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Gene of human vascular endothelial growth factor monoclonal antibody and use thereof

A technology of monoclonal antibody and vascular endothelium, applied in the direction of anti-growth factor immunoglobulin, application, genetic engineering, etc., can solve the problems of cell line instability, high cost, easy loss, etc., and achieve difficult preservation and high production cost , the effect of small molecules

Inactive Publication Date: 2009-07-22
林植华
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to find clinical detection reagents for VEGF-related tumors, the antibody hybridoma cell lines are easy to mutate when used in large-scale production, the cell lines are unstable, easy to lose, expensive and many other shortcomings, the present invention uses genetic engineering methods, Provided a human vascular endothelial growth factor monoclonal antibody light chain variable region gene and heavy chain variable region gene and their expression products, and assembled scFv-type genetically engineered antibody fragments, making it possible to produce antibodies on a large scale and at low cost

Method used

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  • Gene of human vascular endothelial growth factor monoclonal antibody and use thereof
  • Gene of human vascular endothelial growth factor monoclonal antibody and use thereof
  • Gene of human vascular endothelial growth factor monoclonal antibody and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Isolation of variable region genes of antibodies from VEGF165 monoclonal cell line

[0038] Collect the hybridoma cell line ABL-110 that grows well and can secrete VEGF165 monoclonal antibody 7 One (constructed by our laboratory, according to "Current protocol in immunology" Production of Monoclonal Antibodies), the total RNA was extracted with TRIZOL reagent.

[0039] RT-PCR catch VH gene:

[0040] With reference to the method provided by Promega (Access RT-PCRSystem and Access RT-PCRIntroductory System): one-step RT-PCR kits were used to amplify the VH gene:

[0041] The VH gene uses primer VHBack (5'-AGGTSMARCTGCAGSAGTCWGG-3') and primer VHFor (5'-TGAGGAGACGGTGACCGTGGTCCCTTGGCCCCAG-3'),

[0042] The RT-PCR reaction is a total of 50 μl system: 1 μg of total RNA, 10 μl of 5×AMV / Tf1 reaction buffer, 5 μl of primers VH Back and VH For at a concentration of 10 pmol, 1 μl of dNTP at a concentration of 500 uM, and 3 mM MgSO 4 2 μl; 1 μl of 5u AMV reverse trans...

Embodiment 2

[0052] Example 2: Assembly of scFv genes

[0053] To obtain scFv, add a flexible linker between VH and VL (G 4 S) 3 , in order to prevent the introduction of mutations during the amplification process, we use the high-fidelity enzyme Pyrobest (Dalian Bao Biology). The whole assembly schematic diagram is figure 2 , the result is as figure 1 .

[0054] The first round of PCR reaction to amplify the VH:

[0055] The 50 μl PCR reaction system is as follows: 5 μl of 10× reaction buffer; the concentration of 10 μM primer VH P 1 and VH P 2 2.5 μl: 4 μl dNTPs; 35 μl HO 2 O; High-fidelity Pyrobest enzyme 0.5 μl. Denaturation at 95°C for 5min; denaturation at 95°C for 30s, annealing at 55°C for 30s, extension at 72°C for 30s, a total of 26 cycles; extension at 72°C for 5min.

[0056] The second round of PCR reaction amplifies VL:

[0057] The 50 μl PCR reaction system is as follows: 5 μl of 10× reaction buffer; primer VL P at a concentration of 10 μM 1 and VLP 2 2.5 μl: 4...

Embodiment 3

[0060] Example 3: Enzyme digestion and connection of scFv gene

[0061] Double digestion of pET28a:

[0062] EcoRI and HindIII are Takara products. 2 μl of 10×M reaction buffer; 10 μl of vector pET28a; 1 μl each of EcoRI and HindIII; 7 μl of H 2 O. React at 37°C for 6 hours. :

[0063] The third round of amplification product in double enzyme digestion embodiment 2:

[0064] 2 μl of 10×M reaction buffer; 10 μl of the third-round PCR product; 1 μl each of EcoRI and HindIII; 7 μl of H 2 O. React at 37°C for 6 hours. 1% agarose gel electrophoresis, and the product was recovered by tapping the gel.

[0065] Link reaction:

[0066] T4 ligase is a product of Takara. 2 μl of ligation reaction buffer H 2 O; 1 μl each of the products of the last round of enzyme digestion; 16 μl H 2 O. React at 16°C for 12 hours.

[0067] Take 10 μl of the product to transform into competent DH5α. Plate, pick a single clone the next day, identify it by colony PCR, and send it to Shanghai B...

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Abstract

The invention relates to heavy chain and light chain variable region genes of a monoclonal antibody of a human vascular endothelial growth factor, a polypeptide that is encoded by the genes, a carrier that contains the genes, and application of the genes and the polypeptide in the preparation of a clinical testing agent of VEGF-related tumors. The invention utilizes gene engineering means to firstly obtain the heavy chain and light chain variable region genes of the monoclonal antibody from a hybridoma cell strain of the monoclonal antibody with VEGF165 secretion, the heavy chain variable region gene has the nucleotide sequence with SEQ ID NO of 1, the light chain variable region gene has the nucleotide sequence with SEQ ID NO of 3, and the genes are combined with scFv small molecule antibody and cloned into a pET28a expression carrier, have high-efficiency expression in colon bacillus and generate protein that has reserved antibody combination capability and VEGF165 action function. Through the modification with gene engineering techniques, the invention establishes the foundation for biological missile construction, has ultra-convenient antibody production and greatly lowered cost, and is favorable for industrial production.

Description

technical field [0001] The present invention relates to the heavy chain and light chain variable region genes of human vascular endothelial growth factor monoclonal antibody, the polypeptide encoded by the gene, the carrier containing the gene and the gene and polypeptide in the preparation of VEGF-related tumors Application in clinical testing reagents. Background technique [0002] VEGF is a powerful cytokine that can produce a variety of biological effects. It is a type of glycoprotein isolated and purified in the culture medium of bovine pituitary follicular stellate cells by Ferrarra et al. in 1989. It is a family of platelet-derived growth factors. A member of the molecular weight of 34-45kDa, highly conserved sequence, widely distributed in human and animal brain, kidney, liver, spleen, pancreas, and bone and other tissues. Human VEGF is a homodimer formed by two glycoprotein chains. Due to the different cutting methods at the level of gene transcription, VEGF has at...

Claims

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Application Information

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IPC IPC(8): C12N15/13C12N15/70C07K16/22G01N33/577
Inventor 林植华
Owner 林植华
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