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RU-2861323-C1 - SET OF RECOMBINANT PLASMID DNAS ENCODING FULL-LENGTH SURFACE GLYCOPROTEINS OF RECOMBINANT FORM CRF63_02A6 HIV-1, AND SET OF PSEUDOTYPED HIV-1 VIRUSES OBTAINED USING SAID SET OF PLASMID DNAS AND INTENDED FOR DETERMINING ACTIVITY OF PREPARATIONS AGAINST HIV-1

RU2861323C1RU 2861323 C1RU2861323 C1RU 2861323C1RU-2861323-C1

Abstract

FIELD: medicine. SUBSTANCE: invention relates to a set of recombinant plasmid DNAs encoding full-length surface glycoproteins of the recombinant form CRF63_02A6 HIV-1, and to a set of pseudotyped HIV-1 viruses obtained using said set of plasmid DNAs, and can be used in genetic engineering, biotechnology and medicine. A set of pseudotyped viruses of the recombinant form CRF63_02A6 HIV-1 is created, intended for determining the activity against HIV-1 of monoclonal antibodies, vaccines and searching for biologically active compounds against HIV-1, obtained using the set of recombinant plasmid DNAs according to claim 1 and having the amino acid sequences SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 respectively of said plasmid vectors. EFFECT: obtaining thirteen expression plasmid vectors pl6RU12, pl6RU13, pl6RU19, pl6RU20, pl6RU21, pl6RU22, pl6RU23, pl6RU27, pl6RU28, pl6RU35, pl6RU36, p22RUAR13, p22RUAR21, carrying genes encoding full-length variants of the env gene of the recombinant form CRF63_02A6 HIV-1, having the nucleotide sequences SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 respectively. 2 cl, 58 dwg, 4 ex

Inventors

  • Rudometova Nadezhda Borisovna
  • KARPENKO LARISA IVANOVNA
  • Rudometov Andrej Pavlovich
  • SHCHERBAKOV DMITRIJ NIKOLAEVICH
  • Makarova Kristina Petrovna
  • Nizolenko Liliya Filippovna
  • ILICHEV ALEKSANDR ALEKSEEVICH

Dates

Publication Date
20260504
Application Date
20250918

Claims (20)

  1. 1. A set of recombinant plasmid DNAs encoding full-length surface glycoproteins of the recombinant form CRF63_02A6 of HIV-1, used to obtain pseudotyped HIV-1 viruses, including thirteen plasmid constructs:
  2. - the first plasmid construct p16RU12, providing expression of the full-length 16RU12 env gene of the recombinant form CRF63_02A6 HIV-1, encoding the full-length surface glycoprotein Env 16RU12 of the recombinant form CRF63_02A6 HIV-1, in transformed eukaryotic cells HEK293, having the sequence SEQ ID NO: 1, the size of 8383 bp, the molecular weight of 5186274 kilodaltons and containing, in accordance with the physical and genetic map, the following elements: CMV enchancer/promoter, providing efficient expression of the recombinant protein in mammalian cells and having coordinates from 235 to 818 bp; T7 promoter/priming site, providing in vitro transcription/primer landing site for determining the presence/absence of insert by sequencing and having coordinates from 863 to 881 bp; Envelope is the full-length 16RU12 env gene of HIV-1 of the recombinant form CRF63_02A6, having coordinates from 1161 to 3761 bp; V5 tag/epitope, allowing detection of recombinant protein using Anti-V5 or Anti-V5-HRP antibodies and having coordinates from 3880 to 3921 bp; 6 × His is a 6-histidine affinity tag with coordinates from 3931 to 3948 bp. and allowing purification of the recombinant protein on a metal chelate resin such as ProBond™, wherein the C-terminal polyhistidine tag is an epitope for the Anti-His (C-term) antibody and the Anti-His (C-term)-HRP antibody; bGH poly(A) signal is a bovine growth hormone polyadenylation signal having coordinates from 3977 to 4201 bp; f1 origin is the origin of replication of bacteriophage f1 having coordinates from 4247 to 4675 bp; SV40 promoter and origin, providing efficient expression of the neomycin resistance gene and episomal replication in cells expressing the SV40 large T antigen and having coordinates from 4689 to 5019 bp; NeoR/KanR, providing resistance to neomycin, kanamycin and G418 and having coordinates from 5086 to 5880 bp; SV40 poly(A) signal - SV40 polyadenylation signal, having coordinates from 6056 to 6177 bp; Ori - origin of replication ColE1/pMB1/pBR322/pUC, having coordinates from 6628 to 7216 bp; AmpR/AmpR promoter - β-lactamase, providing resistance to ampicillin, carbenicillin and related antibiotics and having coordinates from 7387 to 8352 bp;
  3. - the second plasmid construct p16RU13, providing expression of the full-length 16RU13 env gene of the recombinant form CRF63_02A6 HIV-1, encoding the full-length surface glycoprotein Env 16RU13 of the recombinant form CRF63_02A6 HIV-1, in transformed eukaryotic cells HEK293, having the sequence SEQ ID NO: 3, a size of 8427 bp, a molecular weight of 5214235 kilodaltons and containing, in accordance with the physical and genetic map, the following elements: CMV enchancer/promoter, providing efficient expression of the recombinant protein in mammalian cells and having coordinates from 235 to 818 bp; T7 promoter/priming site, providing in vitro transcription/primer landing site for determining the presence/absence of insert by sequencing and having coordinates from 863 to 881 bp; Envelope is the full-length 16RU13 env gene of HIV-1 recombinant form CRF63_02A6, having coordinates from 1181 to 3766 bp; V5 tag/epitope, allowing detection of recombinant protein using Anti-V5 or Anti-V5-HRP antibodies and having coordinates from 3924 to 3965 bp; 6 × His is a 6-histidine affinity tag with coordinates from 3975 to 3992 bp. and allowing purification of the recombinant protein on a metal chelate resin such as ProBond™, wherein the C-terminal polyhistidine tag is an epitope for the Anti-His (C-term) antibody and the Anti-His (C-term)-HRP antibody; bGH poly(A) signal is a bovine growth hormone polyadenylation signal having coordinates from 4021 to 4245 bp; f1 origin is the origin of replication of bacteriophage f1 having coordinates from 4291 to 4719 bp; SV40 promoter and origin, providing efficient expression of the neomycin resistance gene and episomal replication in cells expressing the SV40 large T antigen and having coordinates from 4733 to 5063 bp; NeoR/KanR, providing resistance to neomycin, kanamycin and G418 and having coordinates from 5130 to 5924 bp; SV40 poly(A) signal - SV40 polyadenylation signal, having coordinates from 6100 to 6221 bp; Ori - origin of replication ColE1/pMB1/pBR322/pUC, having coordinates from 6672 to 7260 bp; AmpR/AmpR promoter - β-lactamase, providing resistance to ampicillin, carbenicillin and related antibiotics and having coordinates from 7431 to 8396 bp;
  4. - the third plasmid construct p16RU19, providing expression of the full-length 16RU19 env gene of the recombinant form CRF63_02A6 HIV-1, encoding the full-length surface glycoprotein Env 16RU19 of the recombinant form CRF63_02A6 HIV-1, in transformed eukaryotic cells HEK293, having the sequence SEQ ID NO: 5, a size of 8446 bp, a molecular weight of 5226020 kilodaltons and containing, in accordance with the physical and genetic map, the following elements: CMV enchancer/promoter, providing efficient expression of the recombinant protein in mammalian cells and having coordinates from 235 to 818 bp; T7 promoter/priming site, providing in vitro transcription/primer landing site for determining the presence/absence of insert by sequencing and having coordinates from 863 to 881 bp; Envelope is the full-length 16RU19 env gene of HIV-1 of the recombinant form CRF63_02A6, having coordinates from 1174 to 3816 bp; V5 tag/epitope, allowing detection of recombinant protein using Anti-V5 or Anti-V5-HRP antibodies and having coordinates from 3943 to 3984 bp; 6 × His is a 6-histidine affinity tag with coordinates from 3994 to 4011 bp. and allowing purification of the recombinant protein on a metal chelate resin such as ProBond™, wherein the C-terminal polyhistidine tag is an epitope for the Anti-His (C-term) antibody and the Anti-His (C-term)-HRP antibody; bGH poly(A) signal is a bovine growth hormone polyadenylation signal having coordinates from 4040 to 4264 bp; f1 origin is the origin of replication of bacteriophage f1 having coordinates from 4310 to 4738 bp; SV40 promoter and origin, providing efficient expression of the neomycin resistance gene and episomal replication in cells expressing the SV40 large T antigen and having coordinates from 4752 to 5082 bp; NeoR/KanR, providing resistance to neomycin, kanamycin and G418 and having coordinates from 5149 to 5943 bp; SV40 poly(A) signal - SV40 polyadenylation signal, having coordinates from 6119 to 6240 bp; Ori - origin of replication ColE1/pMB1/pBR322/pUC, having coordinates from 6691 to 7279 bp; AmpR/AmpR promoter - β-lactamase, providing resistance to ampicillin, carbenicillin and related antibiotics and having coordinates from 7450 to 8310 bp;
  5. - the fourth plasmid construct p16RU20, which provides expression of the full-length 16RU20 env gene of the recombinant form of HIV-1 CRF63_02A6, encoding the full-length surface glycoprotein Env 16RU20 of the recombinant form of HIV-1 CRF63_02A6, in transformed eukaryotic cells HEK293, having the sequence SEQ ID NO: 7, a size of 8527 bp, a molecular weight of 5275806 kilodaltons and containing, in accordance with the physical and genetic map, the following elements: CMV enchancer/promoter, which provides efficient expression of the recombinant protein in mammalian cells and has coordinates from 235 to 818 bp; T7 promoter/priming site, providing in vitro transcription/primer landing site for determining the presence/absence of insert by sequencing and having coordinates from 863 to 881 bp; Envelope is the full-length 16RU20 env gene of HIV-1 recombinant form CRF63_02A6, having coordinates from 1173 to 3773 bp; V5 tag/epitope, allowing detection of recombinant protein using Anti-V5 or Anti-V5-HRP antibodies and having coordinates from 4024 to 4065 bp; 6 × His is a 6-histidine affinity tag with coordinates from 4075 to 4092 bp. and allowing purification of the recombinant protein on a metal chelate resin such as ProBond™, wherein the C-terminal polyhistidine tag is an epitope for the Anti-His (C-term) antibody and the Anti-His (C-term)-HRP antibody; bGH poly(A) signal is a bovine growth hormone polyadenylation signal having coordinates from 4121 to 4345 bp; f1 origin is the origin of replication of bacteriophage f1 having coordinates from 4391 to 4819 bp; SV40 promoter and origin, providing efficient expression of the neomycin resistance gene and episomal replication in cells expressing the SV40 large T antigen and having coordinates from 4833 to 5163 bp; NeoR/KanR, providing resistance to neomycin, kanamycin and G418 and having coordinates from 5230 to 6024 bp; SV40 poly(A) signal - SV40 polyadenylation signal, having coordinates from 6200 to 6321 bp; Ori - origin of replication ColE1/pMB1/pBR322/pUC, having coordinates from 6772 to 7360 bp; AmpR/AmpR promoter - β-lactamase, providing resistance to ampicillin, carbenicillin and related antibiotics and having coordinates from 7531 to 8496 bp;
  6. - the fifth plasmid construct p16RU21, which provides expression of the full-length 16RU21 env gene of the recombinant form of HIV-1 CRF63_02A6, encoding the full-length surface glycoprotein Env 16RU21 of the recombinant form of HIV-1 CRF63_02A6, in transformed eukaryotic cells HEK293, having the sequence SEQ ID NO: 9, a size of 8550 bp, a molecular weight of 5290302 kilodaltons and containing, in accordance with the physical and genetic map, the following elements: CMV enchancer/promoter, which provides efficient expression of the recombinant protein in mammalian cells and has coordinates from 235 to 818 bp; T7 promoter/priming site, providing in vitro transcription/primer landing site for determining the presence/absence of insert by sequencing and having coordinates from 863 to 881 bp; Envelope is the full-length 16RU21 env gene of HIV-1 of the recombinant form CRF63_02A6, having coordinates from 1198 to 3777 bp; V5 tag/epitope, allowing detection of recombinant protein using Anti-V5 or Anti-V5-HRP antibodies and having coordinates from 4047 to 4088 bp; 6 × His is a 6-histidine affinity tag with coordinates from 4098 to 4115 bp. and allowing purification of the recombinant protein on a metal chelate resin such as ProBond™, wherein the C-terminal polyhistidine tag is an epitope for the Anti-His (C-term) antibody and the Anti-His (C-term)-HRP antibody; bGH poly(A) signal is a bovine growth hormone polyadenylation signal having coordinates from 4144 to 4368 bp; f1 origin is the origin of replication of bacteriophage f1 having coordinates from 4414 to 4842 bp; SV40 promoter and origin, providing efficient expression of the neomycin resistance gene and episomal replication in cells expressing the SV40 large T antigen and having coordinates from 4856 to 5186 bp; NeoR/KanR, providing resistance to neomycin, kanamycin and G418 and having coordinates from 5253 to 6047 bp; SV40 poly(A) signal - SV40 polyadenylation signal, having coordinates from 6223 to 6344 bp; Ori - origin of replication ColE1/pMB1/pBR322/pUC, having coordinates from 6795 to 7383 bp; AmpR/AmpR promoter - β-lactamase, providing resistance to ampicillin, carbenicillin and related antibiotics and having coordinates from 7554 to 8519 bp;
  7. - the sixth plasmid construct p16RU22, providing expression of the full-length 16RU22 env gene of the recombinant form of HIV-1 CRF63_02A6, encoding the full-length surface glycoprotein Env 16RU22 of the recombinant form of HIV-1 CRF63_02A6, in transformed eukaryotic cells HEK293, having the sequence SEQ ID NO: 11, the size of 8607 bp, the molecular weight of 5325821 kilodaltons and containing, in accordance with the physical and genetic map, the following elements: CMV enchancer/promoter, providing efficient expression of the recombinant protein in mammalian cells and having coordinates from 235 to 818 bp; T7 promoter/priming site, providing in vitro transcription/primer landing site for determining the presence/absence of insert by sequencing and having coordinates from 863 to 881 bp; Envelope is the full-length 16RU22 env gene of HIV-1 recombinant form CRF63_02A6, having coordinates from 1198 to 3780 bp; V5 tag/epitope, allowing detection of recombinant protein using Anti-V5 or Anti-V5-HRP antibodies and having coordinates from 4104 to 4145 bp; 6 × His is a 6-histidine affinity tag with coordinates from 4155 to 4172 bp. and allowing purification of the recombinant protein on a metal chelate resin such as ProBond™, wherein the C-terminal polyhistidine tag is an epitope for the Anti-His (C-term) antibody and the Anti-His (C-term)-HRP antibody; bGH poly(A) signal is a bovine growth hormone polyadenylation signal having coordinates from 4201 to 4425 bp; f1 origin is the origin of replication of bacteriophage f1 having coordinates from 4471 to 4899 bp; SV40 promoter and origin, providing efficient expression of the neomycin resistance gene and episomal replication in cells expressing the SV40 large T antigen and having coordinates from 4913 to 5243 bp; NeoR/KanR, providing resistance to neomycin, kanamycin and G418 and having coordinates from 5310 to 6104 bp; SV40 poly(A) signal - SV40 polyadenylation signal, having coordinates from 6280 to 6401 bp; Ori - origin of replication ColE1/pMB1/pBR322/pUC, having coordinates from 6852 to 7440 bp; AmpR/AmpR promoter - β-lactamase, providing resistance to ampicillin, carbenicillin and related antibiotics and having coordinates from 7611 to 8471 bp;
  8. - the seventh plasmid construct p16RU23, which provides expression of the full-length 16RU23 env gene of the recombinant form CRF63_02A6 HIV-1, encoding the full-length surface glycoprotein Env 16RU23 of the recombinant form CRF63_02A6 HIV-1, in transformed eukaryotic cells HEK293, having the sequence SEQ ID NO: 13, a size of 8455 bp, a molecular weight of 5231980 kilodaltons and containing, in accordance with the physical and genetic map, the following elements: CMV enchancer/promoter, which provides efficient expression of the recombinant protein in mammalian cells and has coordinates from 235 to 818 bp; T7 promoter/priming site, providing in vitro transcription/primer landing site for determining the presence/absence of insert by sequencing and having coordinates from 863 to 881 bp; Envelope is the full-length 16RU23 env gene of HIV-1 recombinant form CRF63_02A6, having coordinates from 1197 to 3815 bp; V5 tag/epitope, allowing detection of recombinant protein using Anti-V5 or Anti-V5-HRP antibodies and having coordinates from 3952 to 3993 bp; 6 × His is a 6-histidine affinity tag with coordinates from 4003 to 4020 bp. and allowing purification of the recombinant protein on a metal chelate resin such as ProBond™, wherein the C-terminal polyhistidine tag is an epitope for the Anti-His (C-term) antibody and the Anti-His (C-term)-HRP antibody; bGH poly(A) signal is a bovine growth hormone polyadenylation signal having coordinates from 4049 to 4273 bp; f1 origin is the origin of replication of bacteriophage f1 having coordinates from 4319 to 4747 bp; SV40 promoter and origin, providing efficient expression of the neomycin resistance gene and episomal replication in cells expressing the SV40 large T antigen and having coordinates from 4761 to 5091 bp; NeoR/KanR, providing resistance to neomycin, kanamycin and G418 and having coordinates from 5158 to 5952 bp; SV40 poly(A) signal - SV40 polyadenylation signal, having coordinates from 6128 to 6249 bp; Ori - origin of replication ColE1/pMB1/pBR322/pUC, having coordinates from 6700 to 7288 bp; AmpR/AmpR promoter - β-lactamase, providing resistance to ampicillin, carbenicillin and related antibiotics and having coordinates from 7459 to 8424 bp;
  9. - the eighth plasmid construct p16RU27, which ensures the expression of the full-length 16RU27 env gene of the recombinant form CRF63_02A6 HIV-1, encoding the full-length surface glycoprotein Env 16RU27 of the recombinant form CRF63_02A6 HIV-1, in transformed eukaryotic cells HEK293, having the sequence SEQ ID NO: 15, the size of 8607 bp, the molecular weight of 5325683 kilodaltons and containing, in accordance with the physical and genetic map, the following elements: CMV enchancer/promoter, which ensures efficient expression of the recombinant protein in mammalian cells and has coordinates from 235 to 818 bp; T7 promoter/priming site, providing in vitro transcription/primer landing site for determining the presence/absence of insert by sequencing and having coordinates from 863 to 881 bp; Envelope is the full-length 16RU27 env gene of HIV-1 recombinant form CRF63_02A6, having coordinates from 1181 to 3796 bp; V5 tag/epitope, allowing detection of recombinant protein using Anti-V5 or Anti-V5-HRP antibodies and having coordinates from 4104 to 4145 bp; 6 × His is a 6-histidine affinity tag with coordinates from 4155 to 4172 bp. and allowing purification of the recombinant protein on a metal chelate resin such as ProBond™, wherein the C-terminal polyhistidine tag is an epitope for the Anti-His (C-term) antibody and the Anti-His (C-term)-HRP antibody; bGH poly(A) signal is a bovine growth hormone polyadenylation signal having coordinates from 4201 to 4425 bp; f1 origin is the origin of replication of bacteriophage f1 having coordinates from 4471 to 4899 bp; SV40 promoter and origin, providing efficient expression of the neomycin resistance gene and episomal replication in cells expressing the SV40 large T antigen and having coordinates from 4913 to 5243 bp; NeoR/KanR, providing resistance to neomycin, kanamycin and G418 and having coordinates from 5310 to 6104 bp; SV40 poly(A) signal - SV40 polyadenylation signal, having coordinates from 6280 to 6401 bp; Ori - origin of replication ColE1/pMB1/pBR322/pUC, having coordinates from 6852 to 7440 bp; AmpR/AmpR promoter - β-lactamase, providing resistance to ampicillin, carbenicillin and related antibiotics and having coordinates from 7611 to 8576 bp;
  10. - the ninth plasmid construct p16RU28, which ensures the expression of the full-length 16RU28 env gene of the recombinant form of HIV-1 CRF63_02A6, encoding the full-length surface glycoprotein Env 16RU28 of the recombinant form of HIV-1 CRF63_02A6, in transformed eukaryotic cells HEK293, having the sequence SEQ ID NO: 17, the size of 8329 bp, the molecular weight of 5152541 kilodaltons and containing, in accordance with the physical and genetic map, the following elements: CMV enchancer/promoter, which ensures efficient expression of the recombinant protein in mammalian cells and has coordinates from 235 to 818 bp; T7 promoter/priming site, providing in vitro transcription/primer landing site for determining the presence/absence of insert by sequencing and having coordinates from 863 to 881 bp; Envelope is the full-length 16RU28 env gene of HIV-1 recombinant form CRF63_02A6, having coordinates from 1159 to 3735 bp; V5 tag/epitope, allowing detection of recombinant protein using Anti-V5 or Anti-V5-HRP antibodies and having coordinates from 3826 to 3867 bp; 6 × His is a 6-histidine affinity tag with coordinates from 3877 to 3894 bp. and allowing purification of the recombinant protein on a metal chelate resin such as ProBond™, wherein the C-terminal polyhistidine tag is an epitope for the Anti-His (C-term) antibody and the Anti-His (C-term)-HRP antibody; bGH poly(A) signal is a bovine growth hormone polyadenylation signal having coordinates from 3923 to 4147 bp; f1 origin is the origin of replication of bacteriophage f1 having coordinates from 4193 to 4621 bp; SV40 promoter and origin, providing efficient expression of the neomycin resistance gene and episomal replication in cells expressing the SV40 large T antigen and having coordinates from 4635 to 4965 bp; NeoR/KanR, providing resistance to neomycin, kanamycin and G418 and having coordinates from 5032 to 5826 bp; SV40 poly(A) signal - SV40 polyadenylation signal, having coordinates from 6002 to 6123 bp; Ori - origin of replication ColE1/pMB1/pBR322/pUC, having coordinates from 6574 to 7162 bp; AmpR/AmpR promoter - β-lactamase, providing resistance to ampicillin, carbenicillin and related antibiotics and having coordinates from 7333 to 8298 bp;
  11. - the tenth plasmid construct p16RU35, which ensures the expression of the full-length 16RU35 env gene of the recombinant form CRF63_02A6 HIV-1, encoding the full-length surface glycoprotein Env 16RU35peKOM6nHaHTHofi of the form CRF63_02A6 HIV-1, in transformed eukaryotic cells HEK293, having the sequence SEQ ID NO: 19, the size of 8561 bp, the molecular weight of 5297090 kilodaltons and containing, in accordance with the physical and genetic map, the following elements: CMV enchancer/promoter, which ensures efficient expression of the recombinant protein in mammalian cells and has coordinates from 235 to 818 bp; T7 promoter/priming site, providing in vitro transcription/primer landing site for determining the presence/absence of insert by sequencing and having coordinates from 863 to 881 bp; Envelope is the full-length 16RU35 env gene of HIV-1 recombinant form CRF63_02A6, having coordinates from 1190 to 3742 bp; V5 tag/epitope, allowing detection of recombinant protein using Anti-V5 or Anti-V5-HRP antibodies and having coordinates from 4058 to 4099 bp; 6 × His is a 6-histidine affinity tag with coordinates from 4109 to 4126 bp. and allowing purification of the recombinant protein on a metal chelate resin such as ProBond™, wherein the C-terminal polyhistidine tag is an epitope for the Anti-His (C-term) antibody and the Anti-His (C-term)-HRP antibody; bGH poly(A) signal is a bovine growth hormone polyadenylation signal having coordinates from 4155 to 4379 bp; f1 origin is the origin of replication of bacteriophage f1 having coordinates from 4425 to 4853 bp; SV40 promoter and origin, providing efficient expression of the neomycin resistance gene and episomal replication in cells expressing the SV40 large T antigen and having coordinates from 4867 to 5197 bp; NeoR/KanR, providing resistance to neomycin, kanamycin and G418 and having coordinates from 5264 to 6058 bp; SV40 poly(A) signal - SV40 polyadenylation signal, having coordinates from 6234 to 6355 bp; Ori - origin of replication ColE1/pMB1/pBR322/pUC, having coordinates from 6806 to 7394 bp; AmpR/AmpR promoter - β-lactamase, providing resistance to ampicillin, carbenicillin and related antibiotics and having coordinates from 7565 to 8530 bp;
  12. - the eleventh plasmid construct p16RU36, which provides expression of the full-length 16RU36 env gene of the recombinant form CRF63_02A6 HIV-1, encoding the full-length surface glycoprotein Env 16RU36 of the recombinant form CRF63_02A6 HIV-1, in transformed eukaryotic cells HEK293, having the sequence SEQ ID NO: 21, the size of 8578 bp, the molecular weight of 5308030 kilodaltons and containing, in accordance with the physical and genetic map, the following elements: CMV enchancer/promoter, which provides efficient expression of the recombinant protein in mammalian cells and has coordinates from 235 to 818 bp; T7 promoter/priming site, providing in vitro transcription/primer landing site for determining the presence/absence of insert by sequencing and having coordinates from 863 to 881 bp; Envelope is the full-length 16RU36 env gene of HIV-1 recombinant form CRF63_02A6, having coordinates from 1194 to 3761 bp; V5 tag/epitope, allowing detection of recombinant protein using Anti-V5 or Anti-V5-HRP antibodies and having coordinates from 4075 to 4116 bp; 6 × His is a 6-histidine affinity tag with coordinates from 4126 to 4123 bp. and allowing purification of the recombinant protein on a metal chelate resin such as ProBond™, wherein the C-terminal polyhistidine tag is an epitope for the Anti-His (C-term) antibody and the Anti-His (C-term)-HRP antibody; bGH poly(A) signal is a bovine growth hormone polyadenylation signal having coordinates from 4172 to 4396 bp; f1 origin is the origin of replication of bacteriophage f1 having coordinates from 4442 to 4870 bp; SV40 promoter and origin, providing efficient expression of the neomycin resistance gene and episomal replication in cells expressing the SV40 large T antigen and having coordinates from 4884 to 5214 bp; NeoR/KanR, providing resistance to neomycin, kanamycin and G418 and having coordinates from 5281 to 6075 bp; SV40 poly(A) signal - SV40 polyadenylation signal, having coordinates from 6251 to 6372 bp; Ori - origin of replication ColE1/pMB1/pBR322/pUC, having coordinates from 6823 to 7411 bp; AmpR/AmpR promoter - β-lactamase, providing resistance to ampicillin, carbenicillin and related antibiotics and having coordinates from 7582 to 8547 bp;
  13. - the twelfth plasmid construct p22RUAR13, which ensures the expression of the full-length 22RUAR13 env gene of the recombinant form of HIV-1 CRF63_02A6, encoding the full-length surface glycoprotein Env 22RUAR13 of the recombinant form of HIV-1 CRF63_02A6, in transformed eukaryotic cells HEK293, having the sequence SEQ ID NO: 23, the size of 8440 bp, the molecular weight of 5222360 kilodaltons and containing, in accordance with the physical and genetic map, the following elements: CMV enchancer/promoter, which ensures efficient expression of the recombinant protein in mammalian cells and has coordinates from 235 to 818 bp; T7 promoter/priming site, providing in vitro transcription/primer landing site for determining the presence/absence of insert by sequencing and having coordinates from 863 to 881 bp; Envelope is the full-length 22RUAR13 env gene of HIV-1 recombinant form CRF63_02A6, having coordinates from 1198 to 3780 bp; V5 tag/epitope, allowing detection of recombinant protein using Anti-V5 or Anti-V5-HRP antibodies and having coordinates from 3937 to 3978 bp; 6 × His is a 6-histidine affinity tag with coordinates from 3988 to 4005 bp. and allowing purification of the recombinant protein on a metal chelate resin such as ProBond™, wherein the C-terminal polyhistidine tag is an epitope for the Anti-His (C-term) antibody and the Anti-His (C-term)-HRP antibody; bGH poly(A) signal is a bovine growth hormone polyadenylation signal having coordinates from 4034 to 4258 bp; f1 origin is the origin of replication of bacteriophage f1 having coordinates from 4304 to 4732 bp; SV40 promoter and origin, providing efficient expression of the neomycin resistance gene and episomal replication in cells expressing the SV40 large T antigen and having coordinates from 4746 to 5076 bp; NeoR/KanR, providing resistance to neomycin, kanamycin and G418 and having coordinates from 5143 to 5937 bp; SV40 poly(A) signal - SV40 polyadenylation signal, having coordinates from 6113 to 6234 bp; Ori - origin of replication ColE1/pMB1/pBR322/pUC, having coordinates from 6685 to 7273 bp; AmpR/AmpR promoter - β-lactamase, providing resistance to ampicillin, carbenicillin and related antibiotics and having coordinates from 7444 to 8409 bp;
  14. - the thirteenth plasmid construct p22RUK021, which ensures the expression of the full-length 22RUK021 env gene of the recombinant form of HIV-1 CRF63_02A6, encoding the full-length surface glycoprotein Env 22RUK021 of the recombinant form of HIV-1 CRF63_02A6, in transformed eukaryotic cells HEK293, having the sequence SEQ ID NO: 25, the size of 8505 bp, the molecular weight of 5262263 kilodaltons and containing, in accordance with the physical and genetic map, the following elements: CMV enchancer/promoter, which ensures efficient expression of the recombinant protein in mammalian cells and has coordinates from 235 to 818 bp; T7 promoter/priming site, providing in vitro transcription/primer landing site for determining the presence/absence of insert by sequencing and having coordinates from 863 to 881 bp; Envelope is the full-length 22RUAR13 env gene of HIV-1 recombinant form CRF63_02A6, having coordinates from 1198 to 3822 bp; V5 tag/epitope, allowing detection of recombinant protein using Anti-V5 or Anti-V5-HRP antibodies and having coordinates from 4002 to 4043 bp; 6 × His is a 6-histidine affinity tag with coordinates from 4053 to 4070 bp. and allowing purification of the recombinant protein on a metal chelate resin such as ProBond™, wherein the C-terminal polyhistidine tag is an epitope for the Anti-His (C-term) antibody and the Anti-His (C-term)-HRP antibody; bGH poly(A) signal is a bovine growth hormone polyadenylation signal having coordinates from 4099 to 4323 bp; f1 origin is the origin of replication of bacteriophage f1 having coordinates from 4369 to 4797 bp; SV40 promoter and origin, providing efficient expression of the neomycin resistance gene and episomal replication in cells expressing the SV40 large T antigen and having coordinates from 4811 to 5141 bp; NeoR/KanR, providing resistance to neomycin, kanamycin and G418 and having coordinates from 5208 to 6002 bp; SV40 poly(A) signal - SV40 polyadenylation signal, having coordinates from 6178 to 6299 bp; Ori - origin of replication ColE1/pMB1/pBR322/pUC, having coordinates from 6750 to 7338 bp; AmpR/AmpR promoter - β-lactamase, providing resistance to ampicillin, carbenicillin and related antibiotics and having coordinates from 7509 to 8474 bp.
  15. 2. A set of pseudotyped HIV-1 viruses intended for determining the activity of monoclonal antibodies, vaccines and chemical compounds against HIV-1 and obtained using a set of recombinant plasmid DNA according to paragraph 1:
  16. - the first pseudotyped HIV-1 virus having the amino acid sequence SEQ ID NO: 2 of the full-length surface glycoprotein Env 16RU12 of the recombinant form CRF63_02A6 HIV-1;
  17. - the second pseudotyped HIV-1 virus having the amino acid sequence SEQ ID NO: 4 of the full-length surface glycoprotein Env 16RU13 of the recombinant form CRF63_02A6 HIV-1;
  18. - the third pseudotyped HIV-1 virus having the amino acid sequence SEQ ID NO: 6 of the full-length surface glycoprotein Env 16RU19 of the recombinant form CRF63_02A6 HIV-1;
  19. - the fourth pseudotyped HIV-1 virus, having the amino acid sequence SEQ ID NO: 8 of the full-length surface glycoprotein Env 16RU20 of the recombinant form CRF63_02A6 HIV-1;
  20. - the fifth pseudotyped HIV-1 virus, having the amino acid sequence SEQ ID NO: 10 of the full-length surface glycoprotein Env 16RU21 of the recombinant form CRF63_02A6 HIV-1;

Description

The invention relates to a set of recombinant plasmid DNA encoding full-length surface glycoproteins of the recombinant form CRF63_02A6 HIV-1, and a set of pseudotyped HIV-1 viruses obtained using the said set of plasmid DNA, and can be used in genetic engineering, biotechnology and medicine. Pseudotyped HIV-1 viruses assembled using the resulting recombinant plasmids were characterized by sequencing, phylogenetic, and functional analyses. The proposed kits will enable a range of scientific and applied research: investigating the antiviral activity of newly developed chemotherapeutics and assessing the efficacy of HIV-1 vaccines; studying the breadth and spectrum of neutralizing properties of monoclonal broadly neutralizing antibodies; and searching for new broadly neutralizing antibodies by screening the neutralizing properties of HIV-1-infected blood sera; and studying the subtype-specific properties of the recombinant CRF63_02A6 form of HIV-1. The closest analogue (prototype) is a panel of 12 pseudotyped HIV-1 subtype C viruses, which is dominant in South Africa and India [1]. This panel has been characterized using sequencing, phylogenetic analysis, and neutralization assays with monoclonal broadly neutralizing antibodies. The env genes used to generate pseudotyped HIV-1 subtype C viruses represent the genetic and antigenic diversity of HIV-1 among representatives of clade C in South Africa. This panel of 12 pseudotyped HIV-1 subtype C viruses is designed to screen monoclonal broadly neutralizing antibodies, as well as sera from vaccinees in preclinical and clinical trials of developed vaccines against HIV-1 subtype C, which is predominant in South Africa and India. Currently, separate panels of pseudoviruses of the main genetic subtypes of HIV-1 have been created: A, B, C and a number of CRFs [1-4], however, a panel of the recombinant form CRF63_02A6 HIV-1 is missing. It should be noted that in the Russian Federation, the genetic diversity of HIV1 has regional specificities [5]. In the European part of Russia, the HIV1 subtype A6 is dominant [5, 6], while in Siberia, the recombinant form CRF63_02A6 is dominant, which is currently detected in more than 80% of new HIV-1 cases in Siberia [7]. Some data indicate that CRF63_02A6 is more infectious and may have higher replicative activity than other subtypes, which may lead to an increase in the number of people infected with this variant of the virus [8]. Because the recombinant form of HIV-1 CRF63_02A6 continues to actively spread [2] in other regions of Russia, the developed collection of CRF63_02A6 HIV-1 pseudoviruses is an important addition to existing collections worldwide against other HIV-1 subtypes. The use of standardized panels allows for direct comparison of data from various trials and research centers testing drugs specific to different HIV-1 subtypes. The technical result of the claimed invention is the production of a set of expression plasmid vectors p16RU12, p16RU13, p16RU19, p16RU20, p16RU21, p16RU22, p16RU23, p16RU27, p16RU28, p16RU35, p16RU36, p22RUAR13, p22RUAR21, carrying genes encoding thirteen full-length variants of the env gene of the recombinant form CRF63_02A6 HIV-1, amplified by RT-PCR, as well as the production of a set of pseudotyped HIV-1 viruses obtained using the said set of expression plasmid vectors. For each variant of the env gene, one genetic construct with an insert was obtained, used for co-transfection. The specified technical result is achieved by obtaining a set of recombinant plasmid DNA encoding full-length surface glycoproteins of the recombinant form CRF63_02A6 HIV-1, used to obtain pseudotyped HIV-1 viruses, including thirteen plasmid constructs: - the first plasmid construct p16RU12, providing expression of the full-length 16RU12 env gene of the recombinant form CRF63_02A6 HIV-1, encoding the full-length surface glycoprotein Env 16RU12 of the recombinant form CRF63_02A6 HIV-1, in transformed eukaryotic cells HEK293, having the sequence SEQ ID NO: 1 (Fig. 33), a size of 8383 bp, a molecular weight of 5186274 kilodaltons and containing, in accordance with the physical and genetic map (Fig. 1), the following elements: CMV enchancer/promoter, providing efficient expression of the recombinant protein in mammalian cells and having coordinates from 235 to 818 bp; T7 promoter/priming site, providing in vitro transcription/primer landing site for determining the presence/absence of insert by sequencing and having coordinates from 863 to 881 bp; Envelope is the full-length 16RU12 env gene of HIV-1 of the recombinant form CRF63_02A6, having coordinates from 1161 to 3761 bp; V5 tag/epitope, allowing detection of recombinant protein using Anti-V5 or Anti-V5-HRP antibodies and having coordinates from 3880 to 3921 bp; 6 × His is a 6-histidine affinity tag with coordinates from 3931 to 3948 bp. and allowing purification of the recombinant protein on a metal chelate resin such as ProBond™, wherein the C-terminal polyhistidi