RU2571855C2 - Kit of oligonucleotide primers for generation of library of full-length genes coding immunodominant proteins p17 (d11l), p30 (cp204l), p54 (e183l), p60 (cp530r), p72 (b646l) of african swine fever virus - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: object of the invention involves constructing oligonucleotide primers for generating a library of full-length genes coding immunodominant proteins p17, p30, p54, p60, p72 of African swine fever virus.

EFFECT: creating a databank of nucleotide sequences coding immunodominant proteins p17, p30, p54, p60, p72 of African swine fever virus, which are applicable in producing recombinant proteins, studying the biological properties, protein-protein interactions, and creating diagnostic preparations.

3 ex, 1 tbl

Description

The invention relates to biotechnology and for obtaining a library of nucleotide sequences in the composition of the cloning (shuttle) plasmid vector.

African swine fever (ASF) is a contagious, usually acute, swine disease characterized by fever, signs of toxicosis, hemorrhagic diathesis, and high mortality [1]. In 2000, ASF virus was isolated into a separate family Asfarviridae (from the initial letters African swine fever and related viruses), which consists of one genus and one species [4].

African swine fever (ASF) has been known since the beginning of the 20th century; it had the stereotype of a typical natural focal exotic disease with natural circulation of the virus in a population of wild African pigs. When the first cases occurred on domestic pigs, the infection acquired an acute course with a mortality rate of 100%. ASF affects domestic and wild pigs, regardless of breed and age.

The main route of infection is alimentary (with infected food waste), as well as through contact of sick animals with healthy ones. Carriers can be blood-sucking insects (mainly ticks), birds of prey and animals.

In 1957, ASF appeared in Portugal, in 1967 in Spain, in 1964 in France, in 1967 in Italy. In 2008, an ASF outbreak was recorded on the territory of the Russian Federation in the Stavropol, Krasnodar Territory and the Orenburg Region. Currently, the number of regions where ASF outbreaks are recorded is growing.

Specific prophylaxis for ASF has not been developed. This is largely due to the biological properties of the virus. Field strains of the ASF virus are usually heterogeneous polyclonal populations of the virus; they differ in virulence, hemi-absorbing and antigenic properties, which indicates its multi-type variability. All this makes it difficult to fight ASF and the need to improve anti-epizootic measures based on the latest scientific achievements.

ASF virus multiplies in the cytoplasm of cells, but the function of the nucleus is also necessary for its reproduction. 106 virus-specific proteins were detected in infected cells, of which 35 are synthesized before the start of viral DNA replication (early proteins) and 71 after DNA replication (late proteins). During reproduction in the alveolar macrophages of a pig of a highly virulent isolate, 57 acidic and 43 major virus-specific proteins are synthesized [6].

Significant progress in the study of ASFV antigenic variability has been achieved using genetic engineering and molecular biology methods [4, 6].

Obtaining recombinant ASF virus proteins excludes the use of live virus and work with infected animals and allows to obtain antigen in sufficient quantities and high quality.

Currently, more than 120 ASF virus proteins are known to exist, many of which play an important role in the formation of the immune response. Studying the characteristics of these proteins will help to better reveal the mechanisms of the immune response and the interaction of the virus with the host cell, and the preparation of recombinant ASF virus antigens will allow them to be used to diagnose this disease.

In works on the construction of expression systems, genes encoding immunodominant ASF virus proteins are cloned in safe vector systems [2, 3, 5, 7, 8].

To study ASFV virus genes, a library of genes or clones is initially used that encodes immunodominant ASFV virus genes as part of cloning vectors. Thus, recombinant vectors with gene inserts can be a source for obtaining the desired transgene, a source for studying the structure, functions and regulation of individual genes, structure and functions of proteins.

Insertions from the library, thanks to restriction sites, make it possible to clone coding sequences for various promoters (T5, T7, CMV, etc.) and vectors (plasmids, phages, viruses).

Previously, scientists designed and obtained cloning oligonucleotide primers to create the transit expression of viral proteins of African swine fever in the prokaryotic system. A significant drawback of the developed systems was the synthesis of recombinant proteins that underwent heterologous post-translational modifications in the producer cell, which indirectly affected the function of the recombinant protein [2, 3]. The unique advantage of our oligonucleotine primers is that they are designed to design viral protein expression systems under the control of the eukaryotic promoter, which allows the production of authentic recombinant proteins in mammalian cells (for example, COS-I, HEK-293 or CHO cell lines).

The aim of the invention is the construction of recombinant plasmids to obtain a library of full-sized genes encoding the immunodominant proteins p17, p30, p54, p60, p72 of ASF virus. This goal is achieved by the synthesis of specific primers flanking sections of the nucleotide sequences of the ASF genome for cloning at the T / A ends into the plasmid vector pGEM-T-easy and obtaining a clonal library.

Primers were calculated taking into account the uniqueness of the restriction site for a particular gene and observing the open reading frame (ORF) of the protein encoded by the gene. An additional condition for the selection of the restriction site was its presence in the acceptor vector (pN1-GFP). The sequences of these genes were taken from the GeneBank database. The nucleotide sequences of African swine fevervirus Georgia 2007/1 - FR682468.1 were used as the reference strain.

In the table. 1 shows the sequence of specific primers for amplification of full-size copies of genes encoding immunodominant ASF virus proteins.

Table 1 Gene name Primer sequence Length bp GC composition,
% Melting point, Сº Stud melting rate, Сº Restriction site p17 (D117L) F 5'-aattgggatcccatggacactgaaacgt-3 ' 28 46 59 37 Bamhi R 5'-cataaccggttgtgaatgcgcaagttcag-3 ' 29th 48 61 fifty Agei p30 (CP204L) F 5'-cgagcactcgagatggattttattttaaatatatc-3 ' 35 31 59 39 Xhoi R 5'-ttgatttgagctcaatgtaggtgagataaaag-3 ' 32 34 58 fifty Saci p54 (E183L) F 5'-atctcgagctcattatggattctgaattttttc-3 ' 33 33 58 - Saci R 5'-gactgcagaattctcaaggagttttctag-3 ' 29th 41 58 24 EcoRI p60 (CP530R) F 5'-agaagtaatggtaccaatgccctctaatatgaa-3 ' 33 36 59 - KpnI R 5'-aacagggcccgttcttgaagtaacttta-3 ' 28 43 58 - Apai p72 (B646L) F 5'-caatagaattctgcatatggcatcaggaggagc-3 ' 33 45 63 41 EcoRI R 5'-accggtggatccatggtactgtaacgca-3 ' 28 54 62 24 Bamhi

The technical result consists in the creation of a bank of nucleotide sequences encoding immunodominant proteins of the African swine fever virus p17, p30, p54, p60, p72, which can be used to obtain recombinant proteins, study of biological properties, protein-protein interactions and the creation of diagnostic drugs.

The essence of the invention lies in the fact that the resulting recombinant constructs in the clonal library, carrying gene inserts encoding the immunodominant proteins of the ASF virus, contain unique restriction sites for manipulating the nucleotide sequences of the virus genes.

Case Studies

Example 1. Obtaining a gene library includes the following steps.

1. Isolation of DNA from virus-containing material. (Nucleosorption methods and using commercial kits: QIAamp DNA Mini Kit (QIAGEN), QIAamp DNA Blood Mini Kit (QIAGEN, Germany), etc.

2. Amplification of full-sized DNA copies of genes encoding the corresponding proteins.

3. Cloning of amplifiers in a cloning vector.

Pathological material taken from ASF virus infected domestic or wild pigs acts as a source.

Isolation of the DNA pool from the virus-containing material is carried out according to standard methods using the nucleosorption method or using commercial kits (QIAGEN).

PCR assay on DNA obtained. As a DNA-dependent DNA polymerase, polymerases of various manufacturers can be used, introducing a small number of nucleotide substitutions during amplification of extended regions.

The formulation was carried out using the calculated specific primers listed in the table. one.

Prescription PCR mixture:

DNA matrix - 5 μl

10X Buffer (with 2.5 mM MgCl ₂ ) - 2.5 μl

10 mMdNTPmix - 1 μl

10 pMF primer - 1.0 μl

10 pMR primer - 1.0 μl

Taq polymerase - 0.2 μl

DNAse-free H ₂ O - 15.5 μl

Temp-temperature reaction profiles

For amplification of full-sized copies of DNA encoding p17, p30, p54, p72 proteins:

94 ° C - 2 min

40 cycles:

94 ° C - 30 sec

56 ° C - 30 sec

68 ° C - 2 min

72 ° C - 5 min

For amplification of full-sized copies of DNA encoding the p60 protein:

94 ° C - 2 min

40 cycles:

94 ° C - 30 sec

52 ° C - 30 sec

68 ° C - 2 min

72 ° C - 5 min

Detection and confirmation of the molecular weight of PCR products is carried out in a 1.5% TAE or TBE agarose gel with 0.01% ethidium bromide as an intercalating dye.

Molecular masses of PCR products obtained after PCR using ASFV DNA (using isolate No. 4 isolated from wild boar in the Moscow Region as an example)

Amplicons p17 - 354 base pairs (bp);

Amplicons p30 - 606 bp;

Amplicons p54 - 555 bp .;

Amplicons p60 - 1593 bp .;

Amplicons p72 - 1941 bp

The ASF virus gene library is an incomplete library of nucleotide sequences encoding the main immunodominant proteins: p17 (D117L), p30 (CP204L), p54 (E183L), p60 (CP530R), p72 (B646L) as part of the pGEM-T-easy shuttle vector.

A significant difference between the developed oligonucleotide primers is that their sequences contain endonuclease cleavage sites. The primers are highly specific for ASF virus DNA and are optimized for amplification from gene copies at selected temperature conditions (Table 1).

Example 2. Cloning of amplicons p17, p30, p54, p60, p72 of ASF virus (isolate No. 4 isolated from wild boar in the Moscow region) into a plasmid vector.

Due to amplification of DNA copies of genes using Taq polymerase, polyA sites remain at the ends of the fragments, thus the polymerase synthesizes additional nucleotides per 3'amplicon.

Cloning is carried out in the pGEM-T-easy plasmid vector using the commercial pGEM-T-EasyVector System I kit (Promega, USA). A feature of cloning in this vector is that cloning is carried out at the T / A ends, followed by screening for the resistance of the recombinant bacterial clones to ampicillin.

Cloning is carried out according to the manufacturer's instructions with minor modifications.

The calculation of the molar ratio of amplicon and vector is carried out according to the following formula:

The ligation mixture consists of the following components:

10X Buffer;

Vector pGEM;

DNA control;

DNA insert;

T4 DNA ligase;

DNAse-free H ₂ O

Cell transformation is carried out by electroporation of a bacterial culture of E. coli dh5α (NEB, USA) on a Gene Pulser Xcell device (BioRad, USA). Further screening is conducted for colonies grown on Petri dishes on medium (LB agar) with the addition of ampicillin (25 mg per 1 ml of medium).

The fastest method for screening for recombination is the selection of bacterial clones for white and blue colony staining in the presence of X-Gal and IPTG on LB agar. Colonies that do not carry an insert are colored blue. White colonies contain insertion. Additionally, to check the specificity of the insert, PCR analysis was performed using specific oligonucleotides (Table 1). For this, single, isolated colonies are transferred to the prepared PCR mixture (see example 1) using the following temperature conditions:

94 ° C - 2 min

40 cycles:

94 ° C - 30 sec

56 ° C - 30 sec

68 ° C - 2 min

72 ° C - 5 min.

Detection and confirmation of the molecular weight of PCR products is carried out in a 1.5% TAE or TBE agarose gel with 0.01% ethidium bromide as an intercalating dye.

Commercial kits were used to isolate plasmid DNA from a bacterial culture: QIAGEN Plasmid Maxi Kit (QIAGEN, Germany), Axy Prep Plasmid Miniprep Kit (AXYGEN, USA).

Example 3. The use of ASF virus gene library.

Due to the presence of endonuclease cleavage sites, these libraries can act as sources of specific inserts while maintaining reading frames in the expression vector.

These nucleotide sequences of the inserts can be used to produce recombinant protein producers.

The producer is a culture of eukaryotic cells (COS-I, HEK-293 or CHO) with the expression plasmid pN1-GFP p17 / p30 / p54 / p60 / p72. The vector pN1-GFP is restricted to the specific restriction sites of the endonucleases indicated in the table. 1 for each gene.

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

To create constructs expressing in the eukaryotic system, the plasmid vector pN1-GFP was selected. This vector contains a “strong” cytomegalovirus promoter, which ensures the expression of the target protein in various cell cultures.

The resulting recombinant proteins will allow us to study the biological properties of viral proteins and the mechanisms of protein-protein interactions.

Information sources

1. Viral diseases of animals / V.N. Syurin, A.Ya. Samuilenko, B.V. Soloviev, N.V. Fomina. - M.: VNITIBP, 1998 .-- 928 p.

2. Kazakova A.S. Design of producers of recombinant proteins P72, P30 and P54 of the virus of African swine fever: a dissertation for the degree of candidate of biological sciences: 03.02.02: defended 05.16.13 / Kazakova Anna Sergeevna. - Veil, 2013.

3. Kopytov V.O. Cloning and expression of genes of structural proteins p30 and p72 of the virus of African swine fever: dissertation for the degree of candidate of biological sciences: 03.00.06: defended 12.23.04 / Kopytov Valeriy Olegovich. - Veil, 2004.

4. Family Asfarviridae / L.K. Dixon, J.V. Costa, J.M. Escribano, D.L. Rock, E. Vinuela, P.J. Wilkinson [et all.] // Virus taxonomy: Seventh Report of the International Committee on Taxonomy of Viruses. Summers Academic Press, San Diego, 2000 .-- P. 159-165.

5. High level expression of the major antigenic African swine fever virus proteins p54 and p30 in baculovirus and their potential use as diagnostic reagents / J.M. Oviedo, F. Rodriguez, P. Gomez-Puertas, [et all.] // J. Virol. Methods, 1997. - Vol. 64, No. 1. - P. 27-35.

6. Rodriguez, J.M. African swine fever virus-induced polypeptides in porcinealveolar macrophages and in Vero cells: two-dimensional gel analysis / J.M. Rodriguez, M.L. Salas, J.F. Santaren // Proteomics. - 2001. - Vol. 1, No. 11. - P. 1447-1456.

8. Optimization and validation of recombinant serological tests for African Swine Fever diagnosis based on detection of the p30 protein produced in Trichoplusianilarvae / D.M. Perez-Filgueira, F. Gonzalez-Camacho, C. Gallardo, [et all.] // J. Clin. Microbiol., - 2006. - Vol. 44, No. 9. - P. 3114-3121.

9. Serodiagnosis of African swine fever using the recombinant protein p30 expressed ininsect larvae / M.G. Barderas, A. Wigdorovitz, F. Merelo [et al.] // J. Virol. Methods - 2000. - No. 89. - P. 129-136.

Claims (1)

A set of specific oligonucleotide primers with included endonuclease cleavage sites flanking genome sections encoding the full-size genes p17, p30, p54, p60, p72 to obtain a library of African swine fever virus genes having the following nucleotide composition and restriction sites:
Fp17 (BamHI) 5'-aattg ggatcc catggacactgaaacgt-3 '
Rp17 (AgeI) 5'-cata accgg ttgtgaatgcgcaagttcag-3 '
Fp30 (XhoI) 5'-cgagca ctcgag atggattttattttaaatatatc-3 '
Rp30 (SacI) 5'-ttgattt gagctc aatgtaggtgagataaaag-3 '
Fp54 (SacI) 5'-atctc gagctc attatggattctgaattttttc-3 '
Rp54 (EcoRI) 5'-gactgca gaattc tcaaggagttttctag-3 '
Fr60 (KpnI) 5'-agaagtaat ggtacc aatgccctctaatatgaa-3 '
Rp60 (ApaI) 5'-aaca gggccc gttcttgaagtaacttta-3 '
Fp72 (EcoRI) 5'-caata gaattc tgcatatggcatcaggaggagc-3 '
Rp72 (BamHI) 5'-accggt ggatcc atggtactgtaacgca-3 '