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Hybridoma cell line and anti-human erythrocyte surface H antigen monoclonal antibodies generated thereof

A hybridoma cell line, monoclonal antibody technology, applied in the direction of anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, fusion cells, cells modified by introducing foreign genetic material, etc., can solve the problem of genetically engineered antibodies. Reduced affinity, affecting the binding activity of genetically engineered antibodies, and not being completely faithful

Inactive Publication Date: 2010-07-07
FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the degeneracy of the primers, the N and C ends of the variable region cannot be completely faithful to the original sequence, and the change of some amino acid residues can affect the binding activity of the genetically engineered antibody, resulting in a decrease in the affinity of the genetically engineered antibody

Method used

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  • Hybridoma cell line and anti-human erythrocyte surface H antigen monoclonal antibodies generated thereof
  • Hybridoma cell line and anti-human erythrocyte surface H antigen monoclonal antibodies generated thereof
  • Hybridoma cell line and anti-human erythrocyte surface H antigen monoclonal antibodies generated thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1. Obtaining of hybridoma cell line 2E8Z and identification of the monoclonal antibody 2E8 produced against human erythrocyte surface H antigen

[0065] The inventors of the present invention have prepared a hybridoma cell strain capable of producing anti-human erythrocyte H antigen, named 2E8Z, and the cell strain has been preserved in the General Microbiology Center of the China Microbiological Culture Collection Management Committee in Zhongguancun, Beijing on February 08, 2007 , the preservation number is CGMCC No.1942, and the specific construction method can be found in the literature (Li Hui, Pan Jichun, Liu Zi, Liu Jinghan, Zhang Jingang. Development and characteristic identification of non-aggregating monoclonal antibody against human erythrocyte membrane antigen[J]. Journal of Molecular Immunology 2005, 21(4):473-475). The monoclonal antibody produced by the cell line was named 2E8, the agglutination titer of the monoclonal antibody was 1:1024, and the...

Embodiment 2、2E8

[0066] Cloning of Example 2, 2E8 Monoclonal Antibody Variable Region Gene

[0067] Use the following method to clone the heavy chain variable region and light chain variable region encoding genes of the 2E8 monoclonal antibody. The specific process includes the following steps:

[0068] 1. Extraction of total RNA

[0069] The total RNA of the hybridoma cell line 2E8Z CGMCC No.1942 obtained in Example 1 that can produce anti-human erythrocyte surface H antigen was extracted with the TRIzol kit of Invitrogen and referring to the kit instructions.

[0070] 2. Synthesis of cDNA

[0071] Using the total RNA obtained in step 1 as a template, oligo(dT) 15 For primers, use Invitrogen’s SuperScript TM -II reverse transcriptase and perform reverse transcription according to the instructions, 50 μL reaction system and reaction conditions: total RNA 10 μL, ol igo(dT) 15 1 μL, 2.5mmol / L dNTPs 4μL, incubate at 65°C for 5min, then ice bath for 5min, microcentrifuge, then add 5× reverse t...

Embodiment 3

[0084] Embodiment 3, the cloning of fusion protein gene 2E8R3 and the construction of the Escherichia coli expression vector containing this fusion gene

[0085] 1. Cloning of fusion protein gene 2E8R3

[0086] Cloning by flexible peptide (Gly4Ser) using the following method 3 (SEQ ID NO: 5 in the sequence listing) The fusion protein (named 2E8R3) gene 2E8R3 of the monoclonal antibody 2E8 heavy chain variable region and light chain variable region obtained by connecting the anti-human erythrocyte surface H antigen obtained in Example 2 , the specific process includes the following steps:

[0087] 1. Design primers

[0088] Heavy chain variable region and light chain variable region encoding gene and flexible peptide (Gly4Ser) of the monoclonal antibody 2E8 cloned according to Example 2 3 Design primers for the coding sequence, the primer sequences are as follows:

[0089] 2E8H BACKNEW (upstream primer): 5'- ggatcc caggtgcagttgaaggagtcagga-3' (SEQID NO: 14, the underline...

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Abstract

The invention discloses a hybridization tumor cell stem 2E8Z CGMCC NO.1942 and monoclonal antibody of human red blood cell surface H antigen thereof. The monoclonal antibody 2E8 of human red blood cell surface H antigen genereated by hybridization tumor cell stem has advantages of high effect, strong differentia, extensive distribution of antigen. The flexible peptide is easy to be bended, so mixed albumen of the invnetion is feld correctly and without influence each activity combination area of the mixed albumen. Experiment evidence, the mixed albumen can specially combine with human red blood cell surface H antigen; moreover, the mixed albumen with high expression amount, easy to be purified, short production cycle, large production scale and low cost. Base on these advantages, the invention plans to use gene engineering method to produce double function molecule and establish good base of treating based on red blood cell and testing platform, having more actual sense and wider application foreground in medical testing field.

Description

technical field [0001] The invention relates to a hybridoma cell line and the monoclonal antibody against H antigen on the surface of human erythrocyte produced by the hybridoma cell line. Background technique [0002] There are many antigen molecules on the surface of human red blood cells, which have different structures and various functions. Among them, the surface antigen molecules (such as: A, B, O blood group antigens) that make up the blood group system are mainly closely related to blood transfusion safety. ABO blood group antigen genes directly encode specific glycosyltransferases, and then these enzymes synthesize ABO antigens with oligosaccharide properties. Type A epitope is N-acetyl galactose, type B is galactose, and type O is H epitope, which is fucose. The H antigen epitope is the precursor of the AB blood group antigen. [0003] Except for the rare Bombay red blood cells, the H antigen is distributed on the surface of all human red blood cells and belong...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/12C07K16/28C12N15/13C12N15/63C07K19/00C12N15/62
Inventor 章金刚邵长利吕茂民
Owner FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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