Oligonucleotide primers for production of library of full-length genes encoding immunodominant proteins gn, gc and n of rift valley fever virus

FIELD: biotechnology.

SUBSTANCE: proposed primers comprise endonuclease cleavage sites, flanking genomic regions encoding the glycoproteins Gn and Gc, and the nucleoprotein N, for obtaining libraries of genes encoding glycoproteins Gn and Gc and N nucleoprotein of Rift Valley fever virus.

EFFECT: invention can be used in creating a bank of nucleotide sequences encoding immunodominant proteins of Rift Valley fever virus Gn, Gc and N, which can be used for creation of diagnostic and vaccine preparations based on recombinant technologies.

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The invention relates to biotechnology and relates to a library of nucleotide sequences, comprising klonirovanija plasmid vector.

Rift valley fever (RVF) is an acute vector-borne zooantroponoznyh arbovirus disease included in the group of diseases subject to international health regulations 2005 on national and regional level [1]. The pathogen belongs to the family Bunyaviridae genus Phlebovirus.

Rift valley fever is causing enormous socio-economic damage, accompanied by large losses of livestock during the epidemic, diseases and deaths, the cost of quarantine and preventive measures and also hospitalization and treatment of people.

In parts of Africa animals vaccinated with a live vaccine (strain Smithburn"), but in non-endemic regions, the use of live vaccines is prohibited. Currently in development, or in the testing stage, a number of candidate live, recombinant and DNA vaccines. For use among people developed inactivated vaccine. However, this vaccine is not licensed and is not for sale. It is used for experimental purposes to protect veterinarians and laboratory workers at high risk of infection LDR [3].

The emergence of RVF outside the African contin�that poses a real threat for Europe and Asia, where there are competent vectors. Given the danger of the emergence of the pathogen in new areas and the uncertainty of vaccination, there is a need to develop new scientific approaches for the prediction, diagnosis and prevention of this disease.

So, in the work of foreign researchers L. Liu, Cr. Celma and P. Roy "Rifit Valley fever virus structural proteins: expression, characterization and assembly of recombinant proteins described a process of obtaining recombinant proteins Gn, Gc and N, and their subsequent characterization and analysis.

Some success and there are design and recombinant DNA vaccines. So N. Bhardwaj et al. show the induction of high titers of neutralizing antibodies after inoculation of mice with plasmid expressing chimeric protein Gn-C3d [5].

G. Lorenzo al (2010) have received protection, immunosera different DNA constructs. Positive results received N. Boshra al (2012) who have achieved protection from the control of infection with virulent virus after DNA immunization design expressing ubiquitination nucleoprotein N virus LDR.

In the works for the construction of recombinant vaccines with genes encoding protective proteins LDR, cloned in a safe viral vectors. Similar designs obtained Elena Lopez-Gil and co-authors (2013), based on modified virus vacciniaAnkara (rMVA), on the basis of Newcastle disease virus Kortekaas J, with co-authors (2010).

For this research, as well as address various theoretical problems (e.g., determination of the nucleotide sequence of the genome of strains, to study the structure, function and regulation of individual genes, structure and functions of proteins) receive a library of genes, kourouma immunodominant proteins of the virus rift valley fever, which can be used to create diagnostic and vaccine products based on recombinant technology. Inserts from the library due to the restriction sites to allow perekodirovat coding sequence under different promoters (T5, T7, CMV, etc.) and vectors (plasmids, phages, viruses).

Therefore, the aim of the invention is to design oligonucleotide primers for obtaining a full-sized library of genes encoding immunodominant proteins Gn, Gc and N, of the virus of rift valley fever.

This goal is achieved by the synthesis of specific primers flanking the sites of the nucleotide sequences of the genome of LDR with the library.

In table.1 shows the sequences of specific primers for amplification of full-sized copies of the cDNA encoding the immunodominant proteins of the virus LDR.

name and �connected site restriction the nucleotide sequence of*long, nt.flanked plot
F-RVFV-Gn EcoRI5'-CTCGAATTCATGCCTCATCTCAGAAACAGACCA-3'33M segment 474...2067 nt nt 1626
F-RVFV-Gc EcoRI5'-CGCGAATTCATGTGTTCAGAACTAATTCAGGCA-3'33M segment 2079...3474 nt 1428 nt
F-RVFV-Np Sail5'-CCCGTCGACTTAGGCTGCTGTCTTGTAA-3'28S segment 906 1635...757 nt nt
* the restriction site is indicated by underlining.

The technical result consists in the creation of a Bank of nucleotide sequences encoding the immunodominant proteins of the virus rift valley fever Gn, Gc and N that can be used when �the create diagnostic and vaccine preparations based on recombinant technology.

The essence of the invention consists in the fact that the specific primers designed to produce libraries of genes encoding immunodominant proteins of the virus rift valley fever Gn, Gc and N and contain restriction sites for further manipulation of nucleotide sequences of the inserts.

Examples of specific performance

Example 1. Obtaining a library of genes comprises the following steps:

1. Refreshment and life viral material (Mouse-sucker and/or sensitive cell culture).

2. The allocation of RNA from virusdatabases material (Methods using 4M GTZ, Trizol reagent©, or using commercial kits: GeneJET RNA Purification Kit (Thermo Scientific), etc.).

3. Statement FROM PCR on the matrix RNA pool (using a random hexameric or specific primers).

4. Amplification of full-sized copies of the cDNA encoding corresponding proteins.

5. Cloning amplification in klonirovana vector (plasmids, phages, etc.).

As the source can be virussecurity material (infected culture cells, pathological material, lyophilized tab) vaccine or epizootic of rift valley fever virus strain. If necessary, the virus refresh and improve:

The virus titre is not less than 3.0 lg refresh the intracerebral Sara�of diurnal mice pigs: the dose of 0.1 ml per head. 48-72 hours away pups with obvious clinical signs: photophobia, cyanotic mucous membranes, paralysis and other nervous phenomena. These specimens aseptically taken away the brain, and prepare a 10% suspension in physiological solution and transferred to a -40°C.

The virus titer is not below 4.0 lg refresh sensitive cell culture: kidney saiga "PS", green monkey kidney CV-1", kidney Syrian hamster "VNK", etc., the Dose of infection is 10-3-10-4TCID50/CL. 3-5 a day, when you reach the JRS about 40-50% of the culture fluid decanted, and the monolayer pour THE buffer pH 7.0 and transferred to a -40°C.

The allocation of the total pool of RNA from virusdatabases of material is carried out by standard techniques using the 4M GTZ, Trizol reagent, etc., or with the use of commercial kits GeneJET RNA Purification Kit (Thermo Scientific), etc. Firostefani the above materials were prepared samples.

Statement FROM PCR is performed according to the selected passport revertase, using a random or specific primers as given in table.1. As RNA-dependent DNA polymerase may make AMV and M-MLV of revertase different manufacturers (Promega, Fermentas, α enzyme). The obtained cDNA can be stored at -40°C during the month.

The PCR on cDNA obtained. As a DNA-dependent DNA polymerase m�gut to speak polymerases from different manufacturers, making a small number of nucleotide substitutions when the amplification of long sections.

Formulation was conducted using specific primers as given in table. 1.

The PCR recipe mix

matrix to DNA 0.5 ál

10X Buffer (with 2.5 mM MgCl2) To 2.0 ál

10 mM dNTP mix - 0,4 ál

10 PM F-primer and 1.0 μl

10 RM R-primer - 1,0 ál

Pfu polymerase and 0.5 µl (1,25 u)

DNAse-free H2O - 14,6 ál

Time-temperature profiles of the reaction

For amplification of full-sized copies of the cDNA encoding the glycoproteins Gn and Gc are:

95°C - 2 min.

22 cycle:

95°C - 30 sec.

55°C - 30 sec.

72°C 3 min 20 sec.

72°C - 7 min.

For amplification of full-sized copies of the cDNA encoding the nucleoprotein N:

95°C - 2 min.

22 cycle:

95°C - 30 sec.

55°C-30 sec.

72°C 1 min 35 sec.

72°C - 3 min.

Detection and confirmation of the molecular mass of the PCR products is carried out in a 1.2% TAE or TBE agarose gel with EtBr as intercalating dye.

The molecular weight of PCR products obtained after the PCR FROM the genome matrix LDR strain "1974 Vniivvim":

Amplificare Gn - 1626 bp;

Amplificare Gc - 1428 bp;

Amplificare N - 757 bp.

A library of genes of the virus rift valley fever, is an incomplete library of nucleotide sequences that encode the major immunodominant proteins: �glycoprotein Gn and Gc, as well as the nucleoprotein (N) in the composition klonirovannikh vectors.

The essential difference data klonirovannikh primers is that in the sequence of the primers included sites endonuclease cleavage with annealing temperature for glycoproteins 57°C, for nucleoprotein - 55°C.

Example 2. Cloning amplification Gn and Gc, the nucleoprotein N virus PI strain "1974-Vniivvim" in the plasmid vector

Due to the amplification of cDNA copies with the use of Pfu polymerase and the ends of the fragments remain "dumb", i.e., the polymerase does not put additional nucleotides at the ends.

Cloning is carried out in the plasmid vector pJET1.2, using a commercial kit CloneJET PCR Cloning Kit. (Thermo Scientific). The feature of cloning in this vector is that the cloning was carried out by the "blunt" ends, with negatively lethal screening (box disturbs reading frame of the lethal gene ESOC in the composition of plasmids).

Cloning is carried out according to the standard recipe set. To do this, 1 ál of crude Reaction Buffer, PCR product made in a mixture consisting of 10 µl 2X Buffer, 1 ál of pJET1.2/Blunt, DNAse-free H2O - 7 ál. Mix on vortex for 3-5 seconds then make a 5u of T4 DNA Ligase. Incubated at 22°C for 10 min.

The transformation is carried out using reagents TransformAid Bacterial Transformation Kit (thermo Scientific). The competent cells of E. Coli you�RAS strain KRX (Promega).

Further screening is carried out in the ratio of colonies grown on Petri dishes in medium with addition of ampicillin.

The easiest method is PCR analysis. For this single, isolated colony is divided in two, half to make 2 ml of medium SOB with the addition of ampicillin, the other half into the prepared PCR mixture:

10X Buffer (with2,5 mM MgCl2) To 2.0 ál

10 mM DNTP mix - 0,4 ál

10 PM F-primer and 1.0 μl

10 RM R-primer - 1,0 ál

Tag polymerase and 0.5 µl (2.5 u)

DNAse-free H2O - 15,0 ál

The time-temperature profile of the reaction:

For screening clones containing the insert of glycoproteins Gn or Gc:

98°C - 2 min.

25 cycles:

95°C - 30 sec.

57°C - 30 sec.

72°C 1 min 45 sec.

72°C - 4 min.

For screening clones containing the insert of nucleoprotein No. 98°C 2 min.

25 cycles:

95°C - 30 sec.

57°C - 30 sec.

72°C - 48 h.

72°C - 2 min.

Detection and control of molecular weight of the PCR products is carried out in a 1.2% TAE agarose gel with the addition of EtBr.

The clones in the samples which are PCR products of a given molecular weight are being generated.

After outstanding plasmid and sent for sequencing using pJET1.2 sequencing primers". According to the results of sequencing selected clones carrying plasmids with intact reading frame, this clonetech lay aliquots.

Example 3. �primenenie library of genes of the virus PI strain "1974-Vniivvim"

Due to the presence of sites endonucleases splitting (in the case of glycoproteins Gn and Gc - Sail and EcoRI, the nucleoprotein N - Sail and StuI) these libraries can act as sources of specific inserts with sobraniem reading frames in plasmid vector rate (nucleoprotein is in pET32b), pET20b, pCI-neo, etc.

The data of the nucleotide sequence of the inserts can be used for producing recombinant proteins, in particular, work on obtaining chromatographically purified preparation of recombinant nucleoprotein N virus LDR [methods in chromatographic purification of recombinant nucleoprotein N virus rift valley fever (RVF). Malysheva V. I., Kazakov A. S., Imaginou I. R., 2013].

As a producer performs cell culture of E. Coli KRX strain with expressing the plasmid pET32b/RVFV/Np/3/9b. For perchlorovinyl insert plasmid library containing the nucleotide sequence encoding nucleoprotein N, restrictively sites endonucleases StuI cleavage and Sail. Vector 32b restricion Sail and EcoRV.

Ligation, the metamorphosis and screening of clones were performed according to standard procedures (M. R. Green and J. Sambrook, Molecular Cloning A Laboratory Manual, 4th Edition, 2012) and can be performed by any known to the researcher.

The data of the nucleotide sequence of the inserts may be required�us to create a candidate DNA vaccines.

The prospects for regarding DNA immunization with LDR showed in his works a number of foreign researchers (W. Sheng, W. HLW, 2007; N. Lagerqvist, J. Naslund, 2009; Lorenzo G, R. Martin-Folgar, 2010).

To generate constructs expressing in a eukaryotic system, was selected plasmid vector pCI-neo (Promega). This vector contains "strong cytomegalovirus promoter that ensures expression of a target protein in various cell cultures.

For perchlorovinyl glycoproteins Gn and Gc, plasmids from the library containing the nucleotide sequence encoding the glycoprotein glycoprotein Gn and Gc, respectively, were restrictively sites endonucleases splitting EcoRI and Sail. Vector pCI-neo restricion Sail and EcoRI.

Ligation, the metamorphosis and screening of clones was carried out according to standard methods (M. R. Green and J. Sambrook, 2012, (Molecular Cloning, A Laboratory Manual, 4th Edition) and can be performed by any known to the researcher.

After immunization of mice isotonic plasmid pCI-neo/Gn/1 and pCI-neo/Gc/1 for 14 days observed an increase in the titer of specific antibodies, which was 1:160-1:320.

Sources of information

1. The world health organization. International health regulations (2005). - 2nd ed. - Switzerland, 2008. 82 p. - ISBN 978 92 4 458041 7.

2. Newsletter No. 207. Who. "Fever Rift Valley". http://www.who.int/mediacentre/factsheets/fs207/ru/index.htl.

3. Project Epidemiologist.ru. "Fever Rift Valley". http://view-source:www.epidemiolog.ru/prof/1582.html.

4. Public Health England. "Rift Valley Fever Advice for Health Professionals, 23 May 2011". http://www.hpa.org.uk/web/HPAweb&Page&HPAwebAutoListName/Page/1274087829165.

5. Bhardwaj N, Pierce BR, Ross TM (2012) Immunization with DNA Vaccine Expressing Gn Coupled to C3d Prevents Clinical Symptoms of Infection and Protects Mice against an Aerosol Rift Valley Fever Virus Infection. J Bioterr Biodef S3:006. doi: 10.4172/2157-2526.S3-006.

Specific oligonucleotide primers enabled sites endonuclease cleavage flanking areas of the genome that encode glycoproteins Gn and Gc, nucleoprotein N, to obtain libraries of genes encoding glycoproteins Gn and Gc, nucleoprotein N virus rift valley fever, which had the following nucleotide composition and restriction sites:


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