WO2023284889A1 - 一种cld蛋白突变体及应用 - Google Patents

一种cld蛋白突变体及应用 Download PDF

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WO2023284889A1
WO2023284889A1 PCT/CN2022/113737 CN2022113737W WO2023284889A1 WO 2023284889 A1 WO2023284889 A1 WO 2023284889A1 CN 2022113737 W CN2022113737 W CN 2022113737W WO 2023284889 A1 WO2023284889 A1 WO 2023284889A1
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cld
protein
hiv
mutant
recombinant
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PCT/CN2022/113737
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French (fr)
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胡勤学
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杜涛
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成都维瑾柏鳌生物医药科技有限公司
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Priority to US17/931,395 priority Critical patent/US20240059745A1/en
Publication of WO2023284889A1 publication Critical patent/WO2023284889A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/15—Retroviridae, e.g. bovine leukaemia virus, feline leukaemia virus human T-cell leukaemia-lymphoma virus
    • C07K14/155—Lentiviridae, e.g. human immunodeficiency virus [HIV], visna-maedi virus or equine infectious anaemia virus
    • C07K14/16—HIV-1 ; HIV-2
    • C07K14/162—HIV-1 ; HIV-2 env, e.g. gp160, gp110/120, gp41, V3, peptid T, CD4-Binding site
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70514—CD4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1774—Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/178—Lectin superfamily, e.g. selectins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/7056—Lectin superfamily, e.g. CD23, CD72
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • This application relates to the field of genetic engineering, more specifically to a CLD protein mutant and its application.
  • HIV-1 Human immunodeficiency virus
  • AIDS acquired immunodeficiency syndrome
  • the target cell types infected by HIV-1 include T cells, macrophages and some types of DC cells, whose common feature is the expression of CD4 molecules and co-receptor molecules on the cell surface.
  • HIV-1 can be divided into R5 and X4 viruses, and some subtypes of HIV also have an intermediate type R5X4 virus that uses CCR5 and CXCR4.
  • the process of HIV-1 infection of target cells is actually the process of viral envelope protein recognition and binding to CD4 and co-receptors. But no matter it is R5, X4 or R5X4 virus, CD4 can be used to complete the infection process. Binding of the HIV-1 envelope protein to CD4 and coreceptors is sufficient for the virus to infect cells. Therefore, targeting the HIV-1 envelope protein CD4 binding site can block HIV-1 infection of cells.
  • DC-SIGN is a lectin recognition protein expressed on the surface of DC cells, which can enrich viruses by binding to polysaccharides on the surface of envelope proteins, and soluble DC-SIGN can inhibit the binding of HIV-1 envelope proteins to DC cells.
  • the present application has improved the fusion recombinant protein by mutating the cysteine at position 60 of the CD4 domain of the recombinant fusion protein to serine, removing the HIS histidine sequence used in prokaryotic expression, and making it available in the eukaryotic system
  • the activity of the obtained recombinant protein CLD mutant has been greatly improved, and it also has a strong broad-spectrum, and it is very promising to become a new generation of anti-HIV-1 drugs .
  • the purpose of this application is to provide a kind of CLD protein mutant, and described CLD protein mutant is SEQ ID NO.4.
  • Another object of the present application is to provide a CLD protein mutant composition.
  • Another object of the present application is to provide a composite immunogen.
  • Another purpose of the present application is to provide the application of CLD protein mutants or compositions or compound immunogens in the preparation of anti-HIV-1 drugs.
  • the last purpose of this application is to provide the application of the compound immunogen in the preparation of anti-HIV-1 medicine.
  • a CLD protein mutant is SEQ ID NO..4, compared with the primary CLD mentioned in CN 102617738A, the applicant mutated the cysteine at position 60 of CD4 to serine.
  • the encoded product can greatly improve the binding efficiency with HIV-1 virus and maintain the stability of the protein.
  • a CLD protein mutant composition said composition is SEQ ID NO.4, and any one, two, three or four combinations of SEQ ID NO.1, 2, 3 and 5.
  • the application of CLD protein mutants in the preparation of anti-HIV-1 drugs is to use any one of the proteins in SEQ ID NO.1 to SEQ ID NO.5, or any combination thereof, as the only main active ingredient, or One of the components used to prepare anti-HIV-1 drugs.
  • a composite immunogen composed of recombinant CLD protein and HIV-1 envelope protein, including any one protein in SEQ ID NO.1 ⁇ SEQ ID NO.5 and "HIV-1gp160, HIV-1gp140 or HIV-1gp120 .
  • the present application also provides a composite immunogen composed of recombinant CLD protein and HIV-1 envelope protein, and the recombinant CLD protein is any protein in SEQ ID NO.6-SEQ ID NO.8.
  • the eukaryotic expression of the recombinant protein CLD mutants increased the inhibitory ability of the tested HIV-1 strains by 2-3 orders of magnitude, and significantly improved the neutralization ability of the virus.
  • -1T/F (transmitter/founder) virus is more prominent, showing a good application prospect.
  • prokaryotically expressed CLD and eukaryotically expressed CLD non-mutant recombinant fusion proteins eukaryotically expressed CLD mutants are more stable in solution and have little change in the ability to inhibit HIV-1.
  • the inhibitory activity of the eukaryotically expressed CLD mutant recombinant fusion protein on some strains is 3-5 times higher than that of the non-mutant.
  • the series of CLD mutant proteins obtained in the present application have good inhibitory effect on HIV.
  • the CLD mutant protein forms a tetrameric form of the bifunctional energy domain, and the DC-SIGN functional domain in the multimerized CLD mutant binds to the envelope protein, increasing the tetramerized CD4
  • the local concentration of the molecule binds to the CD4 binding site on the envelope protein, thereby increasing the neutralization ability of HIV-1.
  • the complexes of a series of CLD mutant proteins obtained in this application and HIV-1 envelope protein, compared with CD4 or DC-SIGN mixed HIV-1 envelope protein or envelope protein alone, can induce the body to produce A stronger immune response targeting the gp120V1V2 epitope produced a weaker immune response targeting the gp120 V3C3 epitope.
  • the primers used in this example are as follows:
  • P1-R TTAAACGGGCCCTCTAGACTCGAGCTACGCAGGAGGGGGGTTTGGGGTG.
  • P2-F ATGGACCGGGCCAAGCTGCTGCTCCTGCTCCTGCTGCTGCTCCTGCCTCTCTGC
  • P3-R GTCCCAAAGGCTTCTTCTTGAGTCACGCGCGATC.
  • pET28a-C25D (CN 102617738A) as a template to carry out PCR with primers P1-F and P1-R, gel recovery after nucleic acid electrophoresis.
  • pCDNA3.1 as a template to carry out PCR with primers P2-F and P2-R, and gel recovery after nucleic acid electrophoresis. Homologous recombination was performed using Novizyme homologous recombination kit.
  • PCR amplification was performed using Takara circular PCR kit.
  • pCDNA-C25NDC60S encodes CD4D1D2 N-terminal 178 aa part, DC-SIGNNECK and CRD part; 25 amino acid linker; CD460 amino acid is mutated from cysteine to serine.
  • pET28a-C30D (CN 102617738A) as a template to carry out PCR with primers P1-F and P1-R, and gel recovery after nucleic acid electrophoresis.
  • Use pCDNA3.1 as a template to carry out PCR with primers P2-F and P2-R, and gel recovery after nucleic acid electrophoresis. Homologous recombination was performed using Novizyme homologous recombination kit.
  • the Takara circular PCR kit was used for PCR amplification.
  • pCDNA-C30NDC60S encodes CD4D1D2 N-terminal 178 aa part and DC-SIGNNECK and CRD part; 30 amino acid linker; CD4 amino acid 60 is mutated from cysteine to serine.
  • pET28a-C35D (CN 102617738A) as a template to carry out PCR with primers P1-F and P1-R, and gel recovery after nucleic acid electrophoresis.
  • Use pCDNA3.1 as a template to carry out PCR with primers P2-F and P2-R, and gel recovery after nucleic acid electrophoresis. Homologous recombination was performed using Novizyme homologous recombination kit.
  • PCR amplification was performed using Takara circular PCR kit.
  • pCDNA-C35NDC60S encodes CD4 D1D2 N-terminal 178 aa part, DC-SIGNNECK and CRD part; 35 amino acid linker; CD460 amino acid is mutated from cysteine to serine.
  • pET28a-C40D (CN 102617738A) as a template to carry out PCR with primers P1-F and P1-R, gel recovery after nucleic acid electrophoresis.
  • pCDNA3.1 as a template to carry out PCR with primers P2-F and P2-R, and gel recovery after nucleic acid electrophoresis. Homologous recombination was performed using Novizyme homologous recombination kit.
  • the Takara circular PCR kit was used for PCR amplification.
  • pCDNA-C40NDC60S encodes CD4 D1D2 N-terminal 178 aa part, DC-SIGNNECK and CRD part; 40 amino acid linker; CD460 amino acid is mutated from cysteine to serine.
  • pET 28a-C45D as a template to carry out PCR with primers P1-F and P1-R, and gel recovery after nucleic acid electrophoresis.
  • Use pCDNA3.1 as a template to carry out PCR with primers P2-F and P2-R, and gel recovery after nucleic acid electrophoresis. Homologous recombination was performed using Novizyme homologous recombination kit.
  • the Takara circular PCR kit was used for PCR amplification.
  • pCDNA-C45NDC60S encodes CD4 D1D2 N-terminal 178 aa part, DC-SIGNNECK and CRD part; 45 amino acid linker; CD4 amino acid 60 is mutated from cysteine to serine.
  • Subculture at a cell density of 600,000-700,000/ml, with a total volume of 30ml.
  • 293F cell density reaches 1.2-1.5 million cells/ml, collect the cells (centrifuge at 1200 rpm for 5min), and resuspend them with 15ml of medium for transfection. dye.
  • the plasmid used for transfection per million cells was 1-1.5 ⁇ g; 750 ⁇ l normal saline + 37.5 ⁇ g plasmid; 750 ⁇ l normal saline + 150 ⁇ l PEI (1 mg/ml). After mixing separately and standing for 5 minutes, mix and gently mix, let stand at room temperature for 10 minutes (less than 20 minutes), then add the plasmid-liposome mixture into the shaker flask, put it into a shaker after mixing (8% carbon dioxide, 37°C, 125rpm). Add 15ml medium after 4-6h. After 4 days, the cell supernatant was collected, then concentrated by ultrafiltration with a 50KD ultrafiltration tube, and finally concentrated about 80 times, added with 10% glycerol, and stored in -80°C for future use.
  • the protein expressed by eukaryotic expression plasmid pCDNA-C25NDC60S is called C25NDC60S (shown in SEQ ID NO.1)
  • the protein expressed by pCDNA-C30NDC60S is called C30NDC60S (shown in SEQ ID NO.2)
  • pCDNA-C35NDC60S expresses
  • the protein expressed by pCDNA-C45NDC60S is called C35NDC60S (shown in SEQ ID NO.3)
  • the protein expressed by pCDNA-C40NDC60S is called C40NDC60S (shown in SEQ ID NO.4)
  • the protein expressed by pCDNA-C45NDC60S is called C45NDC60S (shown in SEQ ID NO.5 ).
  • Rabbit-derived anti-DC-SIGN monoclonal antibody was coated on a 96-well plate with 5 ⁇ g/ml, 50 ⁇ l/well, and left overnight at room temperature;
  • the concentration of the recombinant protein CLD mutant prepared by the above method was 100 ⁇ g/ml.
  • the pCDNA3.1(+) plasmids (Centralized Facility for AIDS Reagents) containing different HIV-1env genes and the pSG3 (Centralized Facility for AIDS Reagents) framework plasmids lacking HIV-1env genes were passed through liposomes (LipofectamineTM 2000, Invitrogen Corporation) 293T cells were co-transfected. After 48 hours of transfection, the virus-containing culture medium supernatant was filtered with a 0.45 ⁇ m filter membrane and added with 10% volume of fetal bovine serum, then packed into 1.5ml centrifuge tubes and stored at -80°C for preparation; luciferase (commercially available, promega company) to determine the virus titer.
  • the different HIV-1env genes contained in the above different pCDNA3.1(+) plasmids are: MSW2, CH811, 700010040.C9.4520, PRB958_06.TB1.4305, WEAUd15.410.787, 62357_14.D3.4589, R EJ O.D 1 2.1 97 2, S C 0 5.8C 1 1.23 4 4, 1 0 5 9_0 9.A 4.1 4 6 0, 6 24 0_0 8.T A 5.46 2 2, 700010058.A4.4375, 1058_11.B11.1550, SC45 .4B5.2631, 62615_03.P4.3964.
  • plasmids containing the full genome of HIV-1 were transfected into 293T cells via liposomes (LipofectamineTM 2000, Invitrogen Corporation). After 48 hours of transfection, the virus-containing medium supernatant was filtered with a 0.45 ⁇ m filter membrane, and 10% volume of fetal bovine serum was added, and then packed into 1.5ml centrifuge tubes and stored at -80°C for preparation; luciferase (commercially available, promega company) to determine the virus titer.
  • T/F strains include THRO.c/2626, CH077.t/2627, CH040.c/2625, pCH058.c/2960, WITO.c/2474, SUMA .c/2821, CH164, CH185, CH198.
  • E. adopt luciferase (commercially available, promega company) to measure the fluorescence value
  • C25ND and C35ND are the CLD recombinant protein of the prokaryotic expression reported in CN 102617738A:
  • the C35NDS60C protein is derived from "Bifunctional CD4–DC-SIGN Fusion Proteins Demonst rate Enhanced Avidity to gp120 and Inhibit HIV-1 Infection and Dissemination".
  • Experimental group recombinant protein CLD and HIV-1 envelope protein (5 ⁇ g) were mixed at a ratio of 3:1 (molar ratio), and incubated at 4°C for 24 hours, a total of 100 ⁇ l;
  • the recombinant protein CLD is any one of claims 1-8 in the CN 102617738A application.
  • Control group 1 a mixture of CD4 and HIV-1 envelope protein (5 ⁇ g) mixed at a ratio of 12:1 (molar ratio), and incubated at 4°C for 24 hours, a total of 100 ⁇ l;
  • Control group 2 a mixture of DC-SIGN and HIV-1 envelope protein (5 ⁇ g) mixed at a ratio of 12:1 (molar ratio), and incubated at 4°C for 24 hours, a total of 100 ⁇ l;
  • the recombinant protein CLD exists in the form of tetramers, and one CLD tetramer contains four CD4s and four DC-SIGNs, so the molar ratio of 12:1 was used for the control.
  • the HIV-1 envelope protein is gp140 protein of HIV-1 CN54.
  • Immune mice subcutaneously inject 100 ⁇ l of the reagents of each group in step 1), immunize 3 times in total, with an interval of 3 weeks between each two immunizations, kill the mice on the 7th day after the last immunization, and collect serum and spleen ;
  • the antibodies above were serially diluted and incubated at 37°C for 1 h. After washing three times with TBST, the cells were incubated with HRP-labeled goat anti-mouse secondary antibody (diluted 1:5000) at 37°C for 1 h. After 5 washes, TMB was added to incubate at room temperature in the dark for 5 min, and then 2M concentrated sulfuric acid was added to terminate the reaction. Finally, the OD value was detected with a microplate reader, with 450 nm as the experimental wavelength and 570 nm as the reference wavelength.
  • mice in the experimental group were taken, and the lymphocytes were separated.
  • BD Biosciences cytokine kit was used to detect the amount of IL-2, IL-4, IL-5, IFN- ⁇ and TNF- ⁇ in the supernatant.
  • the recombinant protein CLD in the CN 102617738A application affects the binding of mAbs (17b, 19b, 447-52D, 39F, b12, F105) to HIV-1gp140.
  • the complex composed of the recombinant protein CLD and the HIV-1 envelope protein compared with the HIV-1 envelope protein mixed with CD4 or DC-SIGN or the envelope protein alone, can induce the body to produce more A strong antibody response targeting the gp120 V1V2 epitope produced a weaker antibody response targeting the gp120 V3C3 epitope.
  • the complex composed of the recombinant protein CLD and the HIV-1 envelope protein compared with the HIV-1 envelope protein mixed with CD4 or DC-SIGN or the envelope protein alone, can induce the body to produce different gp140-specific Th1/Th2 cellular immune response.
  • gp140-specific cells expressing IL-4, IL-5 and IFN- ⁇ were significantly reduced; cells expressing TNF were also reduced, But there is no significant difference.
  • CLD protein mutant and HIV-1 envelope protein (5 ⁇ g) were mixed at a ratio of 3:1 (molar ratio), and incubated at 4°C for 24 hours, a total of 100 ⁇ l;
  • the CLD protein mutant is the protein shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO.5.
  • Control group 1 a mixture of CD4 and HIV-1 envelope protein (5 ⁇ g) mixed at a ratio of 12:1 (molar ratio), and incubated at 4°C for 24 hours, a total of 100 ⁇ l;
  • Control group 2 a mixture of DC-SIGN and HIV-1 envelope protein (5 ⁇ g) mixed at a ratio of 12:1 (molar ratio), and incubated at 4°C for 24 hours, a total of 100 ⁇ l;
  • CLD exists in the form of tetramers, and one CLD tetramer contains four CD4s and four DC-SIGNs, so the molar ratio of 12:1 was used for the control.
  • the HIV-1 envelope protein is gp140 protein of HIV-1 CN54.
  • Immune mice subcutaneously inject 100 ⁇ l of the reagents of each group in step 1), immunize 3 times in total, with an interval of 3 weeks between each two immunizations, kill the mice on the 7th day after the last immunization, and collect serum and spleen ;
  • the above antibodies serially diluted were incubated at 37°C for 1 h. After washing three times with TBST, the cells were incubated with HRP-labeled goat anti-mouse secondary antibody (diluted 1:5000) at 37°C for 1 h. After 5 washes, TMB was added to incubate at room temperature in the dark for 5 min, and then 2M concentrated sulfuric acid was added to terminate the reaction. Finally, the OD value was detected with a microplate reader, with 450 nm as the experimental wavelength and 570 nm as the reference wavelength.
  • mice in the experimental group were taken, and the lymphocytes were separated.
  • BD Biosciences cytokine kit was used to detect the amount of IL-2, IL-4, IL-5, IFN- ⁇ and TNF- ⁇ in the supernatant.
  • the CLD mutants of this application affect mAbs (17b, 19b, 447-52D, 39F, b12, F105) to bind HIV-1gp140 more strongly.
  • the complex composed of CLD protein mutants and HIV-1 envelope protein in this application can induce the body to produce stronger Antibody responses targeting the gp120V1V2 epitope were generated, while antibody responses targeting the gp120 V3C3 epitope were weaker. And compared with the recombinant protein CLD in Example 4, the difference produced is more obvious.
  • the complex composed of CLD mutants and HIV-1 envelope protein in this application can induce the body to produce different gp140 as an immunogen Specific Th1/Th2 cellular immune response.
  • gp140-specific cells expressing IL-4, IL-5, TNF and IFN- ⁇ were significantly reduced.
  • the difference produced is more obvious.

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Abstract

本申请涉及基因工程领域，更具体涉及一种CLD蛋白突变体及应用。所述的蛋白所述的CLD蛋白突变体为SEQ ID NO.4，申请人将原代CLD重组蛋白中的CD4 60位处的半胱氨酸突变为丝氨酸，获得的CLD蛋白突变体对测试的HIV-1毒株抑制能力提高2-3个数量级，显著提高了对病毒的中和能力，这种差异在测试的HIV-1T/F(transmitter/founder)virus中更加显著，CLD蛋白能够大幅提高与HIV-1病毒的结合效率；CLD蛋白突变体或重组CLD蛋白与包膜蛋白形成的复合物可以作为一种免疫原，更好诱导出针对包膜蛋白V1V2区的抗体，表现出良好的应用前景。

Description

一种CLD蛋白突变体及应用
本申请要求于2021年07月12日提交中国专利局，申请号为202110786982.1，申请名称为“一种CLD蛋白突变体及应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本申请涉及基因工程领域，更具体涉及一种CLD蛋白突变体及应用。
背景技术
人类免疫缺陷病毒(HIV-1)是艾滋病(AIDS，获得性免疫缺陷综合征)的病原。由于HIV-1的高度变异及对人类免疫的机制了解的缺乏，目前尚未开发出成功的HIV-1疫苗。对HIV-1感染者的治疗及预防，主要依赖于抗HIV药物。但由于HIV-1高度的变异性，临床药物长期使用必然导致病毒抗、耐药性。开发新型抗病毒药物对HIV-1治疗的可持续性具有迫切性。
HIV-1感染的靶细胞类型包括T细胞、巨噬细胞及部分类型的DC细胞，其共同特征是细胞表面表达CD4分子及辅助受体分子。根据HIV-1在感染细胞过程中使用的是CCR5还是CXCR4辅助受体可将HIV-1分为R5和X4病毒，有些亚型艾滋病毒还存在使用CCR5和CXCR4的中间类型R5X4病毒。HIV-1感染靶标细胞的过程实是病毒包膜蛋白识别、结合CD4和辅助受体的过程。但无论是R5，X4还是R5X4病毒都要利用CD4才能完成感染过程。HIV-1包膜蛋白与CD4及辅助受体的结合足以使病毒感染细胞。因此，以HIV-1包膜蛋白CD4结合位点为靶标可以阻断HIV-1感染细胞。
DC-SIGN是一种表达在DC细胞表面的凝集素识别蛋白，可以通过结合包膜蛋白表面的多糖富集病毒，可溶性DC-SIGN可以抑制HIV-1包膜蛋白与DC细胞的结合。
早期申请人尝试将CD4与DC-SIGN融合后在原核细胞内表达(CN 102617738A)，结果显示设计的重组蛋白CLD具有较好的抗病毒活性，测试的病毒中和能力达到微克水平，但申请人在后期的实验中发现，该重组蛋白对大部分T/F毒株效果不佳。
针对上述问题，本申请对融合重组蛋白进行了改进，将重组融合蛋白CD4结构域的60位的半胱氨酸突变为丝氨酸，去掉了原核表达中使用的HIS组氨酸序列，在真核系统中进行了表达，所获得的重组蛋白CLD突变体相对原核表达的第一代CLD活性得到了极大的提高，也具有很强的广谱性，极有希望成为新一代的抗HIV-1药物。
申请内容
本申请的目的在于提供了一种CLD蛋白突变体，所述的CLD蛋白突变体为SEQ ID NO.4。
本申请的另一个目的在于提供了一种CLD蛋白突变体组合物。
本申请的另一个目的在于提供了复合免疫原。
本申请的还有一个目的在于提供了CLD蛋白突变体或组合物或复合免疫原在制备抗HIV-1药物中的应用。
本申请的最后一个目的在于提供了复合免疫原在制备抗HIV-1药物中的应用。
为了达到上述目的，本申请采取以下技术措施：
一种CLD蛋白突变体，所述的突变体为SEQ ID NO..4，与CN 102617738A中提到的原代 CLD相比，申请人将CD4 60位处的半胱氨酸突变为丝氨酸。编码产物能够大幅提高与HIV-1病毒的结合效率，并且保持蛋白的稳定性。
一种CLD蛋白突变体组合物，所述组合物为SEQ ID NO.4，以及SEQ ID NO.1、2、3和5中的任意一个、两个、三个或四个的组合。CLD蛋白突变体在制备抗HIV-1药物中的应用，是利用SEQ ID NO.1～SEQ ID NO.5中的任一一个蛋白，或其任何组合以唯一主效成分，或是主效成分之一，用以制备抗HIV-1的药物。
一种重组CLD蛋白与HIV-1包膜蛋白组成的复合免疫原，包括SEQ ID NO.1～SEQ ID NO.5中的任一一个蛋白与“HIV-1gp160、HIV-1gp140或HIV-1gp120。
本申请还提供重组CLD蛋白与HIV-1包膜蛋白组成的复合免疫原，所述重组CLD蛋白为SEQ ID NO.6～SEQ ID NO.8中的任意一个蛋白。
本申请的保护范围还包括，上述复合免疫原在制备抗HIV-1药物中的应用。
本申请与现有技术相比，具有以下优点和效果：
与原核重组CLD相比，真核表达的重组蛋白CLD突变体对测试的HIV-1毒株抑制能力提高2-3个数量级，显著提高了对病毒的中和能力，这种差异在测试的HIV-1T/F(transmitter/founder)virus中更加显著，表现了良好的应用前景。相比原核表达的CLD及真核表达的CLD非突变体重组融合蛋白，真核表达的CLD突变体在溶液中更加稳定，抑制HIV-1的能力变化小。在抗HIV-1感染实验中，真核表达的CLD突变体重组融合蛋白对部分毒株的抑制活性相对于于非突变体提高3-5倍。
本申请所得的系列CLD突变蛋白对HIV具有良好的抑制作用。CLD突变体蛋白在溶液状态下，形成双功能能结构域的四聚体形式，多聚化CLD突变体中的DC-SIGN功能结构域与包膜蛋白结合后，增加了四聚体化的CD4分子与包膜蛋白上CD4结合位点的局部浓度，从而提高对HIV-1中和能力。
本申请所得的一列CLD突变蛋白与HIV-1包膜蛋白组成的复合物，与CD4或DC-SIGN混合的HIV-1包膜蛋白或单独的包膜蛋白相比，作为免疫原可以诱导机体产生更强的靶向gp120V1V2表位的免疫反应，而产生的靶向gp120 V3C3表位的免疫反应则更弱。
具体实施方式
本申请所述技术方案，如未特别说明，均为本领域的常规方案；所述试剂或材料，如未特别说明，均来源于商业渠道。
实施例1：
真核表达载体pCDNA-C25NDC60S、pCDNA-C30NDC60S、pCDNA-C35NDC60S、pCDNA-C40NDC60S和pCDNA-C45NDC60S的构建
本实施例所用到的引物如下：
P1-F:GAATTCCCTGCTGCTGCTCCTGCCTCAGGCCCAGGCTGTGAAGAAAGTGGTG
CTGGGCAAAAA AGGGGATACAGTGGAACTGACCTGTA；
P1-R:TTAAACGGGCCCTCTAGACTCGAGCTACGCAGGAGGGGGGTTTGGGGTG。
P2-F:ATGGACCGGGCCAAGCTGCTGCTCCTGCTCCTGCTGCTGCTCCTGCCTCTGC
AGATATCCAGC ACAGTGG；
P2-R:GAGGCAGGAGCAGCAGCAGGAGCAGGAGCAGCAGCTTGGCCCGGTCCATG
AATTCCACCACACTGGACTAGTGG。
P3-F:GATCGCGCTGACTCAAGAAGAAGCCTTTGGGAC；
P3-R:GTCCCAAAGGCTTCTTCTTGAGTCAGCGCGATC。
(1)pCDNA-C25NDC60S的构建：
以pET28a-C25D(CN 102617738A)为模板用引物P1-F和P1-R进行PCR，核酸电泳后胶回收。以pCDNA3.1为模板用引物P2-F和P2-R进行PCR，核酸电泳后胶回收。用诺唯赞公司的同源重组试剂盒进行同源重组。取15μl重组体系加入到100μl大肠杆菌DH5α感受态，混匀，42℃热激转化，加入800μl LB培养液，37℃振荡培养1h，将菌液涂布在卡那霉素抗性的LB培养基上，37℃培养过夜，从转化的平板上挑取6个菌落接种至5ml含卡那霉素的LB，37℃振荡培养过夜，用质粒提取试剂盒提取质粒并测序验证，构建成功的命名为pCDNA-C25ND。
以构建好的pCDNA-C25ND为模板，P3-F、P3-R为引物对，使用Takara环状PCR试剂盒进行PCR扩增。每50μl PCR体系加入2μl的dpnI于37℃消化2小时，取15μl加入到100μl大肠杆菌DH5α感受态，混匀，42℃热激转化，加入800μl LB培养液，37℃振荡培养1h，将菌液涂布在卡那霉素抗性的LB培养基上，37℃培养过夜，从转化的平板上挑取6个菌落接种至5ml含卡那霉素的LB，37℃振荡培养过夜，用质粒提取试剂盒提取质粒并测序验证，构建成功则被命名为pCDNA-C25NDC60S。pCDNA-C25NDC60S编码包含CD4D1D2 N端178 aa部分和DC-SIGNNECK以及CRD部分；25个氨基酸的linker；CD460位氨基酸由半胱氨酸突变为丝氨酸。
(2)pCDNA-C30NDC60S的构建：
以pET28a-C30D(CN 102617738A)为模板用引物P1-F和P1-R进行PCR，核酸电泳后胶回收。以pCDNA3.1为模板用引物P2-F和P2-R进行PCR，核酸电泳后胶回收。用诺唯赞公司的同源重组试剂盒进行同源重组。取15μl重组体系加入到100μl大肠杆菌DH5α感受态，混匀，42℃热激转化，加入800μl LB培养液，37℃振荡培养1h，将菌液涂布在卡那霉素抗性的LB培养基上，37℃培养过夜，从转化的平板上挑取6个菌落接种至5ml含卡那霉素的LB，37℃振荡培养过夜，用质粒提取试剂盒提取质粒并测序验证，构建成功的命名为pCDNA-C30ND。
以构建好的pCDNA-C30ND为模板，P3-F、P3-R为引物对，使用Takara环状PCR试剂盒进行PCR扩增。每50μl PCR体系加入2μl的dpnI于37℃消化2小时，取15μl加入到100μl大肠杆菌DH5α感受态，混匀，42℃热激转化，加入800μl LB培养液，37℃振荡培养1h，将菌液涂布在卡那霉素抗性的LB培养基上，37℃培养过夜，从转化的平板上挑取6个菌落接种至5ml含卡那霉素的LB，37℃振荡培养过夜，用质粒提取试剂盒提取质粒并测序验证，构建成功则被命名为pCDNA-C30NDC60S。pCDNA-C30NDC60S编码包含CD4D1D2 N端178 aa部分和DC-SIGNNECK以及CRD部分；30个氨基酸的linker；CD4 60位氨基酸由半胱氨酸突变为丝氨酸。
(3)pCDNA-C35NDC60S的构建：
以pET28a-C35D(CN 102617738A)为模板用引物P1-F和P1-R进行PCR，核酸电泳后胶回收。以pCDNA3.1为模板用引物P2-F和P2-R进行PCR，核酸电泳后胶回收。用诺唯赞公司的同源重组试剂盒进行同源重组。取15μl重组体系加入到100μl大肠杆菌DH5α感受态，混匀，42℃热激转化，加入800μl LB培养液，37℃振荡培养1h，将菌液涂布在卡那霉素抗性的LB培养基上，37℃培养过夜，从转化的平板上挑取6个菌落接种至5ml含卡那霉素的LB，37℃振荡培养过夜，用质粒提取试剂盒提取质粒并测序验证，构建成功的命名为pCDNA-C35ND。
以构建好的pCDNA-C35ND为模板，P3-F、P3-R为引物对，使用Takara环状PCR试剂盒进行PCR扩增。每50μl PCR体系加入2μl的dpnI于37℃消化2小时，取15μl加入到100μl大肠杆菌DH5α感受态，混匀，42℃热激转化，加入800μl LB培养液，37℃振荡培养1h，将菌液涂布在卡那霉素抗性的LB培养基上，37℃培养过夜，从转化的平板上挑取6个菌落接种至5ml 含卡那霉素的LB，37℃振荡培养过夜，用质粒提取试剂盒提取质粒并测序验证，构建成功则被命名为pCDNA-C35NDC60S。pCDNA-C35NDC60S编码包含CD4 D1D2 N端178 aa部分和DC-SIGNNECK以及CRD部分；35个氨基酸的linker；CD460位氨基酸由半胱氨酸突变为丝氨酸。
(4)pCDNA-C40NDC60S的构建：
以pET28a-C40D(CN 102617738A)为模板用引物P1-F和P1-R进行PCR，核酸电泳后胶回收。以pCDNA3.1为模板用引物P2-F和P2-R进行PCR，核酸电泳后胶回收。用诺唯赞公司的同源重组试剂盒进行同源重组。取15μl重组体系加入到100μl大肠杆菌DH5α感受态，混匀，42℃热激转化，加入800μl LB培养液，37℃振荡培养1h，将菌液涂布在卡那霉素抗性的LB培养基上，37℃培养过夜，从转化的平板上挑取6个菌落接种至5ml含卡那霉素的LB，37℃振荡培养过夜，用质粒提取试剂盒提取质粒并测序验证，构建成功的命名为pCDNA-C40ND。
以构建好的pCDNA-C40ND为模板，P3-F、P3-R为引物对，使用Takara环状PCR试剂盒进行PCR扩增。每50μl PCR体系加入2μl的dpnI于37℃消化2小时，取15μl加入到100μl大肠杆菌DH5α感受态，混匀，42℃热激转化，加入800μl LB培养液，37℃振荡培养1h，将菌液涂布在卡那霉素抗性的LB培养基上，37℃培养过夜，从转化的平板上挑取6个菌落接种至5ml含卡那霉素的LB，37℃振荡培养过夜，用质粒提取试剂盒提取质粒并测序验证，构建成功则被命名为pCDNA-C40NDC60S。pCDNA-C40NDC60S编码包含CD4 D1D2 N端178 aa部分和DC-SIGNNECK以及CRD部分；40个氨基酸的linker；CD460位氨基酸由半胱氨酸突变为丝氨酸。
(5)pCDNA-C45NDC60S的构建：
以pET 28a-C45D为模板用引物P1-F和P1-R进行PCR，核酸电泳后胶回收。以pCDNA3.1为模板用引物P2-F和P2-R进行PCR，核酸电泳后胶回收。用诺唯赞公司的同源重组试剂盒进行同源重组。取15μl重组体系加入到100μl大肠杆菌DH5α感受态，混匀，42℃热激转化，加入800μl LB培养液，37℃振荡培养1h，将菌液涂布在卡那霉素抗性的LB培养基上，37℃培养过夜，从转化的平板上挑取6个菌落接种至5ml含卡那霉素的LB，37℃振荡培养过夜，用质粒提取试剂盒提取质粒并测序验证，构建成功的命名为pCDNA-C45ND。
以构建好的pCDNA-C45ND为模板，P3-F、P3-R为引物对，使用Takara环状PCR试剂盒进行PCR扩增。每50μl PCR体系加入2μl的dpnI于37℃消化2小时，取15μl加入到100μl大肠杆菌DH5α感受态，混匀，42℃热激转化，加入800μl LB培养液，37℃振荡培养1h，将菌液涂布在卡那霉素抗性的LB培养基上，37℃培养过夜，从转化的平板上挑取6个菌落接种至5ml含卡那霉素的LB，37℃振荡培养过夜，用质粒提取试剂盒提取质粒并测序验证，构建成功则被命名为pCDNA-C45NDC60S。pCDNA-C45NDC60S编码包含CD4 D1D2 N端178 aa部分和DC-SIGNNECK以及CRD部分；45个氨基酸的linker；CD4 60位氨基酸由半胱氨酸突变为丝氨酸。
实施例2：
实施例1制备的各真核表达载体的重组蛋白CLD突变体表达：
1.细胞培养
以60～70万/ml的细胞密度进行传代，总体积30ml，当293F细胞密度达到1.2～1.5百万个细胞/ml，收集细胞(1200转离心5min)，并用15ml培养基重悬用于转染。
2.转染：
每一百万个细胞的转染所用质粒为1～1.5μg；750μl生理盐水+37.5μg质粒；750μl生理盐水+150μl PEI(1mg/ml)。分别混匀后静置5min后，混合并温和的混匀，室温静置10min(低 于20min)，然后将质粒-脂质体混合液加入摇瓶中，混匀后放入摇床(8％二氧化碳，37℃，125rpm)。4～6h后补加15ml培养基。4天后收集细胞上清，然后用50KD超滤管超滤浓缩，最终浓缩约80倍，加入10％甘油，分装存于-80℃备用。
在本申请中真核表达质粒pCDNA-C25NDC60S表达的蛋白称为C25NDC60S(SEQ ID NO.1所示)，pCDNA-C30NDC60S表达的蛋白称为C30NDC60S(SEQ ID NO.2所示)，pCDNA-C35NDC60S表达的蛋白称为C35NDC60S(SEQ ID NO.3所示)，pCDNA-C40NDC60S表达的蛋白称为C40NDC60S(SEQ ID NO.4所示)，pCDNA-C45NDC60S表达的蛋白称为C45NDC60S(SEQ IDNO.5所示)。
3.ELISA检测重组蛋白CLD突变体的浓度
(1)兔源抗DC-SIGN单克隆抗体以5μg/ml包被96孔板，50μl/孔，室温过夜；
洗板机洗脱5次，加入含1％BSA的PBS封闭液封，200μl/孔，于37℃封闭一小时；
(2)洗板机洗脱5次，孵育标准品(原核表达的CLD)/重组蛋白样品，50μl/孔，37℃孵育一小时，标准品用封闭液稀释；
(3)洗板机洗脱5次，加入课题组免疫小鼠所得的抗CLD血清，用封闭液1：1000稀释，50μl/孔，37℃孵育一小时；
(4)洗板机洗脱5次，加入HRP标记的羊抗鼠IgG的二抗，用封闭液1：10000稀释，50μl/孔，37℃孵育一小时；
(5)洗板机洗脱5次，加入提前放置室温的TMB底物，50μl/孔，避光室温孵育5分钟；
(6)终止反应：加入2M H 2SO 4溶液，50μl/孔，使用酶标仪进行读数；
(7)绘制标准曲线，计算重组蛋白CLD突变体浓度。
利用上述方法制备的重组蛋白CLD突变体浓度为100μg/ml。
实施例3：
CLD重组蛋白及重组蛋白CLD突变体在制备治疗或预防HIV-1病毒的药物中的应用：
1)HIV-1假病毒的制备：
含有不同HIV-1env基因的pCDNA3.1(+)质粒(Centralized Facility for AIDS Reag ents)与缺失HIV-1env基因的pSG3(Centralized Facility for AIDS Reagents)框架质粒经脂质体(LipofectamineTM 2000，Invitrogen Corporation)共转染293T细胞。转染48h后，含病毒的培养基上清用0.45μm滤膜过滤后加入10％体积的胎牛血清，分装到1.5ml离心管并于-80℃保存备；采用luciferase(市售，promega公司)测定病毒滴度。
上述不同的的pCDNA3.1(+)质粒含有的不同HIV-1env基因为：MSW2、CH811、700010040.C9.4520、PRB958_06.TB1.4305、WEAUd15.410.787、62357_14.D3.4589、R EJ O.D 1 2.1 97 2、S C 0 5.8C 1 1.23 4 4、1 0 5 9_0 9.A 4.1 4 6 0、6 24 0_0 8.T A 5.46 2 2、700010058.A4.4375、1058_11.B11.1550、SC45.4B5.2631、62615_03.P4.3964。
2)HIV-1真病毒的制备：
包含HIV-1全基因组的不同质粒经由脂质体(LipofectamineTM 2000，Invitrogen Corpor ation)转染293T细胞。转染48h后，含病毒的培养基上清用0.45μm滤膜过滤后加入10％体积的胎牛血清，分装到1.5ml离心管并保存于-80℃保存备；采用luciferase(市售，promega公司)测定病毒滴度。
上述不同质粒为实验室适应株NL4-3和BaL；T/F株包含THRO.c/2626、CH077.t/2627、CH040.c/2625、pCH058.c/2960、WITO.c/2474、SUMA.c/2821、CH164、CH185、CH198。
3)各重组蛋白抑制HIV-1感染TZM-bl细胞系：
A.将各重组蛋白溶液稀释至1μM，并以此为起始浓度，以3为稀释系数向下稀释11个梯度，最后加上一个不含重组蛋白的培养基作为对照；
B.将病毒稀释到200TCID50；
C.60μl稀释好的病毒与60μl各重组蛋白的稀释液混合，37℃孵育1小时；
D.取100μl病毒-重组蛋白混合液加入到提前铺至96孔板的TZM-bl细胞中，随后加入100μl含DEAE(40μg/ml)的完全培养基，于二氧化碳培养箱中培养48小时；
E.采用luciferase(市售，promega公司)测定荧光值；
F.计算抑制率，以不加CLD孔的读数作为0％抑制率。
结果如下表所示：
C25ND和C35ND为CN 102617738A中报道的原核表达的CLD重组蛋白：
所述的C35NDS60C蛋白来源于《Bifunctional CD4–DC-SIGN Fusion Proteins Demonst rate Enhanced Avidity to gp120 and Inhibit HIV-1 Infection and Dissemination》。
Figure PCTCN2022113737-appb-000001
Figure PCTCN2022113737-appb-000002
上述表格的空白表明并未做该项数据。
结果表明CLD蛋白突变体与对各类型HIV-1病毒抑制效果均比CN 102617738A中报道的原核表达的CLD重组蛋白要好。
实施例4：
CN 102617738A申请中的8个重组蛋白CLD任一一个与HIV-1包膜蛋白制备混合免疫原在制备预防HIV-1病毒药物中的应用；
1)重组蛋白CLD、CD4和DC-SIGN与HIV-1包膜蛋白混合免疫原的制备：
实验组：重组蛋白CLD与HIV-1包膜蛋白(5μg)按照3：1(摩尔比)的比例混合，并于4℃孵育24小时，一共100μl；
所述的重组蛋白CLD为CN 102617738A申请中权利要求1-8中的任一蛋白。
对照组1：CD4与HIV-1包膜蛋白(5μg)按照12：1(摩尔比)的比例进行混合的混合物，并于4℃孵育24小时，一共100μl；
对照组2：DC-SIGN与HIV-1包膜蛋白(5μg)按照12：1(摩尔比)的比例进行混合的混合物，并于4℃孵育24小时，一共100μl；
重组蛋白CLD是以四聚体形式存在的，一个CLD四聚体含有四个CD4和四个DC-SIGN，所以对照使用12：1的摩尔比。
所述的HIV-1包膜蛋白为HIV-1CN54的gp140蛋白。
2)免疫小鼠：皮下注射100μl步骤1)的各组别试剂，共免疫3次，每两次免疫之间间隔3周，最后一次免疫后的第7天处死小鼠，并收集血清和脾脏；
3)实验方法
A.为了探究CD4结合gp140能否影响其构象改变并暴露CD4i表位，本次实验我们选用了17b(CD4i)、19b(CD4i)、447-52D(V3)、39F(V3)、12b(CD4BS)和F105 6个识别gp120不同靶位的单克隆抗体进行ELISA实验。用gp140或者gp140与重组蛋白CLD混合物包被96孔 板(0.25μg/孔)在室温下过夜，TBST洗涤三次，接着用含1％BSA的TBST在37℃封闭1h。连续梯度稀释的上述抗体在37℃孵育1h。TBST三次洗涤后用HRP标记的山羊抗小鼠二抗(1:5000稀释)在37℃孵育1h。经过5次洗涤后，加入TMB室温避光孵育5min，然后加入2M浓硫酸终止反应。最后用酶标仪检测OD值，以450nm为实验波长，以570nm为参比波长。
B.为了探究ELISA检测血清中gp140特异性抗体滴度。用gp140或gp140与sCD4混合物包被96孔板(0.25μg/孔)在室温下过夜，TBST洗涤三次，接着用含1％BSA的TBST在37℃封闭1h。连续梯度稀释的样本在37C孵育1h。TBST三次洗涤后，用,HRP标记的山羊抗小鼠二抗(1:5000稀释)在37℃孵育1h。经过5次洗涤后,加入TMB室温避光孵育5min，然后加入2M浓硫酸终止反应。最后用酶标仪检测OD值，以450nm为实验波长，以570nm为参比波长。
C.细胞因子检测
取实验组小鼠脾脏，分离淋巴细胞。以每孔3×107细胞数铺到24孔板中，用gp140(20μg/孔)或者CLD-gp140(35μg/孔)刺激，5天后收上清，用0.22um滤器过滤，分装后存-80℃备用。用BD Biosciences细胞因子试剂盒检测上清中IL-2,IL-4,IL-5,IFN-γ和TNF-α的量。
4)实验结果
CN 102617738A申请中重组蛋白CLD影响mAbs(17b,19b,447-52D,39F,b12,F105)结合HIV-1gp140。
CN 102617738A申请中重组蛋白CLD与HIV-1包膜蛋白组成的复合物，与CD4或DC-SIGN混合的HIV-1包膜蛋白或单独的包膜蛋白相比，作为免疫原可以诱导机体产生更强的靶向gp120 V1V2表位的抗体反应，而产生的靶向gp120 V3C3表位的抗体反应则更弱。
CN 102617738A申请中重组蛋白CLD与HIV-1包膜蛋白组成的复合物，与CD4或DC-SIGN混合的HIV-1包膜蛋白或单独的包膜蛋白相比，作为免疫原可以诱导机体产生不同的gp140特异性的Th1/Th2细胞免疫反应。CLD突变体与HIV-1包膜蛋白组成的复合物免疫的小鼠脾脏细胞中，gp140特异性的表达IL-4、IL-5和IFN-γ的细胞显著降低；表达TNF的细胞也有降低，但是没有显著性差异。
实施例5：
CLD蛋白突变体与HIV-1包膜蛋白混合免疫原在预防HIV-1病毒中的应用：
1)CLD蛋白突变体、CD4和DC-SIGN与HIV-1包膜蛋白混合免疫原的制备：
实验组：CLD蛋白突变体与HIV-1包膜蛋白(5μg)按照3：1(摩尔比)的比例混合，并于4℃孵育24小时，一共100μl；
所述的CLD蛋白突变体为SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4或SEQ ID NO.5所示蛋白。
对照组1：CD4与HIV-1包膜蛋白(5μg)按照12：1(摩尔比)的比例进行混合的混合物，并于4℃孵育24小时，一共100μl；
对照组2：DC-SIGN与HIV-1包膜蛋白(5μg)按照12：1(摩尔比)的比例进行混合的混合物，并于4℃孵育24小时，一共100μl；
CLD是以四聚体形式存在的，一个CLD四聚体含有四个CD4和四个DC-SIGN，所以对照使用12：1的摩尔比。
所述的HIV-1包膜蛋白为HIV-1CN54的gp140蛋白。
2)免疫小鼠：皮下注射100μl步骤1)的各组别试剂，共免疫3次，每两次免疫之间间隔3 周，最后一次免疫后的第7天处死小鼠，并收集血清和脾脏；
3)实验方法
A.为了探究CD4结合gp140能否影响其构象改变并暴露CD4i表位，本次实验我们选用了17b(CD4i)、19b(CD4i)、447-52D(V3)、39F(V3)、12b(CD4BS)和F105等6个识别gp120不同靶位的单克隆抗体进行ELISA实验。用gp140或者gp140与重组蛋白CLD混合物包被96孔板(0.25μg/孔)在室温下过夜，TBST洗涤三次，接着用含1％BSA的TBST在37℃封闭1h。连续梯度稀释的上述抗体在37℃孵育1h。TBST三次洗涤后用HRP标记的山羊抗小鼠二抗(1:5000稀释)在37℃孵育1h。经过5次洗涤后，加入TMB室温避光孵育5min，然后加入2M浓硫酸终止反应。最后用酶标仪检测OD值，以450nm为实验波长，以570nm为参比波长。
B.为了探究ELISA检测血清中gp140特异性抗体滴度。用gp140或gp140与sCD4混合物包被96孔板(0.25μg/孔)在室温下过夜，TBST洗涤三次，接着用含1％BSA的TBST在37℃封闭1h。连续梯度稀释的样本在37C孵育1h。TBST三次洗涤后，用HRP标记的山羊抗小鼠二抗(1:5000稀释)在37℃孵育1h。经过5次洗涤后，加入TMB室温避光孵育5min，然后加入2M浓硫酸终止反应。最后用酶标仪检测OD值，以450nm为实验波长，以570nm为参比波长。
C.细胞因子检测
取实验组小鼠脾脏，分离淋巴细胞。以每孔3×10 7细胞数铺到24孔板中，用gp140(20μg/孔)或者CLD-gp140(35μg/孔)刺激，5天后收上清，用0.22um滤器过滤，分装后存-80℃备用。用BD Biosciences细胞因子试剂盒检测上清中IL-2,IL-4,IL-5,IFN-γ和TNF-α的量。
4)实验结果
相比实施例4中重组蛋白CLD，本申请CLD突变体影响mAbs(17b,19b,447-52D,39F,b12,F105)结合HIV-1gp140更强。
本申请CLD蛋白突变体与HIV-1包膜蛋白组成的复合物，与CD4或DC-SIGN混合的HIV-1包膜蛋白或单独的包膜蛋白相比，作为免疫原可以诱导机体产生更强的靶向gp120V1V2表位的抗体反应，而产生的靶向gp120 V3C3表位的抗体反应则更弱。而且相较于实施例4中重组蛋白CLD，产生的差异更明显。
本申请CLD突变体与HIV-1包膜蛋白组成的复合物，与CD4或DC-SIGN混合的HIV-1包膜蛋白或单独的包膜蛋白相比，作为免疫原可以诱导机体产生不同的gp140特异性的Th1/Th2细胞免疫反应。CLD突变体与HIV-1包膜蛋白组成的复合物免疫的小鼠脾脏细胞中，gp140特异性的表达IL-4、IL-5、TNF和IFN-γ的细胞显著降低。而且相较于实施例4中重组蛋CLD，产生的差异更明显。

Claims (6)

  1. 一种CLD蛋白突变体，其特征在于，所述的CLD蛋白突变体为SEQ ID NO.4。
  2. 一种CLD蛋白突变体组合物，其特征在于，所述组合物为SEQ ID NO.4，以及SEQ ID NO.1、2、3和5中的任意一个、两个、三个或四个的组合。
  3. 一种复合免疫原，其特征在于，包括SEQ ID NO.1～SEQ ID NO.5中的任一一个蛋白与“HIV-1 gp160、HIV-1 gp140或HIV-1 gp120。
  4. 权利要求1所述的CLD蛋白突变体或权利要求2所述的组合物或权利要求3所述的复合免疫原在制备抗HIV-1药物中的应用。
  5. 重组CLD蛋白与HIV-1包膜蛋白组成的复合免疫原，其特征在于，所述重组CLD蛋白为SEQ ID NO.6～SEQ ID NO.8中的任意一个蛋白。
  6. 权利要求5所述的复合免疫原在制备抗HIV-1药物中的应用。
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