Genetic construction on basis of vector plasmid pegfp-n1 producing siphk-inhibitors of reproduction of the human immune deficiency virus of the 1 type (its variants)

FIELD: chemistry, organic, viruses.

SUBSTANCE: invention relates to medical and molecular genetics. Three geometric constructions are proposed on the basis of vector plasmid pEGFP-N1, the constructions producing siPHK: inhibitors of reproduction of the human immune deficiency virus of the 1 type. The invention may be used in development of more efficient anti-HIV preparations on the basis of interfering RNAs (siPHK) produced in cells with the aid of administered vector expressing constructions.

EFFECT: obtaining of genetic construction for development of anti-HIV preparations.

3 cl, 1 dwg, 5 tbl, 3 ex

 

The invention relates to medical and molecular genetics, namely, genetic structures, producing siPHK, which are inhibitors of reproduction of the human immunodeficiency virus (HIV) type 1, and can be used in medicine and scientific research.

Currently, the main approach for the treatment of HIV infection is a prescription antiviral chemotherapy, including drugs that act on the key enzymes of HIV-1 reverse transcriptase, integrase, and protease. The most effective of the known compounds are 3'-azido-3'-deoxythymidine (azidothymidine or AZT, zidovudine, Retrovir, Timated"), which are used in medical practice (Mitsuya, H.; Broder, S. Inhivition of the in vitro infectivity and cytiopathic effect of human T-lymphotropic virus type III/lymphoadenopathy-associated virus/HTLV-III(LAV) by 2',3'-dideoxynucleosides. Proc. Nat. Acad. Sci. USA, 1986, 83, 1911-1915), and 2',3'-dideoxycytidine (ddC, zalcitabine, "GUID"), 2',3'-dideoxyinosine (ddI, didanosine, videx"), 3'-deoxy-2',3'-didehydrothymidine (d4T, stavudine, stavudine, zerit) and 3'-thiacytidine (FTC, lamivudine, Epivir").

However, despite the large number of available anti-HIV drugs, there is the issue of the effectiveness of antiviral therapy. The main reason for treatment failure - adaptive mutagenesis of the virus, leading to the emergence of variants of HIV-1, with steadily the TEW to antiviral drugs. Serious problems are also toxicity and high cost of used medicines.

It is known the use of ribozymes and antisense RNA for the treatment of HIV infection [Dorman N. And Lever, A.M. (2001) RNA-based gene therapy for HIV infection. HIV Med., 2, 114-122; Michienzi, A., Conti, L., Varano Century, Prislei S., Gessani S., and Bozzoni I. (1998) Inhibition of human immunodeficiency virus type 1 replication by nuclear chimeric anti-HIVribozymes in a human T lymphoblastoid cell line. Hum. Gene Ther., 9., 621-628; Veres G., Junker, U., Baker J., Barske C., Kalfoglou C., H. Ilves, S. Escaich, Kaneshima H., and Bohnlein, E. (1998) Comparative analisis of intracellularly expressed antisense RNAs as inhibitors of human immunodeficiency virus type 1 replication. J. Virol., 72, 1894-1901.], which block multiple HIV genes, which reduces its activity.

Closest to the claimed technical solution are genetic constructs that give expression siPHK within HIV-infected cells (Application for U.S. patent No. 20030059944, IPC 12N 15/867, publ. 27.03.2003 year).

However, the evaluation steps interfering RNA produced in the data published genetic constructions were carried out using molecular clones of human immunodeficiency virus, which does not allow to predict their impact on the natural pool of viral populations, knowingly possessing genetic diversity that does not allow a more objective evaluation of antiviral efficacy of such genetic constructs.

The technical result of the offer is aemula of the invention is the development of more effective anti-HIV drugs based on interfering RNA (siPHK), produced in cells using the introduced vector expressing constructs containing palindromes designed for education "studs RNA and selected using a viral model - culture of human cells infected with different strains of HIV-1.

The technical result is achieved by creating three options plasmid constructions, producing the corresponding sites of the genes gag, pol and tat of HIV-1:

genetic structure-based vector plasmid pEGFP-N1, producing siPHK inhibitors reproduction of human immunodeficiency virus type 1 containing a fragment of the genome of HIV-1 pr-hp-pEGFP-N1 area length 19 bp of the conservative region of the protease domain (1749...1767): 5' gaattccTGATTCAGATTGGTTGTACttttttgtacaaccaatctgaatcaaccccggatcc 3'.

genetic structure-based vector plasmid pEGFP-N1, producing siPHK inhibitors reproduction of human immunodeficiency virus type 1 containing a fragment of the genome of HIV-1 tat-hp-pEGFP-N1 area length 19 bp from a conservative district domain reverse transcriptase (2116...2134):

5' gaattccTGCATATTTTTCAGTTCCCttttttgggaactgaaaaatatgcatccccggatcc 3'.

genetic structure-based vector plasmid pEGFP-N1, producing siPHK inhibitors reproduction of human immunodeficiency virus type 1 containing a fragment of the genome of HIV-1 RT-hp-pEGFP-N1 area length 19 bp from a conservative district tat domain (5325...534):

5' gaattccAGTAGGACTAATAGTAGCAtttttttgctactattagtcctactatccccggatcc 3',

where (all three versions): lowercase shows the region containing artificial restriction enzymes cut sites for restricted EcoRI (5'-end) and BamHI (3'-ends), and the loop that separates the region, capable of forming a double-stranded duplex with loop, and the area corresponding sense RNA strand of the virus, as shown in the row on the left, and antisense to the right.

Provides genetic constructs provide a purposeful influence on the mRNA of human immunodeficiency virus the first type (HIV-1) in human cells virousspecificakih interfering RNA (siPHK)containing substances that occur in cells. therapeutic result of this effect is the suppression of the reproduction of HIV-1 in human cells.

For human immunodeficiency virus the first type is characterized by a rapid emergence and selection of mutant variants with resistance to various anti-viral drugs. To solve viral variability developed three genetic structure, stably expressing several siPHK. Then, to evaluate the effectiveness of using viral model - culture of human cells infected with different strains of HIV-1, in particular, strains, reproducibility on the type of acute and chronic infection. This approach allows the rowedit research realistic development processes of HIV infection in the body. The use of laboratory strains of HIV-1 (in contrast to molecular clones of the virus in the prototype) provides an opportunity to assess the impact of interfering RNA to the natural pool of viral populations, knowingly possessing genetic diversity. It also allows for more objective evaluation of antiviral efficacy of genetic constructs.

The drawing shows the physical vector map of the plasmid pEGFP-N1.

Example 1. Description of genetic constructs (three options) and how they obtain Plasmid pEGFP-N1 registered GenBank Accession #U55762, catalog number 6085-1, CLONOTECH Laboratories, Inc. (Prasher, D. C., et al. (1992) Primary structure of the Aequorea victoria green fluorescent protein. Gene 111: 229-233), has a size of 4.7 KBP and characterized in that it contains:

1. The early promoter of human cytomegalovirus (PCMV IE): 1-589

Enhancer region: 59-465

TATA box: 554-560

SIG transcription starts: 583

C->G mutation that removes the Sac website I: 569

2. The multiple cloning sites (MSC). Sequences marked the recognition sites for restriction endonucleases involved in the integration of HIV-specific DNA fragments:

3. EGFP reporter gene (1-3), which allows to control the introduction of vector design is UKTI in eukaryotic cells and to optimize transfection (EGFP):

The Kozak sequence (SIG initiation of translation): 672-682

The start codon (ATG): 679-681; Stop codon: 1396-1398

The insertion of Val at position 2: 682-684

Mutations in the gene GFPmutl (Phe-64 to Leu; Ser-65 to Thr): 871-876

His-231 to Leu mutation (A⊘T): 1373

4. Fragment of SV40 mRNA polyadenylation signal (SV40 poly A):

The polyadenylation signal: 1552-1557 & 1581-1586

mRNA 3' end: 1590 & 1602

5. The origin of single-stranded DNA phage f1 (f1 ori): 1649-2104

(Packed non-coding circuit EGFP)

6. Bacterial promoter for expression of the gene of resistance to kanamycin Kanr(R):

- 35 district: 2166-2171; - 10 district: 2189-2194

The start point of transcription: 2201

7. The origin of replication of SV40 (SV40 ori): 2445-2580

8. Early SV40 promoter (PSV40e)

Enhancer (tandem repeat 72-BP): 2278-2349 & 2350-2421

Repeat 21-BP: 2425-2445, 2446-2466, & 2468-2488

The item early promoter: 2501-2507

The main starting point of transcription: 2497, 2535, 2541 & 2546

9. The gene for resistance to kanamycin/neomycin (Kanr/Neor):

The sequence encoding neomycinphosphotransferase:

The start codon (ATG): 2629-2631; stop codon: 3421-3423

G->A mutation that removes the Pst website 1: 2811

C->A (Arg to Ser) mutation to remove the website BssH II: 3157

10. The polyadenylation signal timedancing (TK) of herpes simplex virus (HSV TK poly A): polyadenylation Signal: 3659-3664 & 3672-3677

11. The origin of replication of plasmid pUC (pUC ori): 4008-4651

12. Primers DL the sequencing

EGFP-N: 745-724

EGFP-C: 1332-1353

Vector plasmid pEGFP-N1 encodes the GFP gene, which allows to control the introduction of vector constructs in eukaryotic cells when the cells fluorescent glow and optimization of transferin. The set of unique recognition sites for restriction endonucleases (MSC), located between early promoter of cytomegalovirus person (CMV) and HGFP coding sequence allows embedding and obtain the expression of different genes. The vector contains the SV40 origin, provides replication in mammalian cells. The cartridge resistance to neomycin (neor), consisting of the early SV40 promoter, a resistance gene for kanamycin/neomycin (Kanr/Neor) and the polyadenylation signal of the gene timedancing of herpes simplex virus allows stable transferout eukaryotic cells using the selective pressure of G418. Bacterial promoter, located nearby, provides resistance to kanamycin cells E. Coli. pEGFP-N1 also contains the origin of replication pUC19, to allow time vector plasmids for bacterial culture E. coli and the origin of replication of phage 1 to obtain single-stranded DNA molecules.

Expressing "hairpin" structures contain fragments of the genome of HIV-1 pr-hp-pEGFP-N1, tat-hp-pEGFP-N1, RT-hp-pEGFP-N1. Using the program BLAST was SEL is Ana's most conservative region of the genome of HIV-1, corresponding functionally important regions of the virus - domains of the protease, reverse transcriptase and tat in the sequence AF316544 (HIV-1 subtype A). Target designs, coding siPHK were these areas. The first structure contains the length of 19 bp of the conservative region of the protease domain (1749 1767...):

5' gaattccTGATTCAGATTGGTTGTACttttttgtacaaccaatctgaatcaaccccggatcc 3'.

The second region with a length of 19 bp from a conservative district domain reverse transcriptase (2116...2134):

5' gaattccTGCATATTTTTCAGTTCCCttttttgggaactgaaaaatatgcatccccggatcc 3'.

The third region with a length of 19 bp from a conservative district tat domain (5325...5343):

5' gaattccAGTAGGACTAATAGTAGCAtttttttgctactattagtcctactatccccggatcc 3'. In the above nucleotide texts lowercase letters shows the region containing artificial restriction enzymes cut sites for restricted EcoRI (5'-end) and BamHI (3'-ends), and the loop that separates the region, capable of forming a double-stranded duplex with loop ("hairpin" dsRNA). Regions corresponding to the sense RNA strand of the virus, as shown in the row on the left, and antisense to the right. Double-stranded DNA corresponding to the above texts, were cloned in the EcoRI-BamHI sites of the vector pEGFP-N1.

Example 2. Suppression of reproduction of human immunodeficiency virus in cell cultures MT-4, transfected with various plasmids expressing virusspecific short interfering RNA.

Research spending is on transplantable lines of lymphoid cells MT-4. Cells were cultured in medium RPMI-1640 with the addition of 10% fetal cattle serum, previously inactivated by heating at 56°C for 30 minutes, 300 mg/ml L-glutamine and 100 μg/ml of gentamicin.

The production and separation of DNA plasmids carried out using ammonium acetate according to the developed methodology for the preparation of DNA for transfection of mammalian cells [Saporito-Irwin S.M., Geist R.T. and Gutmann D.H. (1997) Ammonium Acetate Protocol for the preparation of plasmid DNA Sutable for Mammalian Cell Transfections. BioTechniques 23(3), 424-427.]

Introduction expressing plasmids into cells is performed using transfection. The transfection is carried out with the use of lipofectamine (LipofectamineTM2000, Invitrogen). On the third day of culturing cells MT-4 washed in minimal medium (DMEM without phenol red) and diluted to a concentration of 0.5×106cells/ml 5 μg of plasmid DNA was diluted in 50 μl of minimal medium, incubated 5 min at room temperature and mixed with 3 μl of lipofectamine diluted in 50 μl of minimal environment. The mixture is incubated for 20 min at room temperature. 100 μl of the DNA solution/lipofectamine added to 500 μl of cells MT-4. Cells were cultured in 24-hole tablet. Cells incubated 5 hours at 37°in CO2-incubator. Then add 500 μl of growth medium containing 20% fetal serum of cattle. The transfection efficiency is about 30%. Efficiency is ü evaluate the registration of the appearance of green fluorescence using a fluorescence microscope Olimpus". Cells MT-4, transfected with different plasmids, infecting strain of HIV-1/GKV-4046 after 24 and 72 hours. For infection use of the supernatant of infected strain GKV-4046 HIV-1 cells with a multiplicity of infection 2-5×10-1and 2-5×10-2infectious units per cell. Tablet incubated at 37°With four days in an atmosphere of 5% CO2. Daily take samples for the quantitative determination virousspecificakih protein P24 by direct enzyme immunoassay. Concentration and cell viability assessed by exclusion Trypanosoma blue.

The impact of the introduction into cells expressing vector designs on the reproduction of HIV-1 judged by the accumulation virousspecificakih P24 antigen after 24, 48 and 72 hours. The results are shown in tables 1-4.

Table 1
The accumulation of P24 antigen (ng) in the cells MT-4 infected with a multiplicity of infection 2-5×10-1infectious units of HIV-1 cell
12345
24 hours14211126,413061239no
48 hours1523819 909947no
72 hours30981613166418691248

Table 2
The accumulation of P24 antigen (ng) in the cells MT-4 infected with a multiplicity of infection 2-5×10-2infectious units of HIV-1 cell
12345
24 hoursof 174.4193,6219,2212,8no
48 hours222147138149no
72 hours2035117814341293174

Table 3
The percentage of inhibition of HIV-1 in cells MT-4 infected with a multiplicity of infection 2-5×10-1infectious units of HIV-1 cell
2345
24 hours20,78,112,8no
48 hours 46,2of 40.3of 37.8no
72 hours47,946,339,759,7

Table 4
The percentage suppression of reproduction of HIV in cells MT-4 infected with a multiplicity of infection 2-5×10-2infectious units of HIV-1 cell
2345
24 hours-11-25,7-22,2no
48 hoursto 33.8of 37.832,9no
72 hours42,129,536,491,4
Note:

1 - cells MT-4;

2 - cells MT-4, transfected with plasmid RT-hp-pEGFP-NI;

3 - cells MT-4, transfected with plasmid tat-hp-pEGFP-NI;

4 - cells MT-4, transfected with plasmid pr-hp-pEGEP-NI.

5 - cells MT-4, transfected with plasmid RT-hp-pEGFP-NI (infection of the cells was performed after 72 hours).

When infected cells MT-4 human immunodeficiency virus the first type shows the inhibition of the reproduction of the virus in cells transfected with plasmids expressing VI is specificheskie siPHK. A pronounced antiviral effect was observed when infected cells high concentration of HIV-1 (the number of viral particles is equivalent 1081,5 ng P24), tables 1 and 3. The maximum suppression of the virus (60% and 91%) identified in infected cells after 72 hours after introduction of the plasmid.

Example 3. Suppression of reproduction of HIV in culture cells MT-4 by introducing into the cells by electroporation vector constructs expressing short interfering RNA.

Cells MT-4 were cultured as described above for 3 days to a concentration of 1.6 million/ml, washed twice with RPMI medium, suspended in the appropriate volume of medium to a concentration of 1×107cells/ml To 800 μl of cell suspension added to 40 μg of plasmid DNA, incubated 15 min in ice and transferred to a cuvette for electroporation. Electroporation is carried out at 400 watts, 960 áf (Gene PulserRTransfection Apparatus, Bio-Rad). The pulse duration of 31-36 MS. After electroporation, the cells are transferred into RPMI medium with addition of 10% fetal cattle serum. Efficient introduction of plasmid DNA 92-98% (estimate check the appearance of green fluorescence using a fluorescence microscope Olimpus"). Cells infect through 3 days after electroporation with different multiplicity of infection. For infection use of the supernatant of infected strain GKV-4046 HIV-1 cells with a multiplicity of infection 2-5× 10-1, 2-5×10-2and 2-5×10-3infectious units per cell. Tablet incubated at 37°With four days in an atmosphere of 5% CO2. On the fourth day take samples for the quantitative determination virousspecificakih protein P24 by direct enzyme immunoassay. Concentration and cell viability assessed by exclusion Trypanosoma blue. The impact of the introduction into cells expressing vector designs on the reproduction of HIV-1 judged by the accumulation virousspecificakih antigen P24. The results are shown in table 5.

Table 5
The percentage suppression of reproduction of HIV-1 in cells MT-4 during infection with different multiplicity of infection
1234
The infectious dose of HIV-1
0.2-0.536,926,423,4
0.2-0.5×10-175,372,071,6
0.2-0.5×10-2988979
Note:

2 - cells MT-4, transfected with plasmid RT - hp-pEGFP-NI;

3 - cells MT-4, transfected with plasmid tat-hppEGFP-NI; < / br>
4 - cells MT-4, transfected with plasmid pr-hp-pEGEP-NI.

It is shown that in cells containing the vector constructs, there is a significant suppression of the reproduction of HIV-1 compared with atransferrinemia cells (90%). Thus, there is a dependence of the inhibition of the reproduction of the virus from infecting dose of HIV-1.

1. Genetic structure-based vector plasmid pEGFP-N1, producing siPHK inhibitors reproduction of human immunodeficiency virus type 1 containing a fragment of the genome of HIV-1 pr-hp-pEGFP-N1 area length 19 bp of the conservative region of the protease domain (1749 1767...):

5'

gaattccTGATTCAGATTGGTTGTACttttttgtacaaccaatctgaatcaaccc

cggatcc3',

where lower case letters shows the region containing artificial

restriction enzymes cut sites for restricted EcoRI (5'-end) and BamHI (3'-ends), and the loop that separates the region, capable of forming a double-stranded duplex with loop, and the area corresponding sense RNA strand of the virus, as shown in the row on the left, and antisense to the right.

2. Genetic structure-based vector plasmid pEGFP-N1, producing siPHK inhibitors reproduction of human immunodeficiency virus type 1 containing a fragment of the genome of HIV-1 tat-hp-pEGFP-N1 area length 19 bp from a conservative district domain reverse transcriptase (2116...2134):

gaattccTGCATATTTTTCAGTTCCCttttttgggaactgaaaaatatgcatcccc

ggatcc3',

where lower case letters shows the region containing artificial restriction enzymes cut sites for restricted EcoRI (5'-end) and BamHI (3'- ends), and the loop that separates the region, capable of forming a double-stranded duplex with loop, and the area corresponding sense RNA strand of the virus, as shown in the row on the left, and antisense to the right.

3. Genetic structure-based vector plasmid pEGFP-N1, producing siPHK inhibitors reproduction of human immunodeficiency virus type 1 containing a fragment of the genome of HIV-1 RT-hp-pEGFP-N1 area length 19 bp from a conservative district tat domain(5325...5343):

5'

gaattccAGTAGGACTAATAGTAGCAtttttttgctactattagtcctactatccc cggatcc 3',

where lower case letters shows the region containing artificial restriction enzymes cut sites for restricted EcoRI (5'-end) and BamHI (3'-ends), and the loop that separates the region, capable of forming a double-stranded duplex with loop, and the area corresponding sense RNA strand of the virus, as shown in the row on the left, and antisense to the right.



 

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39 cl, 7 dwg, 9 tbl, 5 ex

FIELD: genetic engineering and medicine genetic.

SUBSTANCE: method for detection of gene resistance of subjects to infection by HIV1 is disclosed. Method includes DNA isolation, CCR5 gene PCR-amplification by using two primers complementary to two gene CCR5 sites. Further amplification products are restricted with endonuclease HincII (HindII), sizes of formed amplified fragments are determined, and on the base of obtained data deletion and single-nucleotide mutations are detected. Method of present invention makes it possible to simultaneously diagnose the presence of two mutation in human CCR5 gene, and (if individual has both mutation in heterozygote state) to distinguish cys- and trans-configurations.

EFFECT: method for diagnosis of congenital gene resistance to infection by HIV1.

2 dwg, 1 tbl, 2 ex

FIELD: biotechnology, genetic engineering, molecular biology.

SUBSTANCE: invention proposes recombinant DNA pcDNA-TCI that has molecular mass 4.3 x 103 kDa and size 6 657 nucleotide pairs. The proposed recombinant plasmid DNA provides expression of artificial gene TCI comprising cytotoxic T-cellular epitopes of HIV-1 in eucaryotic cells. Also, invention proposes recombinant attenuated strain of bacterium Salmonella enteritidis E-23 pcDNA-TCI. The proposed strain is obtained by genetic transformation of the strain Salmonella enteritidis E-23 with plasmid pcDNA-TCI. The strain provides delivery DNA-vaccine in eucaryotic cells as the recombinant plasmid DNA pcDNA-TCI. Proposed groups of inventions provide inducing the expressed specific humoral and cellular immune response to HIV-1 in body. Proposed group of inventions can be used in medicine and virology for construction of live DNA-vaccine against HIV-1.

EFFECT: valuable biological and medicinal properties of strain and vaccine.

2 cl, 3 dwg, 3 tbl, 5 ex

The invention relates to the field of biotechnology and medicine, in particular to genetic engineering and immunology

The invention relates to the field of biotechnology and medicine, in particular to genetic engineering and immunology

FIELD: biotechnology, genetic engineering, molecular biology.

SUBSTANCE: invention proposes recombinant DNA pcDNA-TCI that has molecular mass 4.3 x 103 kDa and size 6 657 nucleotide pairs. The proposed recombinant plasmid DNA provides expression of artificial gene TCI comprising cytotoxic T-cellular epitopes of HIV-1 in eucaryotic cells. Also, invention proposes recombinant attenuated strain of bacterium Salmonella enteritidis E-23 pcDNA-TCI. The proposed strain is obtained by genetic transformation of the strain Salmonella enteritidis E-23 with plasmid pcDNA-TCI. The strain provides delivery DNA-vaccine in eucaryotic cells as the recombinant plasmid DNA pcDNA-TCI. Proposed groups of inventions provide inducing the expressed specific humoral and cellular immune response to HIV-1 in body. Proposed group of inventions can be used in medicine and virology for construction of live DNA-vaccine against HIV-1.

EFFECT: valuable biological and medicinal properties of strain and vaccine.

2 cl, 3 dwg, 3 tbl, 5 ex

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