Long-acting HIV-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 limited long-acting effect and loss of activity of HIV membrane fusion inhibitors

Active Publication Date: 2014-04-30
FUDAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, taking HIV membrane fusion inhibitors as an example, the application of traditional polypeptide long-acting technologies (such as PEG chemical modification and serum albumin / Fc fusion technology, etc.) needs to introduce more than 10 times the volume of HIV membrane fusion inhibitor polypeptides. 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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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] 1. Construction and expression of ABT clones

[0033] 1.1 Materials PCR reagent: PrimeSTARDNAPolymerase (Takara) 10Xbuffer (Mg+) (Takara) dNTP (Takara)

[0034] Sterilized water: high pressure deionized water

[0035] PCR primers: synthesized by Shanghai Jieli Company

[0036] Plasmid: PHFT plasmid was donated by Beijing Huajin Ruiqing Company, PGEX‐6P‐1MD1.1‐L35‐CP38 plasmid was constructed by our laboratory Restriction enzymes: BamHI, XhoI, Bglll (Takara)

[0037] T4 ligase: purchased from Takara company

[0038] 30% Bis‐Acr polyacrylamide gel: purchased from Bio‐rad Company Ni purification column: purchased from Qiagen Company Escherichia coli HB101 and BL21(DE)3 Competent cells were purchased from Beijing Tiangen Company, other chemical reagents were domestic analysis pure.

[0039] 1.2 Experimental steps

[0040] 1.2.1 ABT clone build

[0041] In order to obtain the long-acting fusion peptide ABT (SEQ ID NO: 4), we used the method of enzymatic splicing PCR, u...

Embodiment 2

[0061] 2. FN‐PAGE detection of the effects of AB and ABT on the formation of the hexahelix

[0062] 2.1 Materials

[0063] Non-denaturing polyacrylamide gel (PAGE) gel electrophoresis kit: purchased from Beijing Tianenze Company

[0064] N36, C34, FAM‐C34 peptides were synthesized by Biosystems433A protein synthesizer

[0065] 2.2 Experimental process

[0066] (1) Prepare 18% separating gel and 5% stacking gel

[0067] (2) Prepare peptides such as N36, F‐C34, ABT, AB, N36+ABT, and N36+AB. The final concentration of each peptide is 40um, placed at 37 degrees for 30 minutes, at room temperature and protected from light, and electrophoresis 2 Hour.

[0068] (3) Fluorchem8800 (UV) detection

[0069] (4) Coomassie brilliant blue stained PAGE gel

[0070] 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 formation of the hexahelix.

Embodiment 3

[0071] Embodiment 3: the virus inhibition test of HIV‐1 laboratory adaptation strain and primary generation virus strain

[0072] 3.1 Experimental materials

[0073] Cells: MT‐2 cells, M7 cells Medium: 1640, 1640+10%FBS

[0074] Culture plate: 96-well flat culture plate (corning,) 96-well round bottom culture plate (corning) Cell lysate: 5% TritonX‐100

[0075] Viruses: laboratory-adapted strains HIV‐1IIIB, HIV‐1Bal virus and various HIV‐1 primary virus strains

[0076] 3.2 Experimental process

[0077] (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).

[0078](2) Thoroughly mix the virus strain thawed at -80 degrees Celsius, and add 100 times the TCID50 value (that is, add 50% of the tissue infection dose to the well)

[0079] (3) Adjust MT‐2 or M7 cells to 1x10 5 1 / ml concentration, add 100 microliters of c...

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Abstract

The invention relates to a polymer for long-acting inhibition of HIV-1 membrane fusion. The polymer for long-acting inhibition of HIV-1 membrane fusion comprises two or three parts. The first part is a HIV-1 membrane fusion-inhibition polypeptide part and the second part is an antibody mimetic part capable of bonding with serum albumin thereby prolonging a half life of the polymer in vivo. A connection molecule can be arranged between the above two parts and improve HIV-1 inhibition activity of the polymer. The polymer can block virus-induced cell fusion in vivo for a long time.

Description

[0001] priority information [0002] This application relies on the application No. 201310264732.7 submitted on June 28, 2013. field of invention [0003] 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 of the invention [0004] 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. [0005] Many inhibitors against HIV...

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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