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A broad-spectrum neutralizing antibody against HIV and its preparation method and application

An antibody and broad-spectrum technology, applied in the fields of cellular immunology and genetic engineering, can solve problems such as the difficulty of producing antibodies and achieve good application prospects

Active Publication Date: 2021-04-13
SUZHOU HEALTH COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This may be the reason why HIV broad-spectrum neutralizing antibodies are difficult to produce

Method used

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  • A broad-spectrum neutralizing antibody against HIV and its preparation method and application
  • A broad-spectrum neutralizing antibody against HIV and its preparation method and application
  • A broad-spectrum neutralizing antibody against HIV and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086]Example 1 Preparation of an anti-HIV antibody

[0087]1, sorting of mature B cells

[0088]Buffer1: 500ml 1 × PBS + 2mL 0.5m EDTA + 25ml 10% BSA (including 2mm EDTA, 0.5% BSA)

[0089](1) Collect by 200 μl of the blood of the human GTL mouse, 200 μl of BUFFER1;

[0090](2) Add 10x volumes of Ack Lysing Buffer (Fisher / Biowhittaker), 10 min, 1500 rpm for 10min, 1500 rpm;

[0091](3) Abandon the discard, add 10ml buffer 1 washing cells, 1500 rpm centrifuge for 10 min;

[0092](4) Abandon the discard, add 10 μl of Buffer 1 resuscitation; add the following flow antibody, labeled mature B cells:

[0093]Anti-CD5 / Fitc (UCHT2, EBISCIENCE)

[0094]Anti-CD19 / PE (SJ25-C1, BD Pharmingen)

[0095]Anti-CD10 / APC (BC96, EBIoscience)

[0096]Anti-CD27 / PE-CY7 (O323, EBIoscience)

[0097]Anti-IgM / PE-CY5 (G20-127, BD Pharmingen)

[0098](5) FACS SORTER sorted mature B cells (CD5-CD19 + CD10-CD27-IgM +), collected a single cell into a 96-well PCR plate, built-in 4 μl of cell lysate;

[0099](6) The cells selected from the s...

Embodiment 2

[0116]Example 2 Detection of antibody characteristics

[0117]1, self-reactive detection

[0118]Antibody self-reactivity adopts clinical standard antinear antibody detection kit (Quanta LiteTMAnaelisa, Inova Diagnostics, Inc.) Perform a negative reaction sample, a low-positive reaction sample, a high-positive reaction sample in the detection kit. 4E10 is an HIV broad spectrum and antibody, also a self-reactive / multi-reactive antibody, and we have prepared 4E10 SCFV-Fc in an experiment for a positive control antibody. The test steps are as follows:

[0119](1) Package: The ELISA board in the ANA kit has been carried out;

[0120](2) Anti-anti - 50 μg / mL SCFV-Fc protein 50 μl, room temperature reaction for 2 h;

[0121](3) Secondary antibody: Abandon the liquid, PBST was washed 3 times, add HRP-labeled sheep anti-human IgG-Fc antibody, and at room temperature 1 h;

[0122](4) Selective: Abandon the liquid, PBST was washed 3 times, add TMB substrate solution, 100 μL per well, and electrically expan...

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Abstract

The invention discloses an anti-HIV broad-spectrum neutralizing antibody, its preparation method and application. Specifically, the present invention relates to the heavy chain variable region CDR-H1, CDR-H2, CDR-H3 of the amino acid sequence shown in SEQ ID NO.1, 2, 3; and as shown in SEQ ID NO.5, 6, 7 Amino acid sequences of light chain variable regions CDR-L1, CDR-L2, CDR-L3 are shown. The present invention also relates to nucleic acids encoding antibodies, vectors comprising the nucleic acids, cells and pharmaceutical compositions comprising the vectors. The present invention also relates to the use of said antibody for detection and treatment of HIV infection and HIV-related diseases.

Description

Technical field[0001]The present invention belongs to the field of cellular immunology, gene engineering, and is involved in a broad spectrum and antibody thereof and a preparation method and application thereof.Background technique[0002]Acquired Immunodeficiency Syndrome (AIDS), which is caused by HUMAN Immunodeficiency Virus Type, HIV infection. HIV-1 mainly has three propagation paths, which are blood dissemination, maternal and child transmission and sexual dissemination. HIV mainly violated the human body's immune system, and the immune function of the infected person will gradually weaken or even lost, which will make various secondary infections to enter into the human body and ultimately lead to death. AIDS is seriously harmful to the health and safety of the national, and many medical researchers in the world have put a lot of human abortion to overcome this major problem.[0003]HIV belongs to the reverse transcription viral department, and is divided into two subtypes: 1 an...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/10C12N15/13A61K39/395A61P31/18
CPCA61K2039/505A61P31/18C07K16/1045C07K2317/565
Inventor 常宏刘松柏
Owner SUZHOU HEALTH COLLEGE
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