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Anti-human Delta like 4 monoclonal antibody and application thereof

A monoclonal antibody, DNA molecule technology, applied in the direction of antibody, application, anti-animal/human immunoglobulin, etc., can solve the problems of providing blood flow, inability to tumor tissue, lack of fusion of blood vessels, etc.

Active Publication Date: 2016-03-09
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Inside these tumor tissues, although the density of tumor blood vessels increases, these newly generated blood vessels lack effective fusion with mature vascular plexuses and cannot provide blood flow to tumor tissues, so they can effectively inhibit tumor growth.

Method used

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  • Anti-human Delta like 4 monoclonal antibody and application thereof
  • Anti-human Delta like 4 monoclonal antibody and application thereof
  • Anti-human Delta like 4 monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Embodiment 1: Preparation of anti-D114 monoclonal antibody:

[0020] (1) Immunized mice

[0021] The monoclonal antibody was screened and prepared using recombinant human Dll4 (purchased from Beijing Yiqiao Shenzhou) as the immunogen, dissolved the recombinant antigen in PBS, mixed with quickantibody adjuvant in equal volume, and immunized 6-week-old BALB / c mice. The immunization process Follow the instructions of QuickAntibody. After 21 days, a dose of booster immunization will be given in the same way. On the 35th day, the antigen pulse immunization was carried out according to the conventional method. One week after each immunization, 500 μl of blood was collected from the orbit by the capillary blood collection method. After resting at room temperature for 1 hour, the whole blood was centrifuged at 4°C and 4000 rpm for 15 minutes, and the supernatant serum was collected, and the mouse titer was determined by indirect ELISA. The test results showed that the serum...

Embodiment 2

[0026] Example 2: Amplification of Anti-D114 Monoclonal Antibody Variable Region Gene

[0027] Collect 10 each 7 Total RNA was extracted from four kinds of hybridoma cells in the logarithmic growth phase with an RNA extraction kit, dissolved in 20-50 μl RNase-free water, and stored at -70°C. The first-strand cDNA was synthesized by reverse transcription using total RNA as a template.

[0028] Primers for amplifying the 5′ end of the heavy chain variable region:

[0029] 1. cttccggaattcSARGTNMAGCTGSAGSAGTC

[0030] 2. cttccggaattcSARGTNMAGCTGSAGSAGTCWGG

[0031] Primers for amplifying the 3′ end of the heavy chain variable region:

[0032] ggaagatctCTTGACCAGGCATCCTAGAGTCA

[0033] Primers for amplifying the 5′ end of the Kappa light chain variable region:

[0034] gggagctcGAYATTGTGMTSACMCARWCTMCA

[0035] Primers for amplifying the 3′ end of the Kappa light chain variable region:

[0036] ggtgcatgcGGATACAGTTGGTGCAGCATC

[0037] Among the above primers: R=A, G; Y=C, T; ...

Embodiment 3

[0039] Embodiment 3: Preparation of anti-Dll4 monoclonal antibody

[0040] One week in advance, mice were injected intraperitoneally with 0.5ml paraffin oil. One week after sensitization, the vigorously growing hybridoma cells were centrifuged to discard the medium, resuspended in PBS, and injected intraperitoneally for 10 6 Four kinds of anti-Dll4 hybridoma cells in logarithmic growth phase were injected into the peritoneal cavity of mice. One week later, the ascites of the mice were collected separately.

[0041]Centrifuge at 5000g at 4°C for 20min to remove cells and other precipitated substances in the ascites, and then filter with a 0.22μm membrane filter. Purification was carried out according to the manual of Hi-TrapProtein A column of GE Company. After purification, 4 strains of anti-Dll4 monoclonal antibodies were identified by western blotting, and named MMGZ01, MMAH06, MMZL03 and MMCE08 respectively. The results are as follows: figure 1 shown.

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Abstract

By use of hybridoma technology, recombinant human Delta like 4 (rhDll4) is used as an antigen for immunizing a BALB / c mice to obtain a high affinity and biological activity anti-human Delta like 4 monoclonal antibody. The monoclonal antibody is characterized in that: the monoclonal antibody can be combined with rhDll4 specifically, and can block human umbilical vein endothelial cells (HUVEC) proliferation suppression of the rhDll4. Specifically, screening, a preparation method, and nucleotide and amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-human Delta like 4 monoclonal antibody are disclosed, and the nucleotide and amino acid sequences comprise nucleotide and amino acid sequences corresponding to complementarity determining regions CDR1, CDR2 and CDR3.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to a new high-affinity monoclonal antibody that can specifically bind to Deltalike4 (Dll4) on the surface of human neovascular endothelial top cells, and the variable region sequence of the anti-Dll4 monoclonal antibody and its preparation method . Background technique [0002] In 1975, Koehler and Milstein established the in vitro hybridoma technology to obtain mouse-derived monoclonal antibodies, which started a new era of polyclonal antibodies moving towards monoclonal antibodies. Compared with polyclonal antibodies, monoclonal antibodies have unparalleled advantages, such as high specificity, high titer, high purity, uniform physical and chemical properties, strong reproducibility, low cost and mass production. These characteristics of monoclonal antibodies make it a hot spot in future therapeutic research. [0003] Tumor growth and migration require the host's blood vesse...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13A61K39/395A61P35/00
CPCC07K16/28
Inventor 王旻吴旻许卓斌贾雪莲王世静王泽根张娟罗晨
Owner CHINA PHARM UNIV
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