A VEGF antibody, reorganization AAV virus and its application

A technology of viruses and antibodies, applied in the field of biomedicine, can solve problems such as rarely used antibody structures, and achieve high proliferation effects

Active Publication Date: 2022-04-29
SHANDONG XINRUI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In the prior art, the adeno-associated virus is mainly modified to make it more beneficial to the treatment of AMD, and the amino acid sequence of the VEGF antibody is also partially optimized, but the Fab segment of VEGF is mainly expressed, and the entire antibody is rarely used structure

Method used

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  • A VEGF antibody, reorganization AAV virus and its application
  • A VEGF antibody, reorganization AAV virus and its application
  • A VEGF antibody, reorganization AAV virus and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1: VEGF antibody sequence design

[0035] according to figure 1 Design the VEGF antibody structure, connect the heavy chain and light chain of the antibody through the P2A linker, the heavy chain is at the 5' end of the gene sequence, and the light chain is at the 3' end of the gene.

[0036] The P2A linker is composed of the furin restriction site RKRR (arginine-lysine-arginine-arginine) and the P2A protein; after furin digestion, the residual amino acids of P2A remaining on the heavy chain are removed.

[0037] The P2A protein includes GSG (glycine-serine-glycine), which is located at the front of the P2A protein, and GSG (glycine-serine-glycine) is added to the P2A to improve the enzyme cutting efficiency of the P2A.

[0038] The heavy chain and light chain are composed of variable region and constant region, respectively, and the signal peptide is the signal peptide of human serum albumin, which is located at the 5' end of the heavy chain and light chain...

Embodiment 2

[0046] Embodiment 2: Construction of recombinant expression vector

[0047] The sequences of mab (VEGF), ranibizumab, and G6 Fab that were commissioned to be synthesized were digested with NotI and HindIII respectively to obtain the target fragments with cohesive ends. At the same time, the vector pAAV-pCAG-wpre (purchased from addgene) was digested with NotI and HindIII to obtain a linearized vector fragment (see figure 2 ), use T4 ligase to connect the target fragment and the linearized vector fragment, transform it into E.coli (Top10), after the sequence is correct, use the plasmid extraction kit of OMEGA company to extract the plasmid, and obtain the recombinant expression vector pAAV-mab (VEGF) , pAAV-Raybead, pAAV-G6 Fab. The concentration of the recombinant expression vector pAAV-mab (VEGF) extracted in the present invention is 350ng / ul, the concentration of pAAV-Raybead is 325ng / ul, and the concentration of pAAV-G6 Fab is 315ng / ul.

Embodiment 3

[0048] Example 3: Culture and passage of packaging cell lines

[0049] The packaging cell line used in the present invention is 293T cells. Take out the frozen 293T cells from the liquid nitrogen tank, quickly throw them into a 37°C water bath and shake them quickly, try to dissolve the cell solution completely within 1-2 min. Transfer the cell solution to a 50ml centrifuge tube, add physiological saline to make the total volume 50ml, mix well and centrifuge at 1500 rpm for 5 min. Remove the supernatant, add 5 ml of fresh high-glucose DMEM (containing 10% FBS) medium to resuspend the cells, transfer them to T75 flasks, and fill each bottle with 10 ml of high-glucose DMEM (containing 10% FBS) medium. Place the culture flask steadily at 37°C, 5% CO 2 cultured in an incubator. The cell viability was observed the next day, and the medium was replaced. Afterwards, the growth of the cells was observed every day, and the cells were passaged when the cells covered 80%-90% of the b...

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Abstract

The invention provides a VEGF antibody, which is composed of human serum albumin signal peptide Alb sp, heavy chain, P2A linker, human serum albumin signal peptide Alb sp2, and light chain in series; the invention also provides recombinant AAV virus, And the application of the above-mentioned VEGF antibody in the medicine for inhibiting VEGF-mediated endothelial cell proliferation. The amino acid and codon-optimized VEGF antibody of the present invention has a higher antibody concentration, and has a higher inhibitory effect on the proliferation of human umbilical vein endothelial cells than the other two control VEGF antibodies.

Description

technical field [0001] The invention relates to the application of a VEGF antibody, a recombinant AAV virus and a medicine for inhibiting VEGF-mediated endothelial cell proliferation, and belongs to the technical field of biomedicine. Background technique [0002] Age-related macular degeneration (AMD) is an age-related macular degenerative disease mainly caused by genetic and environmental factors. It is the leading cause of blindness in people over 55 years old in developed countries. It occurs in my country The incidence rate also increases year by year, and is close to the incidence rate of the white population. AMD is divided into two types, wet (neovascular) and dry (non-neovascular), with or without neovascularization in the choroid as the main difference. Currently, the treatment of wet AMD is mainly aimed at choroidal neovascularization (Choroidal Neo-Vascularization, CNV). , anti-VEGF) drug treatment; there is currently no effective treatment for dry AMD, although...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28C12N7/01A61K39/395A61P27/02A61P9/10
CPCC07K16/2863C12N7/00A61P27/02A61P9/10C12N2750/14121A61K2039/505
Inventor 刘明录韩庆梅强邦明王立新张传鹏金海锋冯建海卢永灿王亮李希鹏
Owner SHANDONG XINRUI BIOTECH CO LTD
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