RU2313577C2 - Recombinant plasmid expressing vibrio cholerae cloned cef gene (variants) and escherichia coli strain-producer of vibrio cholerae cef (variants) - Google Patents

Abstract

FIELD: biotechnology, genetic engineering, medicinal microbiology.

SUBSTANCE: invention describes novel recombinant plasmids comprising V. cholerae 01 biovars eltor and classicae cloned cef gene, non01/non0139 (0130 and 0139 (Bengal) - pCef61B, pCef69B, pCef49B and pCef31B as components of vector plasmid pBAD18, respectively. Plasmids are prepared by incorporation of a PCR-amplificate of Vibrio choleare cef gene by sites Eco RI - Xba I in polylinker MCS2 of vector plasmid pBAD18 in orientation providing the transcription direction under control of P_BAD-promoter. Strains of E. coli - Jm103pCef61B (KM 190), Jm103pCef69B (KM 191), Jm103pCef49B (KM 189) and Jm103pCef31B (KM 188) are producers of recombinant plasmids and Cef protein prepared by transformation of E. coli strain Jm103 with recombinant plasmids. Product of cef is carried out in induction with arabinose. Recombinant proteins Cef retain all three type of activity: esterase and biological one in vitro and in vivo. Invention can be used for investigation of role of cytotonic factor cef (CHO cell elongating factor) in pathogenesis of cholera, its biochemical and biological activity.

EFFECT: improved and valuable properties of plasmid, gene and protein.

8 cl, 2 dwg, 4 ex

Description

The invention relates to biotechnology, genetic engineering, medical microbiology and can be used to study the properties, biochemical and biological activity of the cytotonic factor cef (CHO cell elongating factor) Vibrio cholerae.

The role of Cef in the pathogenesis of cholera and as an independent virulence factor of non-cholerogenic strains has not been studied. Studies of the Cef factor require the presence of its preparations, the most promising way of obtaining which is to use producers - recombinant strains of E. coli containing the cloned gene.

The known strain Vibrio cholerae JBK 70 biovar eltor (see article McCardell BA, Kothary MH, Hall RH, Sathyamoorthy V. Identification of a CHO cell elongating factor of Vibrio cholerae O1: Microbial Pathogenesis, 29, 2000, 1-8), from cell of extracts of which Cef preparation was isolated, which is a heat-labile protein with a molecular weight (MM) of 85 kDa and a pI of 3.8, having a unique N-terminal amino acid sequence, having esterase activity, the ability to cause fluid accumulation in the intestines of sucker mice and lengthening CHO cells (CHO cell elongating factor), not accompanied by increased levels of cAMP or pro taglandina E _2, indicating that its mechanism of action different from that of cholera toxin.

A disadvantage of the known producer is that cholera vibrios produce many enzymatically and biologically active substances, therefore, to study the properties of Cef, it is necessary to achieve a high degree of purification of the studied protein, which requires the use of a large amount of bacterial mass and expensive equipment, as well as compliance with the operating mode with highly dangerous pathogens infections, which is possible only in specialized laboratories of institutions of the anti-plague system.

A known biovar vaccine strain Vibrio cholerae CVD 103-Hg is available (see Sathyamoorthy V., Hall RH, McCardell BA et al. Purification and characterization of a cytotonic protein expressed in vitro by the live cholera vaccine candidate CVD 103-Hg: Infection and Immunity, 68, 2000, 6062-60650), from the culture fluid of which a preparation of factor Cef, originally designated as S-CEP (secreted CHO cell elongating protein), is a thermolabile protein with a 79 kD MM, whose N-terminal amino acid sequence was only partly homologous to that of Cef vibrio eltor, with the ability to cause fluid accumulation in the intestines of sucker mice and lengthening of CHO cells.

However, to obtain a very small amount of the drug, extremely large volumes of culture fluid are required, and it is also necessary to carry out a multi-stage purification process from associated biologically active factors and to comply with the regime of work with pathogens of especially dangerous infections.

For the prototype, a method was selected for producing producers of recombinant Cef protein cholera vibrios eltor and classical biovar (see McCardell BA, Sathyamoorthy V., Michalsky J. et al .: Microbial Pathogenesis, 32, 2002, 165-172), which consists in cloning genes into Escherichia coli in the vector plasmid pPICZαA and their subsequent expression in the yeast strain Pichia pastoris X-33 transformed with recombinant plasmids PVNS1 and PVNS2.

The disadvantages of the prototype are the formation in a yeast host of a glycosylated product with a molecular weight of 114 kDa, which maintains esterase activity and causes a prolongation of CHO, but loses its ability to cause fluid accumulation in the intestines of sucker mice and cannot be decomposed without loss of activity, as well as the long term culture of the producer ( 3 days) and the need to use extremely toxic methyl alcohol as an inducer. In addition, producers of Cef cholera vibrios of only two O1 serogroup biovars were designed, which limits the possibility of a comparative analysis of the properties of Cef cholera vibrios O1, O139 and neO1 / non O139 serogroups.

The technical task of the invention is the creation of recombinant plasmids and strains of Escherichia coli expressing the cloned gene cef (CHO cell elongating factor) Vibrio cholerae of different biovars and serogroups under the control of the P _BAD promoter.

The problem is solved by creating:

- a new recombinant plasmid pCef61B expressing the cloned cef gene (CHO cell elongating factor) of the cholera vibrio biovar eltor in E. coli strains;

- a new recombinant plasmid pCef69B expressing the cloned cef gene (CHO cell elongating factor) of the classical biovar cholera vibrio in E. coli strains;

- a new recombinant plasmid pCef49B expressing the cloned cef gene (CHO cell elongating factor) of the non-O1 / non O139 (O13) serogroup cholera vibrio in E. coli strains;

- a new recombinant plasmid pCef31B expressing the cloned cef gene (CHO cell elongating factor) of serogroup cholera vibrio 0139 (Bengal) in E. coli strains.

- Escherichia coli strain Jm103pCef61B - producer of Cef (CHO cell elongating factor) cholera vibrio biovar eltor by transforming E. coli strain Jm103 with recombinant plasmid pCef61B.

- Escherichia coli strain Jm103pCef69B - producer of Cef (CHO cell elongating factor) cholera vibrio of a classic biovar by transforming E. coli strain Jm103 with recombinant plasmid pCef69B.

- strain Escherichia coli Jm103pCef49B - producer of Cef (CHO cell elongating factor) cholera vibrio non O1 / non O139 (O13) serogroups by transforming E. coli strain Jm103 with recombinant plasmid pCef49B.

- strain Escherichia coli Jm103pCef31B - producer of Cef (CHO cell elongating factor) cholera vibrio O139 of the serogroup (Bengal) by transformation of E. coli strain Jm103 with the recombinant plasmid pCef31B.

Figure 1 shows the scheme of the vector plasmid pBAD18 (X81838), which is the source for the preparation of recombinant plasmids pCef61B, pCef69B, pCef49B and pCef31B.

Figure 2 presents the design scheme of recombinant plasmids on the example of pCef61B.

Plasmid pBAD18 (see figure 1) carries the gene for resistance to ampicillin (bla) and contains the P _BAD promoter of the arabinose (araBAD) operon and the araC gene, the product of which regulates this promoter: includes transcription in the presence of arabinose and inhibits it in the presence of glucose . The MCS2 polylinker is inserted into the plasmid in such a way that the expression of the cloned genes is carried out under the control of the P _BAD promoter and is subject to strict regulation.

The plasmids pCef61B, pCef69B, pCef49B, and pCef31B are genetic engineering variants obtained by inserting the cef V. cholerae O1 genes of the eltor and classicae, nonO1 / nonO139 (O13) and O139 (Bengal) biovars into the pBAD18 vector, respectively (see Fig. 2 )

When transformed into Escherichia coli strains Jm103, BW, M17, or DH5α, recombinant plasmids express the cloned gene under the control of the P _BAD promoter. Gene expression is suppressed by glucose and induced by arabinose. In the absence of glucose and arabinose, the expression of cloned genes occurs at a low level.

The strains of E. coli Jm103pCef61B, Jm103pCef69B, Jm103pCef49B and Jm103pCef31B are genetically engineered variants obtained by transformation of the corresponding recombinant plasmids into E. coli strain Jm103, and are producers of plasmid DNA and Cef V. choleraeOlée1OléeleeO1OléeleeO1OléeleeOe1e1OléeoreOlée1Oléeore1OléeoreOlée1Oléeore1Oléeore1Oléeo1e1 (O13) and O139 (Bengal), respectively. The strains were deposited in the collection of the museum of living cultures of the Russian anti-plague Institute "Microbe" under the numbers KM 190, KM 191, KM 189 and KM 188.

The resulting producer strains are characterized by the following features.

Cultural and morphological properties

All strains in liquid nutrient media (Hottinger broth, meat and peptone broth) form uniform turbidity, on dense strains round, convex, smooth, white translucent colonies with a smooth edge, a doughy consistency.

Physiological and biochemical properties

All strains decompose with the formation of acid and gas glucose, lactose-negative colonies form on Endo medium. Auxotrophs. When induced by arabinose, they exhibit esterase activity with respect to twins 20, 40, 60, and 80. The strains E. coli Jm103pCef61B (KM 190), Jm103pCef69B (KM 191) and Jm103pCef49B (KM 189) also hydrolyze tributyrin under induction conditions. Strain E. coli Jm103pCef31B (KM 188) does not have activity against tributyrin.

Antibiotic resistance

The strains are resistant to 50 μg / ml streptomycin, which is a property of the original Jm103 strain, and to 50-100 μg / ml ampicillin due to the presence of the bla gene in the vector plasmid pBAD18.

Biological activity

Clarified ultrasonic cell disintegrates, in addition to esterase activity, have the ability to cause elongation of CHO and L929 cells and fluid accumulation in the intestines of sucker mice.

The method of obtaining and using recombinant plasmids and producer strains is illustrated by the following examples.

Example 1. Cloning of the cef gene and obtaining recombinant plasmids.

For the PCR synthesis of the cef gene, primers are used that we designed based on the analysis of the nucleotide sequence of the cef gene VCA0863 (AE004414): direct 5'-TCAC GAATTC TCTGGAGTCGAACATGAAAC-3 'and reverse 5'-AAGC TCTAGA GAGAGTGCGCTTTTGGT.

Since the amplifiers must be inserted into the pBAD18 plasmid vector in an orientation that ensures the direction of transcription under the control of the P _BAD promoter, a restriction site for the endonuclease forming sticky ends is introduced at the 5'-end of each primer: EcoRI for the forward primer and XbaI for the opposite (in the above sequences are shown in bold and underlined) in accordance with the order of the restriction sites in the polylinker of the vector plasmid.

From strains V. cholerae eltor P-9961, V. cholerae classicae 569B, V. cholerae O13 17449 and V. cholerae O139 P-16131 chromosomal DNAs are isolated by the phenolic method, which serve as templates 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 μm 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 (1 min), 63 ° С - annealing (40 sec), 72 ° С - synthesis (1 min). 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 was subjected to electrophoresis on 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, 2473 kb in size, was cut out. , the latter is isolated by elution, purified with a mixture of phenol: chloroform: isopropanol in a ratio of 25: 24: 1 and precipitated with ethyl alcohol.

The PCR amplifications and DNA of the vector plasmid pBAD18 thus obtained are hydrolyzed with restriction endonucleases EcoRI and XbaI according to the recommendations of the enzyme manufacturer, purified on an agarose gel and ligated using phage T4 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. The addition of glucose is essential since at the cloning stage the cef gene product may have toxic effects on E. coli cells. Crops are incubated at 37 ° C.

The next day, the grown ampicillin-resistant colonies were tested using the following primers for detection of the cef gene:

straight - 5'-CTCCCTACTCTGCTCAGCACTCTGG-3 'and

the reverse is 5'-TCGAGCCCAAGGCCTGTTTG-3 ', and positive clones are selected from which plasmid DNA is isolated and the presence of inserts of about 2.5 kb is confirmed. by hydrolysis of these plasmids with restriction endonucleases EcoRI and XbaI followed by agarose gel electrophoresis.

To determine the ability of recombinants to synthesize Cef, cultures of recombinant clones, as well as a control strain containing vector plasmid pBAD18 without insertion, are grown in LB liquid medium containing 50 μg / ml ampicillin for 3-4 hours and then 0.2% arabinose is induced for 1-2 hours at 37 ° C with joking at 150 rpm. Cells are pelleted by centrifugation, resuspended in buffered saline (PBS) and sonicated using 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. Cell debris is separated by centrifugation, and transparent supernatants are used for testing. Protein content is determined by the Lowry method.

Example 2. The study of the enzymatic activity of preparations of recombinant Cef proteins obtained in example 1.

The enzymatic activity of clarified disintegrates of cells of E. coli Jm103pCef61B (KM 190), E. coli Jm103pCef69B (KM 191), E. coli Jm103pCef49B (KM 189) and E. coli Jm103 pCef31B (KM 188) strains is determined at 37 ° C in wells containing 0.5% tween 20, 40, 60 and 80 and 0.01% calcium chloride to visualize the cleavage, as well as tributyrin and 0.01% gum arabic or 0.2% Triton X-100 as an emulsifier. Positive results are taken into account in the formation of zones of fatty acid precipitation around the wells with calcium ions for twins and enlightenment zones for tributyrin.

All recombinants exhibit high esterase activity with respect to twins 20, 40, and 60 (precipitation zones are formed by introducing into the wells dilutions of disintegrates of cells grown without induction and with induction containing 40 and 1 μg of total protein / ml and higher, respectively) and less pronounced - in relation to tween 80 (400 and 4 μg / ml). In the disintegrate of the strain containing the vector plasmid pBAD18 without insertion, regardless of induction, the described effects are absent.

The disintegrates of E. coli Jm103pCef61B (KM 190), E. coli Jm103pCef69B (KM 191) and E. coli Jm103pCef49B (KM 189) strains also cleaved tributyrin in the same dilutions, whereas E. coli Jm103 pCef31B (KM 188 and E. coli Jm103pBAD18 does not have this ability.

Example 3. The study of the biological activity of Cef in vitro.

The biological activity of clarified disintegrates of the recombinant strains of E. coli Jm103pCef61B (KM 190), E. coli Jm103pCef69B (KM 191), E. coli Jm103pCef49B (KM 189) and E. coli Jm103pCef31B (KM 188) are studied in vitro using cell line culture models -K1 and L-929, which are added to 96-well plates in the amount of 10 ³ cells per 90 μl of RPMI-1640 medium (Sigma) containing 1% fetal bovine serum, and 10 μl of the tested preparations are added in dilutions of 1: 10- 1: 800 in the same medium and incubated in a CO ₂ incubator (Sanjo) at 37 ° C. A morphological study of the cultures is carried out for 2 days using an inverted microscope (Reichert).

1-2 days after introducing dilutions of clarified disintegrates of E. coli JM103 cells containing recombinant plasmids, cell elongation occurs in dilutions of test samples with a total protein concentration of ≥25 μg / ml, while in the control wells there is no cell elongation.

Example 4. The study of the biological activity of Cef in vivo.

The biological activity of clarified cell disintegrates of recombinant strains of E. coli Jm103pCef61B (KM 190), E. coli Jm103pCef69B (KM 191), E. coli Jm103pCef49B (KM 189) and E. coli Jm103pCef31B (KM 188) were studied in vivo . For animals weighing 3-4 g (6-10 for each variant), 100 μl of preparations with a total protein concentration of 300 μg / ml are administered through an insulin syringe with a special tip through the anus using an insulin syringe. 5 hours after administration, the animals were sacrificed and FA (ratio of total weight of the stomach and intestines to body weight) was determined. Statistical processing of the results is carried out using the program "Primer of Biostatistics" v.4.03. In the control group (after PBS administration), the average FA value is 0.058 ± 0.010; the preparation from the strain containing the vector plasmid does not cause fluid accumulation (FA = 0.057 ± 0.012), i.e. Own Escherichia coli proteins are not toxic to sucker mice. In the experimental groups, fluid accumulation is observed: the FA value is 0.076-0.079 ± 0.006-0.010. Differences from controls are statistically significant at P <0.005.

Thus, recombinant Cef proteins retain all three types of activity described for them: esterase and biological in vitro and in vivo.

The use of recombinant plasmids pCef61B, pCef69B, pCef49B and pCef31B and strains KM 190, KM 191, KM 189 and KM 188 will allow large-scale studies of the biological activity of Cef Vibrio cholerae as a virulence factor for cholera pathogens of different biovars and serogroups. Further studies of the new V. cholerae Cef toxin using recombinants obtained by the applicants are of fundamental importance for assessing its role in virulence, elucidating its mechanism of action on eukaryotic cells and evolutionary connections with similar toxins of other pathogens. In addition, plasmids are promising for the creation of more active producers based on E. coli strains.

Recombinant E. coli strains are promising as producers of a non-glycosylated toxin for studying its biological effect and the effect on the activity of other virulence factors. Cef is the only toxic product synthesized by these strains, therefore, crude lysates or disintegrates of bacterial cells can be used as preparations.

The advantage of the proposed producers in comparison with cholera vibrios is also that their cultivation does not require compliance with the regime of work with causative agents of especially dangerous infections.

Claims (8)

1. Recombinant plasmid pCef61B expressing the cloned cef gene (CHO cell elongating factor) of the cholera vibrio biovar eltor inserted at EcoRI-XbaI sites into the polylinker MCS2 of the vector plasmid pBAD18 in E. coli strains under the control of the p _BAD promoter. 2. Recombinant plasmid pCef69B expressing the cloned cef gene (CHO cell elongating factor) of the cholera vibrio of the classical biovar inserted at the EcoRI-XbaI sites into the polylinker MCS2 of the vector plasmid pBAD18 in E. coli strains under the control of the P _BAD promoter. 3. The recombinant plasmid pCef49B, expressing a cloned gene cef (CHO cell elongating factor) Vibrio cholerae neO1 / neO139 (O13) serogroup, built by EcoRI-XbaI sites of the polylinker MCS2 vector plasmid pBAD18, strains of E. coli under the control of promoter P _BAD. 4. Recombinant plasmid pCef31B expressing the cloned cef gene (CHO cell elongating factor) of serogroup cholera vibrio O139 (Bengal) inserted at the EcoRI-XbaI sites in the MCS2 polylinker of the vector plasmid pBAD18 in E. coli strains under the control of the P _BAD- promoter. 5. The strain Escherichia coli Jm103pCef61B carrying the plasmid pCef61B according to claim 1 is the producer of Cef (CHO cell elongating factor) Vibrio cholerae eltor. 6. The strain Escherichia coli Jm103pCef69B carrying the plasmid pCef69B according to claim 2 is the producer of Cef (CHO cell elongating factor) Vibrio cholerae classicae. 7. The strain Escherichia coli Jm103pCef49B carrying the plasmid pCef49B according to claim 3 is the producer of Cef (CHO cell elongating factor) Vibrio cholerae nonO1 / non) 139 (O13). 8. The strain Escherichia coli Jm103pCef31B carrying the plasmid pCef69B according to claim 4 is the producer of Cef (CHO cell elongating factor) Vibrio cholerae O139 (Bengal).

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