RU2335540C1 - EXPRESSION PLASMID DNA pBMC - nef (A)-hum - Google Patents

Abstract

FIELD: medicine; biotechnologies.

SUBSTANCE: it is offered a plasmid DNA pBMC - nef (A)-hum for an expression of human Nef protein of immunodeficiency virus of subtype A, containing an artificial gene nef (A)-hum, coding polypeptide Nef (A)-hum. The invention can be used in medicine and allied industries for the rising of an expression coded by a gene nef of a Nef protein or its fragments in eucariotic cells.

EFFECT: increase of expression.

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Description

The invention relates to the field of biotechnology, in particular to a technology for producing vaccine preparations using genetic engineering and immunology methods, and can be used in medicine and related industries to increase the expression of proteins, in particular, human immunodeficiency virus type 1 Nef protein (HIV-1) subtype A or its fragments and synthetic analogues in eukaryotic cells.

One of the global problems facing biotechnology is to increase the yield of substances produced by cells of prokaryotes and eukaryotes. To solve these problems, as a rule, cells are exposed to various external factors: raising the temperature above 42 ° C, reducing the amount of nutrients, such as phosphates or nitrogen, to a level below that required for the survival of microorganisms, toxic effects - for example, the use of dyes, acids or plant exudates, metabolic destruction of cells as a result of changes in the level of ion content affecting the ability of microorganisms to osmoregulation, or the level of the use of vitamins or cofactors that can cause a cessation of metabolism (RU 2179980, 2002).

However, since the impact of the above factors on the yield of specific producers is characterized by high species-specificity, then for each specific case a large amount of research is necessary

The most difficult questions of stimulation of expression are solved in the case of vaccines against human immunodeficiency virus (HIV), for the manufacture of which it is necessary to learn how to achieve the expression of viral proteins in eukaryotic cells without introducing the virus, which would allow the body to develop the necessary immune response. The problem is associated with the features of the sequences of the human immunodeficiency virus genes, in particular with the use of codons in the viral genes that are not characteristic of intensively expressed mammalian genes, in particular humans, as well as the large variability of the viral population, expressed in genetic divergence.

One approach to solving this problem is to use the genomes of retro viruses, adenoviruses, papillomaviruses, and papovaviruses as stimulants for the expression of protective antigens of pathogenic viruses in mammalian cells.

The most detailed studies were conducted using the vaccinia virus (BOB). In particular, recombinant BOBs were constructed that express individual HIV-1 proteins separately: gp160, gp120. Tat and reverse transcriptase (Moss B., and Flexner C. // Ann. Rev. Immunol, 1987, v.5, p.305; Moss B. // Seminars in Virology, 1992, v.3, p.277; Falkner FG, et al. // Virology, 1988, v. 164, p. 450; Flexner C., et al. // Virology, 1988, v. 166, p. 339; Takahashi H, et al. // Proc Natl. Acad. Sci. USA, 1988, v. 85, p. 3105; Walker BD, et al. // Science, 1988, v. 240, p. 64; Willey RL, et al. // J. Virol 1988, v. 62, p. 139). The resulting recombinant BOBs were used to immunize laboratory animals and to identify HIV-1 proteins that could induce virus neutralizing antibodies. However, using recombinant BOB, it was not possible to induce a full-fledged protective immune response against HIV infection. Apparently, this is due to the low immunogenicity of individual viral proteins (RU 2194075, 2002).

To solve the problem of obtaining an immune response to HIV proteins, the TBI protein was created, which includes several HIV-1 cell epitopes. However, when using it, it is not possible to obtain the full range of target products. In addition, the degree of expression of the resulting proteins is not high enough (RU 2237089, 2005).

The closest in technical essence and the achieved effect to the claimed invention is to stimulate the expression of Nef protein by introducing into the cell of higher eukaryotes expression plasmid DNA pBMC-nef (A) m-1 containing the modified human immunodeficiency virus type 1 nef subtype A subtype A (RU 2229518, 2002).

The technical problem solved by the authors was the creation of expression plasmid DNA for expression of the synthetic protein Nef (A) -hum, which allows to increase its yield and immunogenicity.

The technical result was obtained using expression plasmid DNA pBMC-nef (A) -hum containing a modified human immunodeficiency virus nef gene of type 1 subtype A, called nef (A) m-1, the sequence of which is shown in Fig. 1.

The basis of the invention was the idea that in the genes of the virus there are codons in which a lot of adenine and thymine predominate and guanine and cytosine are rare, which are not characteristic of intensively expressed human genes. As a result of the studies conducted by the authors, it was found that by replacing in the nef gene part A and T - rich codons with synonymous G and C - rich codons, it is possible to increase the yield of Nef (A) -hum protein 10-15 times.

The disadvantages of this invention are: the relatively low yield of the target product and the low occurrence of certain amino acids of the synthesized protein Nef (A) -hum in the HIV-1 population circulating in Russia.

According to the present invention, a new plasmid DNA pBMC-nef (A) -hum was obtained by the following method.

The codons in the nef gene fragment encoding Nef HIV-1 protein of subtype A were analyzed. All codons with a high content of adenine and thymine were replaced by synonymous codons with a reduced content of adenine and thymine and, accordingly, a higher content of guanine and cytosine, while the gene composition preferably included codons characteristic of intensely expressed human genes. In addition, the Kozak sequence, initiating and two translation termination codons, was included in the gene sequence. Also, codons coding for some rare amino acids in immunologically important regions of the Nef protein were identified and replaced with codons of amino acids that are more common in the population of HIV-infected people in Russia and the CIS countries. In addition, codons encoding the first two NH ₂ -terminal amino acids of the Nef protein were removed from the sequence, which makes its myrrhization impossible, undesirable when using this protein as an immunogen. As a result, an automated nef (A) -hum gene was generated using automated chemical synthesis, encoding the synthetic Nef (A) -hum protein, which was inserted into the rVMS vector to obtain the expression plasmid pBMC-nef (A) -hum.

The invention is illustrated by drawings, on which:

figure 1 - the nucleotide sequence of the gene nef (A) -hum in comparison with the nucleotide sequence of geneap1 (A) m-1 (prototype);

figure 2 - the nucleotide sequence of the gene nef (A) -hum in comparison with the nucleotide sequence of the natural gene nef (A);

figure 3 - the amino acid sequence of the protein Nef (A) -hum in comparison with the amino acid sequence of the protein Nef (A) (prototype).

Nucleotides and amino acids that are the same for the genes presented are marked with a dot (.) In the compared sequence, absent in the sequences with a tilde (~), differing ones are shown with the corresponding letter, termination codons are indicated (*). The 627-nucleotide nef (A) -hum gene differs from the Nef (A) m-1 prototype in 104 nucleotides and in 175 nucleotides differs from the natural nef (A) gene. Protein Nef (A) -syn with a length of 207 amino acids differs from the prototype Nef (A) in 9 amino acids.

The industrial applicability of the claimed stimulator of protein synthesis and synthetic protein is illustrated by the following examples.

Example 1. Obtaining plasmid DNA, providing expression of the synthetic protein Nef (A) -hum using the artificial gene nef (A) -hum.

The artificial nef (A) -hum gene, the sequence of which is shown in FIG. 1, was obtained by chemical synthesis of overlapping fragments, followed by annealing and ligation. The collected gene was inserted in a known manner into the rVMS plasmid vector to obtain the expression plasmid pBMC-nef (A) -hum. The obtained plasmid pBMC-nef (A) -hum transformed Escherichia coli cells for its subsequent production.

Example 2. The study of the synthesis of synthetic protein Nef (A) -hum in eukaryotic cells.

The 293 human cell culture was transformed with the DNA of the recombinant plasmid pBMC-nef (A) -hum. As a control, a parallel 293 cell culture was transformed with an equal amount of pBMC-nef (A) m-1 plasmid DNA. Transformed cell cultures were incubated in a nutrient medium for 48 hours at a temperature of + 37 ° C and a CO ₂ content of 5% in air, the cell lysate proteins were lysed and analyzed by electrophoresis in SDS page followed by immunoblotting with the serum of a patient infected with HIV-1 subtype A. The color intensity of the bands corresponding to the Nef protein was analyzed using a computerized video system. It was found that in the culture of cells transformed with pBMC-nef (A) -hum plasmid DNA, the level of Nef (A) -hum protein synthesis is at least 6-8 times higher than the synthesis of Nef (A) protein in cells transformed with a control plasmid pBMC-nef (A) m-1.

Example 3. The use of plasmid DNA pBMC-nef (A) -hum for immunization.

BALB / c laboratory mice were injected intramuscularly three times with 10 micrograms of pBMC-nef (A) -hum plasmid DNA, and a similar dose of pBMC-nef (A) m-1 was administered to the control group of mice. After 6 weeks, the titers of antibodies to the natural Nef protein in a known manner were determined in the blood serum of animals. The results are shown in the table.

Table Antibody titers in sera of mice immunized with pBMC-RT (A) -hum and pBMC-RT (A) m-1 plasmid DNA Antibody titers with the introduction of DNA plasmids pBMC-nef (A) -hum 1: 2048 Antibody titers with the introduction of DNA plasmid pBMC-nef (A) m-1 1: 512

The use of the inventive stimulant allows 4-5 times to increase the yield of synthetic Nef protein in comparison with the prototype.

Claims (1)

PBMC-nef (A) -hum expression plasmid DNA for expression of the Nef (A) -hum synthetic protein in eukaryotic cells containing the expression plasmid DNA, characterized in that it contains the artificial nef (A) -hum gene having the nucleotide sequence represented by figure 1, and the coding polypeptide, the amino acid sequence of which is presented in figure 3.

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