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Long-acting HIV-1 (Human Immunodeficiency Virus-1) membrane fusion inhibitor

A single, high-molecular technology, applied in the field of long-acting membrane fusion polypeptide drugs, can solve the problems of loss of activity of HIV membrane fusion inhibitors and limited long-acting effect

Inactive Publication Date: 2013-10-02
FUDAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, taking HIV membrane fusion inhibitors as an example, applying traditional polypeptide long-acting technologies (such as PEG chemical modification and serum albumin / Fc fusion technology, etc.), it is necessary to introduce peptides that are 10 times larger than HIV membrane fusion inhibitors. Unrelated groups or protein molecules will lead to the loss of the activity of HIV membrane fusion inhibitors, and the long-acting effect is still limited

Method used

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  • Long-acting HIV-1 (Human Immunodeficiency Virus-1) membrane fusion inhibitor
  • Long-acting HIV-1 (Human Immunodeficiency Virus-1) membrane fusion inhibitor
  • Long-acting HIV-1 (Human Immunodeficiency Virus-1) membrane fusion inhibitor

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1: Construction and expression of ABT clone

[0029] 1.1 Materials

[0030] PCR reagents: PrimeSTAR DNA Polymerase (Takara), 10X buffer (Mg+) (Takara), dNTP (Takara).

[0031] Sterilized water: high pressure deionized water.

[0032] PCR primers: synthesized by Shanghai Jieli Company.

[0033] Plasmid: PHFT plasmid was donated by Beijing Huajin Ruiqing Company, and PGEX‐6P‐1MD1.1‐L35‐CP38 plasmid was constructed by our laboratory.

[0034] Restriction enzymes: BamHI, XhoI, BglII (Takara)

[0035] T4 ligase: purchased from Takara Company.

[0036] 30% Bis-Acr polyacrylamide gel: purchased from Bio-rad.

[0037] Ni purification column: purchased from Qiagen.

[0038] Competent Escherichia coli HB101 and BL21(DE)3 were purchased from Beijing Tiangen Company, and other chemical reagents were domestic analytical grade.

[0039] 1.2 Experimental steps

[0040] 1.2.1 ABT clone construction

[0041] In order to obtain the long-acting fusion peptide ABT (SEQ ...

Embodiment 2

[0059] Embodiment 2: FN‐PAGE detects the influence of AB and ABT on the formation of six helices

[0060] 2.1 Materials

[0061] Non-denaturing polyacrylamide gel (PAGE) gel electrophoresis kit: purchased from Beijing Tianenze Company.

[0062] N36, C34, and FAM-C34 polypeptides were synthesized by Biosystems 433A protein synthesizer.

[0063] 2.2 Experimental process

[0064] (1) Prepare 18% separating gel and 5% stacking gel.

[0065] (2) Prepare peptides such as N36, F‐C34, ABT, AB, N36+ABT, and N36+AB. The final concentration of each peptide is 40 uM, place at 37 degrees for 30 minutes, and avoid light at room temperature. Under the condition of voltage 125V, electrophoresis 2 hours.

[0066] (3) Fluorchem8800 (ultraviolet) detection.

[0067] (4) Coomassie brilliant blue stained PAGE gel.

[0068] This experiment ( figure 2 ) shows that AB does not compete with C34 or N36 to affect the formation of the six helix. Only ABT competes with C34 or N36 to affect the fo...

Embodiment 3

[0069] Embodiment 3: HIV‐1 laboratory-adapted strain and the virus inhibition test of primary virus strain

[0070] 3.1 Experimental materials

[0071] Cells: MT‐2 cells, M7 cells Medium: 1640, 1640+10% FBS.

[0072] Culture plate: 96-well flat culture plate (corning), 96-well round bottom culture plate (corning).

[0073] Cell lysate: 5% TritonX‐100.

[0074] Viruses: laboratory-adapted strains HIV‐1 IIIB, HIV‐1 Bal virus and various HIV‐1 primary virus strains.

[0075] 3.2 Experimental process

[0076] (1) Doubly dilute ABT, AB, CP38 and T20 polypeptide proteins in a 96-well plate, and set positive control wells (no polypeptide protein wells) and negative control wells (cell control wells and virus control wells).

[0077](2) Thoroughly mix the virus strains thawed at -80 degrees Celsius, and add 100 times the TCID50 value (that is, add 50% of the tissue infection dose to the well).

[0078] (3) Adjust the concentration of MT‐2 or M7 cells to 1x105 cells / ml, and add 10...

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PUM

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Abstract

The invention relates to long-acting inhibiting polymer for HIV-1 (Human Immunodeficiency Virus-1) membrane fusion. The long-acting inhibiting polymer is characterized by comprising two or three parts, wherein the former two parts are respectively a polypeptide part for inhibiting the HIV-1 membrane fusion and a simulative antibody part which can be combined with serum albumin so as to prolong the half-life period of the polymer in vivo; the middle of the two parts can also be additionally provided with a connecting molecule for improving the activity of the polymer in inhibiting HIV-1. The polymer can be used for preventing a cell fusion effect mediated by virus in vivo for a long term.

Description

technical field [0001] The invention relates to the field of human immunodeficiency virus inhibitors, in particular to long-acting membrane fusion polypeptide drugs for treating HIV infection and a design method thereof. Background technique [0002] The AIDS syndrome caused by HIV virus infection is one of the unsolved problems in the world. At present, there is a lack of effective drugs and vaccines to combat HIV infection. So far, the drugs used clinically to treat HIV infection are mainly divided into four categories, namely reverse transcriptase inhibitors, protease inhibitors, integrase inhibitors and HIV membrane fusion inhibitors. These drugs interfere with or block different steps in the viral replication process. HIV membrane fusion inhibitors are the newest of the above four drug classes. [0003] Many inhibitors against HIV are peptides or proteins. Peptide or protein drugs all face the problem of being degraded after taking them. Some traditional peptide pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70A61K38/16A61K47/48A61K48/00A61P31/18
CPCA61K38/16A61K47/64A61P31/18C07K14/005C07K2319/00C07K2319/31C12N2740/16122
Inventor 姜世勃陆路徐巍黄金王瑞
Owner FUDAN UNIV
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