RU2511029C2 - RECOMBINANT STRAIN OF Escherichia coli TG1(pRVMoscow3253G-L) FOR OBTAINING SET OF PCR-STANDARDS AND SET OF PCR-STANDARDS FOR DETERMINATION OF CONCENTRATION OF RABIES VIRUS "Moskva 3253" IN RABIES ANTIGEN - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and deals with recombinant strain of E. coli TG1(pRV_Moscow3253G-L) for obtaining PCR-standards for quantitative determination of cDNA of rabies virus of strain "Moskva 3253".Recombinant strain is created on the basis of strain of E. coli TG1 by transformation with plasmid pRV_Moscow3253G-L. Plasmid is obtained by ligation of fragment G-L of the region of genome of fixed rabies virus of strain "Moskva 3253", which has sequence SEQ ID NO1, into plasmid pGem-T. Also claimed is set of PCR-standards for quantitative determination of cDNA of rabies virus of strain "Moskva 3253" in rabies antigen. Set contains solutions of plasmid pRV_Moscow3253G-L DNA in concentrations 10⁸, 10⁷, 10⁵, 10³ GE/ml. Concentration is determined by method of polymerase chain reaction, with hybridisation-fluorescence account of results.

EFFECT: invention contributes to standardisation of stage of preparation of rabies antigen in production of heterological anti-rabies immunoglobulin.

2 cl, 3 dwg, 2 ex

Description

The invention relates to biotechnology and relates to the construction of a recombinant strain of Escherichia coli carrying the cloned sequence of the G-L fragment of the genome of the rabies virus strain "Moscow 3253" and a set of PNR standards for quantitative PCR, and can be used in the manufacture of rabies immunoglobulin.

The unfavorable epidemiological situation of rabies, which has developed in almost all regions of the Russian Federation due to an increase in the incidence of wild and domestic animals, causes a sharp increase in the number of people who seek anti-rabies help every year, which dictates the need to improve anti-rabies drugs and improve their quality [4].

An anti-rabies immunoglobulin is obtained from hyperimmune serum of producers immunized with a rabies antigen. The lack of quantitative methods for determining the content of rabies virus in rabid antigen during immunization prevents the standardization of antigenic material.

One of the promising approaches to the determination of the fixed rabies virus of the strain Moscow 3253 in the material for immunization is the use of a quantitative polymerase chain reaction with hybridization-fluorescence real-time results using PCR standards with a certain number of gene equivalents of viral particles or fragments of the genome the virus.

Known PCR standards (EBVQ-PCR Standard, cat. No. STD020; HSV1Q-PCR Standard, cat. No. STD031; HSV2Q-PCR Standard, cat. No. STD032; HHV6Q-PCR Standard, cat. No. STD036), which are a plasmid solution with different concentrations for determining the amount of Epstein-Barr virus, herpes simplex virus of the first and second types, other viruses in biological material by PCR using real-time results that are produced by NaNogen Advanced Diagnostics (Italy) [10, 12].

Known PCR standards (Colibrator) for the quantitative diagnosis of hepatitis C (cat. No. 01N30-070), HIV-1 (cat. No. 6L18-70), hepatitis B (cat. No. 02N40-70), etc. by PCR using real-time results produced by Abbott Molecular (USA) [2, 3]. However, PCR standards for the quantitative determination of rabies virus by PCR, taking into account the results in real time, these firms do not produce.

According to the results of a search for information on the presence of PCR standards for the quantitative accounting of the fixed rabies virus of the strain Moscow 3253 in the material for immunization by PCR, taking into account the results in real time, was not found.

To create PCR standards used in quantitative PCR, taking into account the results in real time, a producer strain of a specific fragment of a specific region of the rabies virus genome is required. To date, there is no information on recombinant strains with a specific fragment of the region of the rabies virus genome for its quantitative assessment.

To construct a recombinant strain, the choice of a unique region of the target DNA is required, which has high stability in all passages of the virus required under production conditions and sufficient specificity.

The literature describes the use of the N region of the virus genome as a target DNA. This locus is characterized by high conservatism [1]. Several alleles of this locus were noted that are specific for certain genotypes of lissaviruses [6, 7, 8, 9, 11, 13]. When studying the variability of the N-locus of rabies virus, it was shown that this site cannot be used as a matrix for determining individual strains of the virus using amplification technologies. To detect the fixed rabies virus of the Moscow 3253 strain by PCR with reverse transcription of cDNA, the G locus and GL region of the rabies virus genome are most suitable as the target DNA, characterized by high polymorphism and the frequency of occurrence of mutations [1] and differing in individual strains rabies virus.

According to the results of the patent search and literature data, information about the presence of recombinant strains carrying the G-L fragment of the genome of the rabies virus strain "Moscow 3253" was not found.

The objective of the present invention is the construction of a strain of E. coli carrying a recombinant plasmid with a cloned sequence of a GL fragment of the genome of the fixed rabies virus strain Moscow 3253, to obtain PCR standards and the creation on its basis of a set of PCR standards for the quantitative determination of cDNA of rabies virus strain "Moscow 3253" in the rabbit antigen used in the production of anti-rabies immunoglobulin by PCR using real-time results.

The technical result consists in standardizing the stage of the technological process of preparing a rabbit antigen in the production of heterologous anti-rabies immunoglobulin, by creating a recombinant strain of E. coli KM 229 for the production of PCR standards and a set of PCR standards based on it, allowing quantitative characterization of the rabbit antigen by PCR with hybridization real-time fluorescence detection.

The technical result is achieved by constructing a recombinant E. coli strain carrying the plasmid pRV _Moscow3253 GL containing a fragment of the GL region of the genome of the fixed rabies virus strain Moscow 3253, having a size of 881 bp, the sequence SEQ ID NO1, designed to obtain the kit PCR standards.

The sequence of SEQ ID NO1 is presented in figure 1.

To construct a recombinant strain, the nucleotide sequence of the GL fragment of the genome region of the Moscow 3253 strain of 881 bp, flanked by BeshG 5'-gacttgggtctcccgaactgggg-3 'and BeshL 5'-caaaggagagttgagattgtagtc-3' primers was selected (from 5543 to 46) the sequence of the genome of the strain of the virus RV-97, GenBank NCBI No. EF542830).

The cloning procedure consisted of the following steps.

1. The amplified fragment was purified from mononucleotides not included in the reaction on a Gentry-sep colounins column (Pirseton Separations INC, USA) in accordance with the manufacturer's instructions.

2. The fragment was ligated into the pGem-T plasmid using the commercial pGem-T Vector Sistems kit (Promega USA) according to the manufacturer's instructions.

3. Cells of E. coli TG1 strain were transformed with an aliquot of a subsidized mixture. For this, 200 ± 30 μl of freshly prepared competent cells was added to 20 ± 5 μl of the ligation mixture. After incubation at 42 ° C for 90 seconds, the mixture was kept on ice for 2 minutes and grown for 1 hour at 37 ° C. Cells were plated on agar medium with ampicillin at a concentration of 100 μg / ml in Petri dishes and incubated for 18 ± 2 hours at 37 ° C. .

4. The grown colonies were checked for the presence of a G-L fragment of the genome of the fixed rabies virus of the Moscow 3253 strain, which has the sequence SEQ ID NO1, by PCR with BeshG 5'-gacttgggtctcccgaactgggg-3 'and BeshL 5'-caaaggagagttgagattgtagtc-3 primers.

Colonies of E. coli TG1 (pRV _Moscow3253 GL) were grown on LB liquid medium with ampicillin in test tubes for 18 ± 2 hours at a temperature of 37 ° C with constant shaking on a rocking chair. The resulting biomass was collected by centrifugation in microtubes in a volume of 1.5 ml.

6. A plasmid was isolated from the obtained biomass by membrane filtration using a PureYield Plasmid Miniprep Sistems kit (Promega, USA) or the like.

7. The presence of the fragment was confirmed by PCR with BeshG and BeshL primers, specificity was assessed by sequencing on a genetic analyzer, for example, CeO-800, using standard protocols for sample preparation and instrument software.

The recombinant strain carrying the plasmid pRV _Moscow3253 GL, containing in its composition a GL fragment of the genome of the fixed rabies virus strain Moscow 3253 "size 881 bp, having the sequence of SEQ ID NO1, is characterized by the following features.

Cultural and morphological properties: represented by gram-negative rods, giving on a nutrient medium LB with ampicillin smooth, with a smooth edge of the colony with a diameter of (2 + 0.5) mm.

Physiological and biochemical properties: preserves the main cultural, morphological and biochemical properties of E. coli TG1.

Plasmid pRV _Moscow3253 GL with a cloned GL fragment of the genome region of the fixed rabies virus strain Moscow 3253, having the sequence SEQ ID NO1, is the initial product for obtaining PCR standards used in performing quantitative PCR based on real-time results to determine the amount of fixed rabies virus strain "Moscow 3253" in the rabies antigen in the production of rabies immunoglobulin.

The inventive recombinant strain of E. coli TG1 (pRV _Moscow3253 GL) was deposited in the State collection of pathogenic bacteria at FKUZ RosNIPCHI "Microbe", under the number KM 229.

The resulting recombinant strain of Escherichia coli KM 229 is designed to obtain a set of PCR standards used for quantitative determination in PCR taking into account the real-time results of the fixed rabies virus of the strain Moscow 3253 in the rabies antigen in the production of rabies immunoglobulin.

The technical result is also achieved by obtaining, on the basis of plasmid pRV _Moscow3253 GL, produced by a recombinant strain of E. coli KM 229, a set of PCR standards. PCR standards are a solution of DNA plasmid pRV _Moscow3253 GL at a concentration of 10 ⁸ to 10 ³ GE / ml.

The kit for the quantitative determination of cDNA of the fixed rabies virus of the Moscow 3253 strain in the rabies antigen consists of PCR standards with the abbreviation (Rabies virus) RV1, RV2, RV3, RV4, which indicates different concentrations of plasmid DNA: from RV1 - maximum concentration to RV4 - minimum . To detect high concentrations of rabies virus, a combination of PCR standards RV1, RV2, RV3 is used; to determine low concentrations - a combination of PCR standards RV2, RV3, RV4.

PCR standards RV1, RV2, RV3, RV4 were prepared from a DNA solution pRV _Moscow3253 GL, the concentration of which was determined spectrophotometrically at a wavelength of 260 and 280 nm. The optical density D = 1 corresponds to - 50 μg / ml double-stranded DNA. The purity of the DNA preparation was determined by the ratio of the optical density of the solution at 260 and 280 nm. To prepare the PCR standards, we used the initial DNA solution of plasmid pRV _Moscow3253 GL with a concentration of at least 10 μg / ml with a ratio of D ₂₆₀ / D _{280 of} at least 1.5.

The quantitative characteristic of the PCR standards RV1, RV2, RV3, RV4 is expressed by the number of genome equivalents of the plasmid pRV _Moscow3253 GL in solution, which was calculated by the formula:

K _{pRVMoscow3253G-L} = A × 0.234 × 10 ¹² [GE / ML],

Where A is the concentration of plasmid DNA pRV _Moscow3253 GL in μg / ml.

The PCR standard RV1 contains n × 10 ⁸ GE / ml pRV _Moscow3253 GL

The PCR standard RV2 contains n × 10 ⁷ GE / ml pRV _Moscow3253 GL

The PCR standard RV3 contains n × 10 ⁵ GE / ml pRV _Moscow3253 GL

The PCR standard RV4 contains n × 10 ³ GE / ml pRV _Moscow3253 GL

The kit is intended for the quantitative determination of cDNA of the fixed rabies virus of the strain "Moscow 3253" in virus-containing material at the stages of preparation of the rabies antigen used in the production of heterologous rabies immunoglobulin.

The use of the kit allows you to control and standardize the technological operations carried out at the stage of preparation of the rabies antigen in the production of heterologous rabies immunoglobulin, which meets the GMP requirements for the technological process and helps to improve the quality of the drug.

The practical application of PCR standards based on a recombinant strain is illustrated by the following examples.

Example 1. The quantitative determination of high concentrations of rabies virus strain "Moscow 3253" using a set of PCR standards for the example of rabic antigen of organo-tissue origin.

Rabic antigen of organo-tissue origin was obtained from the brain suspension of rabbits, which previously introduced rabies virus strain "Moscow 3253". Samples for analysis were prepared from antigenic material (brain suspension of rabbits) disinfected according to MU 1.3.2569-09 [5].

Determination of rabies virus concentration was carried out in several stages.

Samples of rabbit antigen of organo-tissue origin were taken in 100 μl and transferred into 5 1.5 ml microcentrifuge tubes. Additionally, 100 μl of TE buffer, a negative control of DNA / RNA (OKV) isolation, was added to a separate 1.5 ml microcentrifuge tube.

RNA isolation. For RNA isolation, we used a “kit of reagents for RNA / DNA isolation from the clinical material“ RIBO-sorb ”manufactured by the company (LLC“ InterLabService ”, Russia). The set of reagents for RNA isolation consists of a lyse solution, a solution for washing 1, a solution for washing 3, a solution for washing 4, a sorbent and an RNA buffer. In 5 microcentrifuge tubes with 100 μl of disinfected rabbit antigen of organo-tissue origin and 1 OKV tube, 450 μl of pre-heated lysing solution was heated at a temperature of (62 ± 2) ° С for 5-10 min and mixed thoroughly. Left in a tripod for 5-8 minutes, stirring 3-4 times. Centrifuged for 5 s at 3000-5000 rpm. The sorbent was carefully resuspended and 30 μl was added to each tube. Stirred, left in a rack for 1 minute, mixed again and left for 5 minutes. Tubes were centrifuged at 10,000 rpm for 45 s. The supernatant was removed, and 400 μl of washing solution 1 was added to the precipitate. The mixture was stirred until the sorbent was completely resuspended, centrifuged for 45 s at 10,000 rpm. After removal of the supernatant, 500 μl of washing solution 3 was added to the tubes. The sorbent was carefully resuspended and the tubes were centrifuged for 45 s at 10,000 rpm. After removal of the supernatant, the operation was repeated again. Then, 400 μl of washing solution 4 was added to the tubes, carefully resuspended, and centrifuged for 45 s at 10,000 rpm. The supernatant was removed and the tubes were placed in a thermostat at a temperature of 60 ° C for 10 min, while the lids were left open.

After drying, 50 μl of RNA buffer was added to each tube, mixed and placed in a solid state thermostat at a temperature of 60 ° С for 3 min. Stirred and centrifuged for 1 min at 13,000 rpm. The supernatant contained purified virus RNA.

Conducting a reverse transcription reaction.

To set up the reverse transcription reaction, a set of reagents was used to obtain cDNA on the Reverta-L RNA template (InterLabService LLC, Russia). The set of reagents for producing cDNA on the RNA matrix contains RT-G-mix-1, RT-mix - 0.125 ml, revertase (MMLv) and DNA buffer.

Before starting work, tubes with RT-mix and RT-G-mix-1 (RT-G-mix-2) were removed from the freezer and the contents of the tubes were thawed at room temperature. Then they were thoroughly mixed and centrifuged for 5 s at 3000-5000 rpm to precipitate drops from the walls. To prepare the reaction mixture, 5 μl of RT-G-mix-1 (RT-G-mix-2) was added to the RT-mix tube, mixed thoroughly, centrifuged for 5 s at 3000-5000 rpm to precipitate drops from the walls. To the resulting solution was added 6 μl of MMLv revertase, gently mixed and centrifuged for 5 s at 3000-5000 rpm to precipitate drops from the walls.

5 microcentrifuge tubes with a volume of 0.6 ml were selected, corresponding to the number of test samples and OKV. 10 μl of the resulting reaction mixture was added to each tube, then 10 μl of the RNA preparation and 10 μl of TE buffer in OKB were added to the tubes. The tubes were incubated in a thermocycler or solid state thermostat for 30 min at a temperature of 37 ° C. 20 μl of DNA buffer was added to all tubes and gently mixed 8-10 times. The obtained cDNA was used for PCR with hybridization-fluorescent results.

Arrangement of PCR.

0.2 ml microtubes were removed from the freezer, containing 1 PCR mixture, 2 PCR mixture, TE buffer, Taq enzyme DNA polymerase with an activity of 5 units / μl, a set of PCR standards RV1, RV2, RV3 for taking into account high concentrations of rabies virus antigen in the sample. The contents of the tubes were completely thawed.

PCR mixture-1 in a volume of 5 μl contained a solution of primers RV8 5'-ACGTACTATGACCTCTACCC-3 ', RV9 5' - CCAGGAACTAATACTAAGGG-3 'and RV10 probe 5'-FAM-CTCCGATGATCCCATGCTTG-BHQ1-3', which provide the GL fragment for the amplification region fixed rabies virus strain Moscow 3253, in the amount of 8 and 4 pMol, respectively, 1 mmol of each of the four dNTPs, 0.025% sodium azide solution and nuclease-free water. PCR mix-1 was transferred 5 μl into 9 0.2 ml microcentrifuge tubes. In each tube, a molten 20% solution of paraffin in mineral oil was layered on the surface of the PCR mixture-1.

PCR mixture-2 in a volume of 10 μl containing 2.5 × Taq buffer with ammonium sulfate, 6.25 mmol of magnesium chloride, Taq enzyme DNA polymerase, nuclease-free water, was mixed on a microcentrifuge / shaker and transferred to 10 μl in 5 microtubes with PCR mixture-1 without damaging the paraffin layer.

In 5 microtubes, 10 μl of a cDNA sample was added, in 3 microtubes 10 μl of each PCR standard RV1, RV2, RV3 was added, and 10 μl from an OKV sample containing TE buffer, the study of which is carried out with the analyzed samples, was introduced into 1 microtube starting at the stage of DNA extraction. 10 μl of TE buffer was added to a 1 microtube with a negative amplification control (K-).

The prepared microtubes were placed in a RotorGene 6000 thermal cycler and PCR was carried out with hybridization-fluorescence results taking into account when annealing the primers at 55 ° C for 10 cycles, then at 51 ° C for 35 cycles. Accounting for fluorescence through the FAM channel was carried out at a temperature of 51 ° C.

Accounting for results.

The rabies virus cDNA content in the antigenic material was quantified by the signal fluorescence intensity by comparing the test sample with the PCR standards RV1, RV2, RV3 using the RotorGene 6000 thermal cycler software, if there was a negative result in the K and OKV samples.

The concentrations of the fixed rabies virus of the strain “Moscow 3253” in 5 test samples of the brain suspension of rabbits ranged from 5.57 × 107 copies / ml to 3.07 × 109 copies / ml (figure 2). The correlation coefficient amounted to 0.99086, the reaction efficiency - 0.85949.

Example 2. Quantitative determination of low concentrations of rabies virus strain "Moscow 3253" on the example of the culture fluid at the stage of growing the virus in cell culture using a set of PCR standards.

For the study, 6 samples of the virus-containing culture fluid obtained by the suspension method of growing Virus fixe on a Vero cell culture before the concentration step were taken. The disinfection of virus-containing material, the isolation of RNA, the reverse transcription reaction and PCR were carried out analogously to example 1, only when performing PCR, a set of PCR standards RV2, RV3, RV4 was used to take into account low concentrations of rabies virus in the sample.

The concentrations of the fixed rabies virus of the strain Moscow 3253 in the studied samples ranged from 3.31 × 104 copies / ml to 1.59 × 106 copies / ml (figure 3). The correlation coefficient amounted to 0.99959, the reaction efficiency - 0.90885.

Thus, the inventive recombinant strain of E. coli TGl (pRV _Moscow3253 GL) to obtain a set of PCR standards and a set of PCR standards for the quantification of cDNA of the fixed rabies virus of strain "Moscow 3253" in the rabies antigen allow us to determine different degrees of concentration of the fixed rabies virus " Moscow 3253 "in virus-containing material, to control and standardize the technological operations carried out at the stage of preparation of the rabid antigen in the production of heterologous rabies immunogen obulina that meets the GMP requirements for the process and improves the quality of the produced product.

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Claims (2)

1. Recombinant E. coli TG1 strain (pRV _Moscow3253 GL) to obtain PCR standards for quantitative determination of cDNA of the Moscow 3253 strain, created on the basis of E. coli TG1 strain by transforming plasmid pRV _Moscow3253 GL obtained by ligation of a GL fragment of a fixed genome region rabies virus strain "Moscow 3253", having the sequence of SEQ ID NO1, in the plasmid pGem-T, deposited in the State collection of pathogenic bacteria "Microbe" under the number KM 229. 2. A set of PCR standards for the quantitative determination of various concentrations of fixed rabies virus of the strain "Moscow 3253" in virus-containing material by polymerase chain reaction with hybridization-fluorescent results, containing DNA solutions of plasmid pRV _Moscow3253 GL carrying the GL fragment of the genome region of the fixed rabies virus strain "Moscow 3253" having the sequence of SEQ ID NO1, in concentrations of 10 ⁸ , 10 ⁷ , 10 ⁵ , 10 ³ GE / ml.

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