RU2744095C1 - Escherichia coli jm 109 1-430 bacterial strain - producer of the recombinant pwnv1protc plasmid, which carries the sequence of the 5'-untranslated area and a section of the west nile virus 1 polyprotein gene of the genotype encoding the capsid protein - Google Patents

Abstract

FIELD: genetic engineering.SUBSTANCE: recombinant strain of Escherichia coli JM 109 1-430 is proposed for use as a positive control in evaluating the efficiency of cDNA amplification of the West Nile virus 1 genotype. The strain is a producer of the recombinant pWNV1protC plasmid, which carries a fragment of the West Nile virus 1 genotype genome with a size of 430 b.p., which is a sequence of the 5'-untranslated area of the genome, and a section of the West Nile virus polyprotein gene encoding a capsid protein. The strain is deposited in the State Collection of Pathogenic Bacteria of the Federal Public Health Institution Russian Anti-Plague Research Institute "Microbe” of Russian Federal State Agency for Health and Consumer Rights, under the number KM 2049.EFFECT: invention provides efficient replication and separation of the recombinant pWNV1protC plasmid with a high yield.1 cl, 2 dwg, 3 ex

Description

The invention relates to the field of genetic engineering, microbiology, virology and consists in the construction of a recombinant bacterial strain Escherichia coli JM 109 1-430, which is the producer of the recombinant plasmid pWNV1protC, carrying the sequence of the 5'-untranslated region and a region of the gene of the West Nile virus polyprotein 1 genotype encoding protein. The strain is intended for obtaining the recombinant plasmid pWNV1protC, which is used as a positive control to assess the efficiency of amplification of West Nile virus 1 genotype cDNA by reverse transcription polymerase chain reaction. The strain was deposited in the State Collection of Pathogenic Bacteria FKUZ Russian Research Anti-Plague Institute "Microbe" Rospotrebnadzor under the number KM 2049.

West Nile virus belongs to the Flaviviridae family, the Flavivirus genus, the Japanese encephalitis virus group. According to the classification of pathogenic biological agents adopted in the Russian Federation, West Nile virus belongs to viruses of the II pathogenicity group.

West Nile virus is the causative agent of a naturally occurring focal arbovirus infection with a vector-borne transmission mechanism called West Nile fever (WNF). Clinical manifestations of WNI vary from influenza-like syndrome to the development of severe complications in the form of meningitis and meningoencephalitis. Mortality in WNV, depending on the immune status of the diseased, is up to 10%. Since 1999, laboratory-confirmed cases of WNV in Russia have been registered annually. The areas of West Nile virus occupy vast territories within the equatorial, tropical and temperate climatic zones in Africa, Europe, America, Asia and Australia. In Russia, the distribution area of West Nile virus covers the territories of the south of the European part, regions of Siberia and the Far East.

According to modern data, West Nile virus strains are divided into 9 genotypes (genetic lines). In Russia, the circulation of 1, 2, and 4 West Nile virus genotypes, which have different epidemic significance, has been reliably registered. Thus, strains of West Nile virus genotypes 1 and 2 are highly pathogenic for humans, and the pathogenicity of West Nile virus 4 genotype for mammals has not yet been established. There is evidence that infection with some West Nile virus strains of subgenotype 1a leads to the emergence of more severe neuroinvasive forms of WNN. This, apparently, indicates a higher virulence of some West Nile virus strains of subgenotype 1a compared to strains of subgenotype 1b and genotype 2.

Genotyping is a comprehensive analysis of a genotype unique to each living organism based on the study of its genome. The circulation of different genetic lines of the virus on the territory of the Russian Federation dictates the need to develop and improve diagnostic methods and test systems for identifying and differentiating strains of the causative agent of West Nile fever. Establishing the genotype of West Nile virus is an important component of epidemiological monitoring to study the pathways of the virus spread and to identify links between its genetic variants and clinical forms of WNV.

One of the promising approaches to detecting the West Nile virus genotype in samples of biological material with high sensitivity and specificity is the use of polymerase chain reaction with reverse transcription in real time (RT-PCR). RT-PCR is a direct method for detecting West Nile virus RNA. The RT-PCR method is based on the sequential implementation of two processes: the reverse transcription reaction and the polymerase chain reaction. The reverse transcription reaction is the synthesis of a complementary DNA strand (cDNA) from an RNA template by the enzyme revertase. The PCR method consists in amplifying the target regions of the obtained cDNA by means of the DNA polymerase enzyme and primers flanking regions of unique cDNA targets that exist only in certain genotypes of the virus.

To control the efficiency of amplification, and, consequently, to objectively assess the results of the study, it is necessary to use positive control samples. The Positive Control ensures that all the components in the reaction mixture are ensuring that the PCR is progressing normally. If, at the stage of recording the PCR results, the absence of accumulation of positive control amplicons is revealed, the results for all samples in the test series are considered unreliable, and negative samples are potentially false negative.

A nucleic acid preparation containing primer annealing targets is used as a positive control. One approach is to use the RNA or DNA of the target pathogen. However, in the case of pathogenic viruses, this approach is associated with a high biological hazard. Thus, the West Nile virus genome is represented by a single-stranded molecule (+) RNA (positive polarity). The (+) RNA molecule is directly capable of participating in translation as a template, and therefore, can be an infectious biological agent outside the viral particle. In addition, RNA molecules, due to the peculiarities of their chemical structure, are less stable than DNA. In this regard, a different approach is used, which consists in constructing positive control samples using genetic engineering methods. For example, by cloning specific regions of the viral genome into vectors, which are plasmids or bacteriophages of E. coli. Such samples meet biological safety requirements, are shelf stable and are used in the production of diagnostic kits.

М. et al. Production of recombinant NS1 protein and its possible use in encephalitic flavivirus differential diagnosis // Protein Expression and Purification 153 (2019) 18-25]. В данной работе авторы использовали для клонирования нуклеотидную последовательность West Nile virus, кодирующую неструктурный белок (NS1). Однако локус NS1 является высококонсервативным для большинства штаммов West Nile virus и больше подходит для идентификации вируса, а не для его типирования.The literature describes an approach to constructing expression vectors based on plasmids with cloned fragments of the genomes of St. Louis and West Nile encephalitis viruses [Lorch M., Collado M.,

M. et al. Production of recombinant NS1 protein and its possible use in encephalitic flavivirus differential diagnosis // Protein Expression and Purification 153 (2019) 18-25]. In this work, the authors used the West Nile virus nucleotide sequence encoding a non-structural protein (NS1) for cloning. However, the NS1 locus is highly conserved for most West Nile virus strains and is more suitable for virus identification rather than typing.

The closest analogue is the genetically modified E. coli TGI pWNAp ^R strain, designed by T.Yu. Krasovskaya. and deposited in the State collection of pathogenic bacteria FKUZ RosNIPCHI "Microbe" Rospotrebnadzor under the number KM 201 [Krasovskaya, T.Yu. Development and testing of a test system for detecting West Nile virus RNA by reverse transcription and polymerase chain reaction: abstract of thesis. … Candidate of medical sciences: 03.00.06 / Krasovskaya Tatyana Yurievna. - Koltsovo: State. scientific. center of virology and biotechnology "Vector", 2007. - 24 p.]. On the basis of this strain, a positive control sample was created for the PCR test system developed by the author and a standard sample of the enterprise used to standardize the procedure for monitoring the sensitivity and specificity of the diagnostic drug "GenNil-REF" (FKUZ RosNIPCHI "Microbe" Rospotrebnadzor, Russia) under production conditions ... There are other known diagnostic reagent kits for the detection of RNA West Nile virus registered in the Russian Federation as medical devices, which include positive control samples: AmpliSens WNV-FL (FBSI Central Research Institute of Epidemiology of Rospotrebnadzor, Russia), OM-screen LZN / LDR-RV "(CJSC SINTOL, Russia). However, all the above kits and the control samples used in their composition are applicable only for the detection of West Nile virus, and not for the differentiation of its genotypes.

The aim of the present invention is to construct a bacterial strain Escherichia coli JM 109 1-430, which is a producer of the recombinant plasmid pWNV1protC, carrying the sequence of the 5'-untranslated region and the gene region of the West Nile virus 1 polyprotein of the genotype encoding the capsid protein, for use as a positive control in the assessment the efficiency of amplification of cDNA West Nile virus 1 genotype.

The goal is achieved by creating a recombinant bacterial strain Escherichia coli JM 109 1-430 (State collection of pathogenic bacteria FKUZ of the Russian Research Anti-Plague Institute "Microbe" Rospotrebnadzor, number KM 2049), intended for use as a positive control in assessing the effectiveness of amplification of cDNA West Nile virus 1 genotype, which is a producer of the recombinant plasmid pWNV1protC carrying a 430 bp fragment of the West Nile virus 1 genome genotype, which is the sequence of the 5'-untranslated region of the genome, as well as a region of the West Nile virus polyprotein gene encoding a capsid protein.

Characteristics of the bacterial strain Escherichia coli JM 109 1-430

The E. coli JM 109 1-430 strain was obtained by transforming the cells of the E. coli JM 109 strain with the recombinant plasmid pWNV1protC carrying a fragment of the West Nile virus 1 genotype genotype 430 bp in size, representing the sequence of the 5'-untranslated region of the genome as well as the region the West Nile virus polyprotein gene encoding a capsid protein. The resulting recombinant E. coli strain JM 109 1-430 retains the basic properties characteristic of the E. coli species.

When inoculated in LB broth (pH 7.2) after 24 hours at 37 ° C, the recombinant E. coli strain JM 109 1-430 grows in the form of a uniform turbidity of the nutrient medium with the formation of a small easily breakable grayish sediment, as well as a parietal ring. On LB-agar (pH 7.2) forms translucent colonies of medium color, easily merging with each other. On medium Endo forms colorless (lactose-negative) colonies. In smears from broth and agar cultures, the cells of the E. coli strain JM 109 1-430 have the form of gram-negative rods.

Recombinant strain E. coli JM 109 1-430 has the following spectrum of biochemical activity: ferments glucose with the formation of acid and gas, does not ferment lactose and urea; does not utilize sodium citrate from the minimal environment; does not possess ornithine decarboxylase, as well as phenylalanine deaminase; forms indole, but does not form hydrogen sulfide.

Recombinant E. coli strain JM 109 1-430 is sensitive to monovalent coli bacteriophage and polyvalent sextaphage pyobacteriophage.

The recombinant E. coli strain JM 109 1-430 does not have hemolytic activity, is not virulent for white mice and golden hamsters ( _{strain LD 50} > 1 × 10 ⁷ mc), is resistant to the antibiotic ampicillin.

The recombinant E. coli strain JM 109 1-430 is stored in a lyophilized state or on semi-liquid LB agar under a layer of petroleum jelly.

The resulting recombinant strain of E. coli JM 109 1-430 has a new (acquired) property - it is a producer of the recombinant plasmid pWNV1protC. The copy number of the recombinant plasmid pWNV1protC is up to 200 genomic copies per cell. Plasmid pWNV1protC contains Ori replication, the antibiotic resistance marker ampicillin (Amp ^R ), the lacZ promoter located immediately in front of the insertion region, and a 430 bp region of the West Nile virus 1 genotype genome, which is a 5'-untranslated sequence regions of the genome, as well as a region of the polyprotein gene encoding the West Nile virus 1 genotype capsid protein.

Examples of specific implementation

Example 1. Construction of a bacterial strain E. coli JM 109 1-430 - the producer of the recombinant plasmid pWNV1protC, carrying a fragment of the genome West Nile virus 1 genotype.

The construction of the bacterial strain E. coli JM 109 1-430 was carried out by genetic engineering methods by cloning in E. coli JM 109 cells a fragment of the West Nile virus 1 genotype genome in the plasmid vector pUC19. As a fragment for insertion into the cloning vector, the sequence of the 5'-untranslated region of the genome was selected, as well as the region of the gene of the West Nile virus 1 polyprotein of the genotype encoding the capsid protein. The calculated length of the cloned fragment was 430 bp. The cloned fragment occupies position 1 to 430 nucleotides in the genome of West Nile virus genotype 1 (GenBank NCBI, Accession number: AY278441).

The source of viral RNA was the West Nile virus 1 isolate of genotype 1115, obtained from the Reference Center for Monitoring the causative agent of West Nile fever, operating on the basis of the Volgograd Research Anti-Plague Institute of Rospotrebnadzor. The West Nile virus genotype in the used RNA sample was confirmed by sequencing. Viral RNA was isolated using the RIBO-prep reagent kit (FBSI Central Research Institute of Epidemiology, Rospotrebnadzor, Russia) in accordance with the manufacturer's instructions. The cDNA strand was synthesized from the template of the isolated viral RNA by means of a reverse transcription reaction using the REVERTA-L reagent kit (FBSI Central Research Institute of Epidemiology, Rospotrebnadzor, Russia).

The nucleotide sequence of a 430 bp fragment of the West Nile virus 1 genome genotype required for insertion into the plasmid vector was obtained by amplification of the West Nile virus 1 genotype cDNA region by PCR with original primers carrying restriction sites for endonucleases at the 5'-ends XbaI and EcoRI having the following structure:

5'-TTATCA-TCTAGA-T-AGTAGTTCGCCTGTGTGAGCTGA-3'-WNV-XbaI-F

5'-TTAACT-GAATTC-T-CAATTCCGGTTTTCCCTCCTCTTTTC-3'-WNV1a-EcoRI-R.

To obtain the cloned fragment, the PCR mixture with a volume of 25 μL included the following components: primers WNV-XbaI-F / WNV1a-EcoRI-R, an equimolar mixture of four dNTPs (Amersham Biosciences, USA), magnesium chloride (Thermo Scientific, Lithuania), Taq buffer with ammonium sulfate (Thermo Scientific, Lithuania), Taq-F-polymerase (FBSI Central Research Institute of Epidemiology of Rospotrebnadzor, Russia), cDNA sample. When carrying out PCR, the following regime was used: preliminary denaturation at 95 ° С - 15 min; cycling (95 ° С - 10 s, 72 ° С - 50 s) × 50 cycles. PCR was performed on a C1000 Touch Thermal Cycler (Bio-Rad, Singapore).

The resulting amplicons were subjected to restriction in order to obtain sticky ends. At the same time, restriction of the vector was carried out, which was the plasmid pUC19 (Thermo Scientific, USA). The enzymes XbaI and EcoRI (Thermo Scientific, USA) were used as endonucleases. The amplicon was ligated with the vector in a 3: 1 ratio using T4 phage ligase (Invitrogen, USA). The resulting recombinant plasmid pWNV1protC was used to transform competent cells of E. coli JM 109 (Promega, USA).

As a result of the genetic engineering manipulations, a clone of the recombinant E. coli strain JM 109 1-430 was obtained, stably replicating the plasmid pWNV1protC. To achieve efficient replication and isolation of the recombinant plasmid pWNV1protC with a high yield, the E. coli JM 109 1-430 strain was cultured in LB medium (Lysogeny broth) according to Lennox (Becton Dickinson, USA) with ampicillin (Sintez, Russia) at a concentration 100 μg / ml at 37 ° C.

The recombinant plasmid pWNV1protC can be isolated from the cells of the recombinant E. coli JM 109 1-430 strain and used as a positive control in constructing diagnostic reagent kits for detecting West Nile virus 1 genotype strains by reverse transcription polymerase chain reaction.

Example 2. Identification of a fragment of the West Nile virus 1 genotype genome in the pWNV1protC plasmid of the recombinant E. coli JM 109 1-430 strain by PCR and sequencing.

To confirm the presence of the cloned sequence of the West Nile virus 1 genotype region in the recombinant plasmid pWNV1protC and to determine its size, the obtained transformants of E. coli were examined by PCR followed by electrophoretic detection.

A daily culture of the obtained recombinant E. coli strain JM 109 1-430 grown on LB agar was used to prepare a suspension in 6 ml of 0.9% sterile sodium chloride solution (MOSFARM LLC, Russia), pH 7.2 ± 0.1, equivalent in concentration 1 × 10 ⁹ MC / ml according to the industry standard turbidity sample of 10 units of the Federal State Budgetary Institution "NTs ESMP" of the Ministry of Health of Russia (OSO 42-28-85-P (10ME)). Then tenfold dilutions of the prepared suspension were carried out in 0.9% sodium chloride solution to a final concentration of 1 × 10 ⁴ MC / ml.

Next, sodium merthiolate (Panreac, Spain) was added to 6 ml of the resulting suspension of E. coli cells at a concentration of 1 × 10 ⁴ m.c./ml to a final concentration of 1: 10000 (0.01%) and heated at 56 ° C in for 30 minutes to inactivate E. coli JM 109 1-430 cells. Then, 100 μl of inactivated suspension of E. coli JM 109 1-430 cells was transferred into a 1.5 ml microcentrifuge tube and DNA extraction was performed using a commercial kit of reagents "RIBO-prep" (FBUN Central Research Institute of Epidemiology of Rospotrebnadzor, Russia) in accordance with the instructions for set.

Then a sample of isolated DNA in a volume of 10 μl was used for PCR amplification. The reaction mixture with a volume of 25 μL for PCR included the following components: universal forward and reverse primers Universal M13, flanking the polylinker of the pUC19 vector (Invitrogen, USA), an equimolar mixture of four dNTPs (Amersham Biosciences, USA), magnesium chloride (Thermo Scientific, Lithuania ), Taq buffer with ammonium sulfate (Thermo Scientific, Lithuania), Taq-F-polymerase (FBSI Central Research Institute of Epidemiology of Rospotrebnadzor, Russia), DNA test. PCR was performed on a C1000 Touch Thermal Cycler (Bio-Rad, Singapore) in the following mode: keeping the temperature at 95 ° С for 15 min; cycling (× 40), including: denaturation at a temperature of 95 ° C - 10 s, annealing at a temperature of 55 ° C - 15 s, elongation at a temperature of 72 ° C - 25 s. The PCR results were taken into account by electrophoresis in a 2% agarose gel. Similarly, a reference sample was investigated, which was 10 μl of pUC19 plasmid DNA.

Figure 1 shows an electropherogram of the results of amplification using universal primers Universal M13 of the original plasmid vector pUC19 and a fragment of the constructed recombinant plasmid pWNV1protC. The number 1 indicates the negative control. Number 2 denotes a 103 bp amplicon corresponding to a fragment of plasmid pUC19 without an insert. Number 3 denotes a 514 bp amplicon corresponding to a fragment of the pWNV1protC plasmid, which contains a fragment of the West Nile virus genotype 1 genome (430 bp), flanked by polylinker regions of the original pUC19 plasmid. Number 4 marks a molecular weight marker (100-1000 bp). The sizes of the specific amplicons indicated that the recombinant plasmid pWNV1protC, produced by the E. coli JM 109 1-430 strain, had an insert of the appropriate length (430 bp).

Determination of the nucleotide sequence of the inserted region of the West Nile virus genome 1 genotype in the recombinant plasmid pWNV1protC was carried out by sequencing the amplicons obtained using universal primers Universal M13. Sample preparation and PCR were performed as described above. Amplicons obtained using the DNA of the plasmid pWNV1protC were purified using chromatographic columns CENTRI-SEP (Princeton Separations, United States) according to the manufacturer's instructions. To carry out the cyclic sequencing reaction, the BigDye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems, USA) was used. Sequencing was performed on an ABI Prism 3130 automatic genetic analyzer (Applied Biosystems, USA). The sequencing results were analyzed using the MEGA 7 computer program (Pennsylvania State University, USA).

The nucleotide sequence obtained as a result of sequencing a region of the recombinant plasmid pWNV1protC isolated from the culture of E. coli JM 109 1-430 strain showed 100% homology with a similar RNA sequence of West Nile virus 1 genotype, which is the sequence of the 5'-untranslated region of the genome , as well as a fragment of the West Nile virus polyprotein gene encoding the West Nile virus 1 genotype capsid protein. This confirmed the presence of a genome fragment of the West Nile virus 1 genotype in the recombinant plasmid pWNV1protC produced by the E. coli JM 109 1-430 strain.

Example 3. Use of the recombinant plasmid pWNV1protC produced by the E. coli JM 109 1-430 strain as a positive control for detecting RNA West Nile virus 1 genotype by RT-PCR in real time.

The possibility of using the recombinant plasmid pWNV1protC produced by the E. coli strain JM 109 1-430 as a positive control was assessed using a set of specific oligonucleotide primers WNV-1type-F / WNV-1type-R and a fluorescently labeled probe WNV-1type designed by specialists of the Volgograd Scientific Research Anti-Plague Institute of Rospotrebnadzor to identify West Nile virus 1 genotype.

Sample preparation was carried out as described in example 2. Reverse transcription and PCR were performed in one tube. The reaction mixture, in addition to the pWNV1protC plasmid DNA, included the developed primers WNV-1type-F / WNV-1type-R and the fluorescently labeled probe WNV-1type-P, as well as an equimolar mixture of four dNTPs (Amersham Biosciences, USA), PCR -buffer (FBUN Central Research Institute of Epidemiology of Rospotrebnadzor, Russia), magnesium chloride solution (Thermo Scientific, Lithuania), MMlv revertase enzyme (Thermo Scientific, USA) and Taq-F-DNA polymerase enzyme (FBUN Central Research Institute of Epidemiology of Rospotrebnadzor, Russia). The volume of the reaction mixture was 25 μl. As a negative control, an equal volume of distilled water was added to the test tube instead of the sample. RNA West Nile virus 1 genotype was used as a comparison sample.

PCR was performed on a Rotor-Gene Q device (QIAGEN, Germany). Reaction conditions: stage of reverse transcription at 50 ° С - 30 min; the stage of preliminary denaturation at 95 ° С - 15 min; first cycling (5 repetitions) - denaturation at 95 ° С - 5 s; annealing of primers at 56 ° С - 25 s; chain elongation at 72 ° С - 15 s; second cycling (40 repetitions) - denaturation at 95 ° C - 5 s; annealing of primers at 56 ° С - 25 s; chain elongation at 72 ° С - 15 s. The detection of the accumulation of reaction products was carried out via the Green channel at the second cycling stage at a temperature of 56 ° C.

When analyzing the RT-PCR data in real time, a positive result was obtained both for the DNA sample of the recombinant plasmid pWNV1protC isolated from the E. coli JM 109 1-430 strain, and for the reference sample, which is West Nile virus 1 genotype RNA, which indicated the possibility of using the recombinant plasmid pWNV1protC as a positive control for RT-PCR. Figure 2 shows a graph of the fluorescence rise curves obtained by RT-PCR. Number 1 designates a DNA sample of the recombinant plasmid pWNV1protC isolated from the E. coli strain JM 109 1-430. Number 2 marks the West Nile virus 1 genotype RNA sample. The negative control is below the threshold line.

Thus, the constructed bacterial strain Escherichia coli JM 109 1-430 is a producer of the recombinant plasmid pWNV1protC, which can be used as a positive control to evaluate the efficiency of amplification of West Nile virus 1 genotype cDNA in clinical laboratory diagnostics. The constructed bacterial strain Escherichia coli JM 109 1-430 can be used as a raw material for the design and production of reagent kits for the detection and differentiation of West Nile virus genotypes.

Claims (1)

The bacterial strain Escherichia coli JM 109 1-430 (State collection of pathogenic bacteria FKUZ of the Russian Research Anti-Plague Institute "Microbe" Rospotrebnadzor, number KM 2049), intended for use as a positive control in assessing the effectiveness of amplification of cDNA West Nile virus 1 genotype, which is a producer of the recombinant plasmid pWNV1protC carrying a genome fragment of West Nile virus 1 genotype 430 bp in size, which is the sequence of the 5'-untranslated region of the genome, and a region of the West Nile virus polyprotein gene encoding a capsid protein.

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