RU2287582C2 - GLYCOPROTEIN GENE (G-GENE) OF RUSSIAN REFERENCE STRAIN ZL-4 OF CARP SPRING VIREMIA VIRUS AND RECOMBINANT PLASMID DNAS pcDNA-G AND pBACarpVax-G EXPRESSING GLYCOPROTEIN GENE AND PROVIDING DEVELOPMENT OF PROTECTIVE IMMUNITY IN FISH AGAINST INFECTION WITH CARP SPRING VIREMIA VIRUS - Google Patents

Abstract

FIELD: genetic engineering, biochemistry, pisciculture.

SUBSTANCE: invention relates to the determined nucleotide sequence encoding glycoprotein of the Russian isolate ZL-4. Based on this sequence DNA-plasmids are constructed that provide expression of glycoprotein of the Russian isolate ZL-4 in fishes. DNA-vaccine constructed on basis of indicated DNA-plasmids provides the development of protective immunity in fishes against the following infection with ZL-4 isolate of carp spring viremia virus. Using the invention provides protection of carp in Russian aquaculture from the following infection with carp spring viremia virus.

EFFECT: valuable properties plasmid DNA and glycoprotein gene.

3 cl, 4 dwg, 3 ex

Description

The invention relates to biotechnology, specifically to genetic engineering, and is a plasmid pcDNA-G and pBACarpVAX-G containing a recombinant full-sized glycoprotein gene (G-gene) of the Russian reference strain ZL-4 of the spring virus viremia of the carp (VVK, SVC) under control CMV promoter or β-actin carp promoter, respectively. The proposed recombinant constructs are candidates for DNA vaccines against spring carp viremia (IHC).

VVC is an acute contagious fish disease, causing significant damage to fish farming in Europe and the countries of the former USSR. Recently, IHC has also been discovered in the United States and China. The causative agent of the disease, known as the spring carp viremia virus (SVCV) or Rhabdovirus carpio, is a rhabdovirus previously assigned to the genus Vesiculovirus [1]. The most susceptible to the disease is carp (Cyprinus carp L.) - the main object of Russian aquaculture. With an outbreak of the disease, about 30-40% of the fish stock usually dies; in severe cases, death can reach 70%. In addition to carp, in vivo the virus can infect white and spotted silver carp, grass carp, goldfish, common catfish and pike, and in artificial conditions, ornamental species of cyprinids [2].

Currently, there are no effective means of prevention and treatment of IHC. Attempts have been made to create an inactivated and attenuated vaccine against the disease [3-5]. However, such vaccines have known disadvantages that limit their widespread use in fish farming practice.

In the last decade, the development of so-called DNA vaccines has become one of the priority areas in modern experimental veterinary medicine. These vaccines have several advantages over traditional vaccines, which makes them attractive to aquaculture. They are cheap, thermostable and easy to use, and a single vaccination can induce a “detailed” immune response with long-term memory [6]. Recombinant technologies currently occupy a dominant position in the construction of "ideal" vaccines.

The immune response against rhabdoviruses is directed primarily to the surface transmembrane glycoprotein (G-protein) of the virion. Neutralizing antibodies and cytotoxic lymphocytes demonstrate exclusive specificity for this protein [7]. Therefore, the recombinant nucleotide sequence encoding a viral glycoprotein is the most suitable candidate for the role of an immunogenic basis for a DNA vaccine against pathogens of the Rhabdoviridae family.

Foreign researchers obtained encouraging results in the work on the creation and study of the immunogenic and protective properties of DNA vaccines against rhabdovirus diseases of fish. The main studies were conducted with DNA vaccines against two diseases of salmonids - infectious necrosis of hematopoietic tissue and viral hemorrhagic septicemia. It has been shown that of experimental DNA vaccines for fish, the most effective constructs are those where expression of the recombinant viral protein is controlled by the cytomegalovirus (CMV) promoter. A single intramuscular injection of DNA vaccines based on the G protein of viruses demonstrates the high efficiency of the induction of a protective immune response against the corresponding disease [6, 8, 9]. Vaccine preparations were effective when used in various concentrations on fish of different species and age groups. Their protective effect against infection by heterologous fish rhabdoviruses was established [10, 11, prototype].

In a typical experiment, 2 groups of rainbow trout of 25-30 ind. fish in each, with an average body weight of 1 g, are vaccinated once with intramuscular injection of 0.01-1 μg of plasmid DNA, and then after 30 days they are infected with a lethal dose of the virus through water or by intraperitoneal injection. Over the next 3-4 weeks, fish mortality in these groups is 1-10%, while in the control groups receiving an empty plasmid vector or buffer, more than 90% die. Kinetics studies have shown the duration of protective immunity (even 2 years after vaccination with 0.1 μg of DNA), which is detected already 4-7 days after vaccination [10,12].

There is a recombinant plasmid pcDNA3-G containing the G gene of the American SVC virus isolate, created in the laboratory of Jo-Ann Leong, Department of Microbiology, University of Oregon, USA. This design was used by researchers to study the non-specific resistance of salmon fish to viruses of infectious necrosis of hematopoietic tissue and viral hemorrhagic septicemia [10]. However, the presence of a cytomegalovirus promoter in a DNA vaccine design can lead to difficulties in passing it through licensing authorities. In this regard, the ability of various promoters to initiate the expression of foreign genes in fish tissues is currently being studied in a number of laboratories abroad.

There is a recombinant plasmid carrying the β-actin carp promoter, with which it was shown that this promoter provides expression of the luciferase gene at a level comparable to that for the CMV promoter. A DNA vaccine against viral hemorrhagic septicemia containing the gene of this virus under the control of the β-actin promoter of the carp ensured the development of protective immunity in vaccinated rainbow trouts, although higher concentrations of the vaccine were required to achieve the necessary protection [13, prototype]. It is assumed that the use of this promoter in the design of a DNA vaccine will simplify the procedure for licensing it.

To obtain the maximum protective effect from vaccination, it is necessary to use the optimal dose of the drug, the coincidence of the time of infection with the peak of the developing immune response of fish, and the maximum homology of antigenic properties between the viral isolate used to create the vaccine and the isolate most common in the region.

In connection with the foregoing technical objective of the invention is the creation of genetic engineering constructs encoding the glycoprotein of the VVK virus of the Russian reference strain ZL-4 and protecting the carp from subsequent infection with a virulent strain of this pathogen, i.e. Candidates for DNA vaccines against IHC in Russian aquaculture.

The problem is solved by establishing the nucleotide sequence of the glycoprotein gene of the Russian isolate ZL-4 virus VVK. To do this, at the first stage, two fragments of the specified gene are cloned, amplified in the polymerase chain reaction (PCR) using the cDNA of this gene obtained by reverse transcription on a genomic viral RNA matrix. Embedding is carried out according to standard methods in the plasmid vector pUC18 (Figure 1) [15]. Recombinant clones are identified by restriction analysis and agarose gel electrophoresis. Sequencing of the obtained DNA sequence of the cloned fragments confirmed that they really encode VVC glycoprotein (Figure 2). The G gene sequence of only the European reference strain Fijan of this virus has been published [14]. When comparing with it in the sequence of the G gene of the Russian strain, 9 nucleotide substitutions were found, three of which are significant, leading to amino acid substitutions in the molecule of the encoded protein (Figure 3). At the second stage, the entire gene sequence was cloned via pBluKS (-) (to obtain the correct orientation of the gene sequence) into pcDNA3.1 plasmid vectors. and pBACarpVAX, which provide gene expression and the production of glycoprotein in eukaryotic cells (Figure 1).

The problem is solved by constructing two recombinant plasmid DNAs: pcDNA-G (6955 bp) and pBACarpVAX-G (5431 bp), which carry the full-sized gene of the glycoprotein strain ZL-4 of the VVK virus and ensure its expression in fish cells under control of the CMV promoter and the β-actin carp promoter, respectively.

The recombinant plasmid DNA pcDNA-G with a molecular weight of 46.1 × 10 ⁶ Yes provides the expression of VVC glycoprotein in fish cells when administered intramuscularly as a DNA vaccine. 6955 bp design consists of the following elements:

- EcoR I - Xho I of the vector DNA fragment of the plasmid pcDNA 3.1. (+) Size 5405 bp [Invitrogen, USA], containing the CMV promoter and the BGH poly A sequence for expression of the G gene in eukaryotic cells; T7 promoter, which ensures the propagation of the target plasmid in the cells of E.coli bacteria;

- EcoR I - Xho I fragment with a size of 1550 bp containing the full-sized gene of the glycoprotein isolate ZL-4 of the VVK virus;

- genetic marker: ampicillin resistance gene;

- unique restriction sites: EcoR I (722), Kpn I (691), Xho I (2278), Xba I (2284).

The recombinant plasmid DNA pBACarpVAX-G with a molecular weight of 36.1 × 10 ⁶ Yes provides expression of the VVC glycoprotein gene in fish cells when administered intramuscularly as a DNA vaccine. The design has a size of 5431 bp and consists of the following elements:

- EcoR I - XhoI of the vector DNA fragment of the plasmid pBACarpVAX 3881 bp (a derivative from pVAX1 Invitrogen, USA, provided by colleagues from the Danish Veterinary Institute) containing a β-actin carp promoter instead of the CMV promoter in pVAX1 (the promoter is replaced by restriction saigas Nru I - EcoR I) and a BGH poly A sequence that provides G- expression gene in eukaryotic cells; T7 promoter, pMB1 ori, ensuring the propagation of the target plasmid in E. coli bacteria cells;

- EcoRI - XhoI fragment with a size of 1550 bp containing the full-sized gene for the glycoprotein of the virus VVK isolate ZL-4;

- genetic marker: kanamycin resistance gene;

- Unique restriction sites: EcoR I (1615), Xho I (3257).

The above genetic engineering constructs are produced in preparative quantities for vaccination of carps by intramuscular injection at a dose of 1 μg of DNA per gram of ichthyomass. The protective effect of vaccines is assessed by the survival of the fish compared to the unvaccinated control group of fish after infection with a lethal dose of the virus of the VVK 10 weeks after vaccination.

The experiments showed that the DNA vaccine based on the plasmid construct pcDNA-G ensures the survival of 82% of the experimental animals, and the construction of pBACarpVAX-G - 74% (Figure 4).

FROM (RPS) = [1 - (% mortality among vaccinated fish /% mortality in control)] × 100%

RPS (relative percent survival) or FM (protection index) for pcDNA-G and pBACarpVAX-G was 71.5 and 57.3% in relation to the fish groups injected with the corresponding control plasmids, or 58 and 40% in relation to the group, which was injected buffer solution

The invention is illustrated by the following graphic materials.

Figure 1. Scheme of creating pBACarpVAX-G and pcDNA-G constructs carrying the glycoprotein gene of the Russian reference strain ZL-4 of the VVK virus.

Figure 2. The nucleotide sequence of the glycoprotein gene of the Russian reference strain ZL-4 of the virus VVK.

Figure 3. Aligned nucleotide sequences of the glycoprotein genes of the reference strains Fijan and GL-4 of the VVK virus.

Figure 4. Survival of fish vaccinated with pBACarpVAX-G and pcDNA-G, expressing the VVC glycoprotein gene.

For a better understanding of the invention, the following are examples of its implementation.

Example 1. Determination of the nucleotide sequence of the glycoprotein gene of the Russian reference strain ZL-4 virus VVK

Isolation of RNA from a virus suspension is carried out according to the method of Chomczynski and Sacchi [16]. Two fragments of the glycoprotein gene are obtained by amplification in a polymerase chain reaction of cDNA synthesized on a genomic viral RNA template using two pairs of oligonucleotide primers. The synthesis of the first cDNA strand on a viral RNA template was carried out at 42 ° C for 2 hours in the following reaction mixture: 0.02 M Tris-HCl, pH 8.0, 0.1 M KCl, 2 mM MgCl ₂ , 1 mM DTT, 0.5 mM dNTP, 15 each mcg primers G1 or G2 and 25 units. M-MuLV reverse transcriptase activity. The reaction is stopped by warming the mixture at 95 ° C for 5 minutes.

To increase the cloning efficiency of the SVCV glycoprotein gene, PCR technology using synthetic primers was used. Since the size of the G gene of the virus is quite large (1530 bp), it is advisable to amplify the sequence of the G gene in parts. To do this, use two pairs of primers that allow amplification of the left and right parts of the gene separately. The selection of primers to initiate the RT-PCR reaction is based on the analysis of the nucleotide sequence of the G gene and the intergenic regions of the Fijan strain of VVK virus:

Primers have restriction enzyme recognition sites, which, after amplification, allows cloning of fragments into vector DNA more efficiently at the “sticky” ends. PCR is carried out in 0.5 ml tubes (Bio Tip Germany). The composition of the reaction mixture per 25 μl:

dNTP 10 mM (SibEnzyme, Russia) - 2 μl

10-fold buffer (SibEnzyme, Russia) - 2 μl

Primers - 50 rM

DNA Polymerase (Taq pol) - 1 U (SibEnzyme, Russia)

The reaction is carried out on a BIS 109 amplifier according to the following program:

PCR fragments G 1-3 (868 bp) and G 2-4 (879 bp) are digested with BamH I-Hind III restriction enzymes and ligated under standard conditions with plasmid pUC18 hydrolyzed with BamH I-Hind III restriction enzymes. Competent E. coli JM103 cells were transformed with a ligase mixture and plasmid DNA was isolated from white clones grown on ampicillin, Xgal and IPTG and subjected to restriction analysis with BstE II-Pvu II, BamH I-Hind III, Hae III enzymes. Correspondence of the nucleotide sequence of the insert in the selected recombinant plasmid is checked by sequencing.

Example 2. Creating the target pcDNA-G and pBACarpVAX-G constructs

To assemble the full-sized gene and the correct orientation of the gene sequence, both fragments are cloned at the EcoR I-Nru I (from pUC-G-1-3) and Nru I-Hind III (from pUC-G-2-4) restriction sites into the pBluKS plasmid (-). The resulting full-length gene sequence is cloned at the sticky ends of EcoR I - XhoI into eukaryotic pcDNA3.1 expression vectors. and pBACarpVAX (Figure 1). Competent E. coli JM103 cells are transformed with a ligase mixture and from clones grown on medium with ampicillin (in the case of pcDNA-G) or kanamycin (in the case of pBACarpVAX-G), plasmid DNA is isolated and subjected to restriction analysis with BstE II - Pvu II enzymes EcoR I - Hind III, Hae III. Correspondence of the nucleotide sequence of the insert in the selected recombinant plasmid is checked by sequencing.

Example 3. The study of the ability of target genetically engineered structures to induce a protective immune response of vaccinated carps against infection with a virulent strain of the virus VVK

Preparation of DNA preparations for vaccination of carp

E. coli JM103 cells containing pcDNA-G or pBACarpVAX-G are grown as follows: a single colony is seeded in 250 ml of YT medium containing ampicillin (100 μg / ml) or kanamycin (50 μg / ml) and incubated for 16 hours at 37 ° C. Plasmid DNA was isolated by the standard alkaline method [15].

Carp immunization and infection schedule

The experiments use yearlings of carp (Cyprinus caprio) with an average body weight of 20-30 g (30 fish per group) contained in 90-liter aquariums with running aerated water at a temperature of about 8 ° C. Fishes are injected intramuscularly with the above preparations at a dose of 1 μg of DNA per gram of ichthyomass. After a day, the water temperature is gradually increased to 13-15 ° C.

After 10 weeks, the fish is infected intraperitoneally with a virulent strain M2 of the VVK virus at a dose of about 10 ⁸ TCID ₅₀ per fish.

The experiments showed that the DNA vaccine based on the plasmid construct pcDNA-G ensures the survival of 82% of the experimental animals, and based on the construction of pBACarpVAX-G - 74% (Figure 4).

Thus, the created genetic engineering constructs with the intramuscular administration of carps provide the development of protective immunity in fish against infection with a virulent strain of spring carp viremia virus. The proposed recombinant plasmid DNA can be used as a DNA vaccine against IHC in domestic fish farming.

Bibliography

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Claims (3)

1. Polynucleotide encoding the surface transmembrane glycoprotein of the spring viremia virus of the carp of Russia reference isolate ZL-4, essentially characterized by the nucleotide sequence shown in figure 2. 2. Recombinant plasmid DNA pcDNA-G size 6961 bp and a molecular weight of 46.1 · 10 ⁶ daltons, which ensures the development of protective immunity in fish against infection with the virulent isolate of the spring viremia virus of the carp of Russian aquaculture in the form of a DNA vaccine with intramuscular injection, including: - EcoR I - Xho I fragment with a size of 1556 bp containing a polynucleotide encoding the surface transmembrane glycoprotein (G gene) of the spring viremia virus of the carp of the reference isolate ZL-4 for Russia described in paragraph 1; - EcoR I - Xho I vector DNA fragment of plasmid pcDNA 3.1. (+) 5405 bp in size [Invitrogen, USA], containing the CMV promoter and the BGH poly A sequence for expression of the G gene in eukaryotic cells; T7 promoter and ampicillin resistance gene, providing selection and reproduction of the target plasmid in E. coli cells; - Unique recognition sites by restriction endonucleases having the following coordinates: EcoR (722), Kpn I (691), Xho I (2278), Xbal (2284). 3. Recombinant plasmid DNA pBACarpVax-G with a size of 5437 bp and a molecular weight of 36.1 · 10 ⁶ daltons, which ensures the development of protective immunity in fish against infection with the virulent isolate of the spring viremia virus of the carp of Russian aquaculture in the form of a DNA vaccine with intramuscular injection, including: - EcoR I - Xho I fragment with a size of 1556 bp containing a polynucleotide encoding the surface transmembrane glycoprotein (G gene) of the spring viremia virus of the carp of the reference isolate ZL-4 for Russia described in paragraph 1; - EcoR I - Xho I vector fragment of plasmid pBACarpVax 3881 bp [derivative from pVax1 Invitrogen, USA], containing the β-actin carp promoter at the Nru I restriction sites — EcoR I and the BGH poly A sequence, which provide expression of the G gene in eukaryotic cells; T7 promoter, pMB1 ori, and kanamycin resistance gene, which select and reproduce the target plasmid in E. coli bacteria cells; - Unique recognition sites by restriction endonucleases: EcoR I (1615), Xho I (3257).

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