Recombinant plasmid dna rwms-nef(a)m-1, expressing the nef protein of human immunodeficiency virus type 1 in eukaryotic cells

 

The invention relates to the field of biotechnology and medicine, in particular to genetic engineering and immunology. The proposed recombinant plasmid DNA rwms-nef(A)m-1, which contains the nef gene of human immunodeficiency virus type 1 subtype a, in the sequence of nucleotides in which the codons with a predominance of adenine and/or thymine adenine and/or thymine partially replaced by guanine and/or cytosine. Plasmid provides the expression of the NEF protein and can be used for immunization against human immunodeficiency virus. 1 table, 3 Il.

The invention relates to the field of biotechnology and medicine, in particular to genetic engineering and immunology, and can be used for the expression of nef protein of human immunodeficiency virus type 1 (HIV-1) subtype a in eukaryotic cells.

Known recombinant plasmid DNA: pGEX-2T-nef 27, pYEULCBX-nef 27, pVL1393-nef 27, containing the DNA fragment of human immunodeficiency virus type 1, size 624 base pairs that encodes a viral protein NEF (see Ahmed A. Azad, Paul Failla, Anna Lucantoni, John Bentley, Chris Mardon, Andrew Wolfe, Kerri Fuller, Dean Hewish, Shomik Sengupta, Sonia Sankovich, Elizabeth Grgacic, Dale McPhee, lan Macreadie. Large-scale production and characterization of recombinant human immunodeficiency virus type 1 NEF. Journal of General Virology (1994), 75, 651-655).deficit human type 1, in the form of a recombinant protein in a bacterial, yeast cells and in cultured insect cells. However, the synthesis of NEF HIV-1 in mammalian cells is not performed.

As follows from the above analysis of the prior art, close analogues, which could be taken as a prototype of the present invention, is not revealed.

The present invention laid the task of creating a recombinant plasmid DNA, which provides the expression of the NEF protein encoded nef gene of HIV-1 subtype a in mammalian cells, and thereby immunization of the human body.

According to the invention this problem is solved due to the fact that recombinant plasmid DNA pBMC-nef(A)m-1, expressing the nef protein of human immunodeficiency virus type 1 in eukaryotic cells that contains the nef gene of human immunodeficiency virus type 1 subtype a, shown in Fig.2, in the sequence of nucleotides nef gene in codons with a predominance of adenine and/or thymine adenine and/or thymine partially replaced by guanine and/or cytosine, and also due to the introduction of this fragment sequence Kozak, initiating and two termination codons.

The invention is illustrated by drawings, which not only>is a of Fig.2 is a nucleotide sequence modified according to the present invention nef gene Russian variant of subtype a HIV-1. (replacement bases are underlined);

in Fig.3 - structure of plasmid DNA pBMC-nef(A)m-1.

Designations in Fig.3: nef(A)m-l - DNA fragment containing the modified nucleotides, bla - gene beta-lactamase, Ori - Replicator, R - promoter predannih protein gene of cytomegalovirus, a Kozak sequence, BGH - site termination of transcription and polyadenylation.

Plasmid DNA isolation pBMC-nef(A)m-1 as follows.

The DNA fragment encoding nef gene of HIV-1 subtype a, was isolated using polymerase chain reaction matrix which served as DNA from peripheral blood lymphocytes of a patient infected with human immunodeficiency virus first type subtype A. This fragment was cloned in the vector rvms receiving plasmid pBMC-nef(A) containing natural nef gene. It was further allocated to the obtained plasmid pBMC-nef(A) and introduced in its sequence in a known manner fragments containing the Kozak sequence, initiation and termination codons with obtaining plasmids rwms-nef(A)-1. Next was isolated plasmid pBMC-nef(A)-1 and produced in the nef gene required replacement nucleated cells occurs in the following way.

Cell culture ZTZ mouse transformed DNA plasmids rvms, pBMC-nef(A)-1 and pBMC-nef(A)m-1 (1 micrograms of each plasmid DNA), after which cells were incubated for 48 hours at a temperature of +37And the content of CO₂in the air, equal to 5%, literally and analyzed proteins in the cell lysates by electrophoresis in SDS page followed by Western blot turns with the serum of a patient infected with HIV-1 subtype A. the intensity of the color areas corresponding to the NEF protein was analyzed using computerized video system. It is established that in the culture of cells transformed by the plasmid pBMC-nef(A)m-1 nef protein synthesis 10-15 times higher than in cells transformed by the plasmid rwms-nef(A)-1.

The use of plasmid DNA pBMC-nef(A)m-1 for immunization of the body is illustrated by the following example. Laboratory mice BALB/c mice were injected intramuscularly with 10 and 25 micrograms of plasmid DNA pBMC-nef(A)-1 and pBMC-nef(A)m-1 according to the present invention. Immunization was repeated after four weeks and after another two weeks, we collected blood serum of immunized animals and defined them in the titer of antibodies to recombinant NEF protein subtype a known manner. The results are shown in the table.