained in the vector plasmid pQE30.

The method of obtaining and using a recombinant plasmid and producer strain is illustrated by the following examples.

Example 1. Cloning of the ace gene and obtaining a recombinant plasmid

For PCR synthesis of the ace gene, primers are used designed by the applicants based on the analysis of the nucleotide sequence of the ace gene (AF 220606, AF 414369):

straight - 5'-TTTTGGATCCGTGATGCTTATGATGGACAC-3 'and

the reverse is 5'-AAGAAAGCTTTAGGTTTAACGCTCGCAGGG-3 '.

Since amplitudes must be inserted into the pQE30 plasmid vector in an orientation that ensures the direction of transcription under the control of the T5 promoter, a restriction site for the endonuclease forming sticky ends is inserted at the 5'-end of each primer: BamHI for the forward primer and HindIII for the opposite (in the sequences cited) in bold and underlined) in accordance with the order of the restriction sites in the polylinker of the vector plasmid.

From strain V. cholerae eltor 15500, chromosomal DNA is isolated by the phenolic method, which serves as a matrix for the synthesis of the desired gene.

300 μl of the reaction mixture for the polymerase chain reaction contain 0.5 ng of the DNA template and the following components in the indicated concentrations: 2.5 μl of each primer, 2.5 mm of all four deoxynucleotide triphosphates, 3 units Taq polymerase and 0.1 volume of 10-fold buffer attached to it. The mixture is poured into 30 μl into 0.5-ml plastic tubes and the reaction is carried out according to the following scheme: 94 ° С - denaturation (40 sec), 60 ° С - annealing (40 sec), 72 ° С - synthesis (40 sec). In total, 30 cycles of amplification are carried out, in the last cycle the synthesis time is increased to 30 minutes. At the end of the reaction, the mixture is subjected to electrophoresis in a 0.7% agarose gel in Tris-acetate buffer in the presence of 0.5 μg / ml ethidium bromide, then viewed in ultraviolet light, a portion of the gel with an amplified fragment of 311 bp is cut out, the last is isolated elution, purified with a mixture of phenol: chloroform: isoamyl alcohol in a ratio of 25: 24: 1 and precipitated with ethyl alcohol.

Thus obtained PCR amplification and DNA of the vector plasmid pQE30 are hydrolyzed with restriction endonucleases BamHI and HindIII according to the recommendations of the enzyme manufacturer, purified on an agarose gel and ligated using T4 phage DNA ligase and the buffer attached to it according to the manufacturer's recommendations.

Ligase mixtures transform competent E. coli Jm103 cells prepared the day before by treatment with calcium chloride. After the standard transformation procedure (0 ° C - 40 min, 42 ° C - 2 min, 0 ° C - 5 min), the cells are diluted 10 times with LB medium with 0.5% glucose, grown for 1 h and plated on LB agar containing 50 μg / ml ampicillin and 0.5% glucose. Crops are incubated at 37 ° C.

The next day, the grown ampicillin-resistant colonies were tested using the above primers and positive clones were selected from which plasmid DNA was isolated and confirm the presence of inserts 0.3 kb in length. hydrolysis of these plasmids with restriction endonucleases BamHI and HindIII, followed by agarose gel electrophoresis.

Plasmid DNA was isolated from E. coli strain Jm103pAce90 and competent E. coli M15 cells [pREP4] were transformed with it. Recombinant clones were selected on LB agar containing 50 μg / ml ampicillin and 25 μg / ml kanamycin.

Example 2. The study of the expression of the cloned ace gene in E. coli

To identify the ability of recombinants to synthesize Ace, the recombinant E. coli strain M15 [pREP4] pAce90 (KM 194), as well as a control strain containing the vector plasmid pQE30 without insertion, was grown in LB liquid medium containing 50 μg / ml ampicillin for 3 -4 h at 37 ° C with joking at 150 rpm and then induce 1 mm IPTG for 1-2 hours, the cells are precipitated by centrifugation, lysed in a buffer containing 65 mm Tris-HCl pH 6.8, 1% SDS and 10 mm 2-mercaptoethanol, at a temperature of 96 ° C for 15 minutes The lysate is subjected to electrophoresis in 16.5% SDS page and the gel is stained with silver nitrate. A major protein band in the 13 kDa region, absent in the lysate of the control strain, is detected in the lysate, which corresponds to the size of the desired recombinant protein containing a hexahistidine block at the N-terminus. The percentage of 6His protein is determined using the Quantity One program; it makes up 7-10% of the total cellular proteins.

Example 3. The study of the biological activity of the preparation of the recombinant protein Ace obtained in example 1, in vivo

The sediment of cells grown with the induction of IPTG is resuspended in physiological solution, and destroyed by ultrasound on a PG-100 MSE 150 W disintegrator (England) using a conical rod at an oscillation frequency of 20 kHz and an amplitude of 12-16 μm for 1 min. The cell residues are separated by centrifugation, and the total protein content is determined by the Lowry method in transparent supernatants (clarified disintegrates), the concentration is adjusted to 500 μg / ml and tested on a model of sucker mice.

For animals weighing 3-4 g (8 for each variant), 100 μl of the preparations (50 μg of total protein per animal) is administered through the anus. 5 hours after administration, the animals were sacrificed and FA (ratio of stomach and intestinal weight to body weight) was determined. Statistical processing of the results is carried out using the program "Primer of Biostatistics" v.4.03. The difference between the FA control and experimental groups should be statistically significant at P <0.005.

The use of the recombinant plasmid pACe90 and strain E. coli KM 194 will speed up the process of obtaining the protein Ace Vibrio cholerae for large-scale studies of its properties and biological activity. Further studies of Ace using the producer obtained by the applicants are of fundamental importance for assessing its role in virulence and elucidating the mechanism of action on eukaryotic cells.

The recombinant E. coli strain KM 194 is promising as a producer of Ace cholerae vibrios for studying its biological effect and the effect on the activity of other virulence factors. Ace is the only toxin synthesized by this strain, therefore, crude lysates or disintegrates of bacterial cells can also be used as drugs.

The advantage of the proposed producer in comparison with cholera vibrios is also that its cultivation does not require compliance with the regime of work with pathogens of especially dangerous infections.

Claims (2)

1. Recombinant plasmid pACe90 expressing the cloned ACE gene (accessory cholera enterotoxin) Vibrio cholerae, inserted at the BamHI-HindIII sites in the polylinker of the vector plasmid pQE30, under the control of the T5 promoter. 2. The strain Escherichia coli M15 [pREP4] pAce90 (KM 194), carrying the recombinant plasmid pACe according to claim 1 - producer of accessory cholera enterotoxin (ACE) Vibrio cholerae.

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