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Heavy chain and light chain variable region gene of human B lymphocyte stimulation factor monoclonal antibody and its application

A technology of B lymphocytes and monoclonal antibodies, applied in the direction of anti-cytokine/lymphokine/interferon immunoglobulin, application, genetic engineering, etc., can solve the problems of cell line instability, high cost, easy loss, etc., to achieve Not easy to save, high production cost, good for industrial production

Inactive Publication Date: 2005-08-17
NANJING NORMAL UNIVERSITY
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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 BLyS-related autoimmune diseases, and aiming at many shortcomings such as easy mutation of antibody hybridoma cell lines in large-scale production, unstable cell lines, easy loss, and high cost, the present invention uses gene Engineering means, providing a human B lymphocyte stimulating factor monoclonal antibody light chain variable region gene and heavy chain variable region gene and their expression products, and assembling scFv-type genetically engineered antibody fragments, enabling large-scale and low-cost production Antibodies made possible

Method used

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  • Heavy chain and light chain variable region gene of human B lymphocyte stimulation factor monoclonal antibody and its application
  • Heavy chain and light chain variable region gene of human B lymphocyte stimulation factor monoclonal antibody and its application
  • Heavy chain and light chain variable region gene of human B lymphocyte stimulation factor monoclonal antibody and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: The variable region gene of antibody isolated from BLyS monoclonal cell line ABL-1 is collected and grows well, the hybridoma cell line ABL-110 that can secrete BLyS monoclonal antibody 7 One (constructed by our laboratory, according to "Current protocol in immunology" Production of Monoclonal Antibodies), total RNA was extracted with TRIZOL reagent.

[0034] RT-PCR catch VH gene:

[0035] With reference to the method provided by Promega (Access RT-PCR System and Access RT-PCR Introductory System): one-step RT-PCR kits were used to amplify the VH gene respectively:

[0036] The VH gene uses primer VH Back (5'-AGGTSMARCTGCAGSAGTCWGG-3') and primer VH For (5'-TGAGGAGACGGTGACCGTGGTCCCTTGGCCCCAG-3'),

[0037] 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...

Embodiment 2

[0047] Example 2: Assembly of scFv genes

[0048] 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 .

[0049] The first round of PCR reaction to amplify the VH:

[0050] 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.

[0051] The second round of PCR reaction amplifies VL:

[0052] 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

[0056] Example 3: Enzyme digestion and connection of scFv gene

[0057] Double digestion of pET28a:

[0058] 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. :

[0059] The third round of amplification product in double enzyme digestion embodiment 2:

[0060] 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.

[0061] 1% agarose gel electrophoresis, and the product was recovered by tapping the gel.

[0062] Link reaction:

[0063] 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.

[0064] Take 10 μl of the product to transform into competent DH5α. Plate the plate, pick a single clone the next day, identify it by colony PCR, and s...

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Abstract

The present invention relates to human B lymphocyte exciting factor monoclonal antibody heavy chain and light chain variable region gene, the gene coded polypeptide, vector containing the gene, the application of the gene and the polypeptide in preparing clinical detecting reagent for BLyS relative diseases and autoimmune diseases, and the preparation process. The heavy chain variable region gene has full length of 342 bp, nucleotide sequence shown in <400>1, and coded amino acid shown in <400>3; and the light chain variable region gene has full length of 321 bp, nucleotide sequence shown in <400>2, and coded amino acid shown in <400>4. The gene engineering expression produced protein has the antibody combining ability maintained and small scFv molecule, is easy to combine with other effector molecule via gene engineering process. The present invention lays foundation for constituting biological missile, has low cost and is favorable to industrial production.

Description

technical field [0001] The present invention relates to the heavy chain and light chain variable region genes of human B lymphocyte stimulating factor monoclonal antibody, the polypeptide encoded by the gene, the carrier containing the gene and the gene and polypeptide in the preparation of BLyS-related self The application in the clinical detection reagent of immune disease, and its preparation method. Specifically, the heavy chain and light chain variable region genes of the present invention come from BALB / C mouse hybridoma cell ABL-1. Background technique [0002] Human B lymphocyte stimulating factor (hBLyS) is a cytokine closely related to human immune regulation newly discovered by Moore PA et al. (TNF) superfamily is a type II transmembrane protein, which is mainly expressed in human peripheral blood mononuclear cells, spleen, lymph nodes, bone marrow and other tissues, and can be expressed in the extracellular soluble part (hsBLyS) under the action of certain metal...

Claims

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Application Information

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IPC IPC(8): C07K16/24C12N15/13C12N15/63C12N15/70C12P21/08G01N33/53
Inventor 张双全曹鹏
Owner NANJING NORMAL UNIVERSITY
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