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Monoclonal antibody of human carboxypeptidase A inhibition factor Latexin and use thereof

A monoclonal antibody, transferase technology, applied in anti-animal/human immunoglobulin, biochemical equipment and methods, instruments, etc., can solve the problem of complex tumor cell mechanism

Active Publication Date: 2009-08-05
BEIJING CANCER HOSPITAL PEKING UNIV CANCER HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the heterogeneity of tumor cells and the complexity of the mechanism of occurrence, the currently known molecular markers can only solve the problem of early diagnosis and treatment indicators for some patients. Diagnosis and early treatment provide more and more reliable target molecules, which is an important part of tumor research

Method used

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  • Monoclonal antibody of human carboxypeptidase A inhibition factor Latexin and use thereof
  • Monoclonal antibody of human carboxypeptidase A inhibition factor Latexin and use thereof
  • Monoclonal antibody of human carboxypeptidase A inhibition factor Latexin and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1. Acquisition and identification of the full-length reading frame sequence of Latexin gene

[0055] 1. RACE method to amplify the 5′ end of Latexin

[0056] 1. Preparation of total cellular RNA: use Trizol LS Reagent (Cat. No. 10296-010), a product of Invitrogene Company, and operate according to the product instruction.

[0057] 2. Use SMART TM RACE cDNA amplification kit (purchased from Clontech Company) obtained Latexin 5' end fragment:

[0058]2.1 Synthesis of the first strand of cDNA

[0059] 10mM Poly Oligo * (dT) 1μl

[0060] Total cellular RNA 3μl (1μg)

[0061] SMART II A Oligo ** (10mM) 1μl

[0062] ↓

[0063] 70°C for 5 minutes, ice bath for 2 minutes

[0064] ↓

[0065] 5×first strand synthesis solution 2μl

[0066] DTT (20mM) 1μl

[0067] dNTP (10mM) 1μl

[0068] PowerScript TM Reverse transcriptase 1 μl

[0069] ↓

[0070] 1.5 hours at 42°C

[0071] ↓

[0072] When terminating the reaction, add 100ml Tricine-EDTA buffer solution...

Embodiment 2

[0093] Example 2. Construction of Latexin prokaryotic expression vector pGEX / Latexin, prokaryotic expression and purification of antigen

[0094] 1. Construction of Latexin prokaryotic expression vector pGEX / Latexin

[0095] pcDNA3-Latexin was digested with EcoRI, recovered and purified to cut out a 756bp fragment (Latexin cDNA194-887 (positions 194-887 shown in SEQ ID No. 1), in addition to containing a small sequence from the pcDNA3 vector), which Clone into pGEX-4T-3 (purchased from Promega) to construct a prokaryotic expression vector of pGEX-4T-3-Latexin, transform it into BL21 expression bacteria, and spread LB Amp+ Disk, clone picking, plasmid extraction, enzyme digestion identification, automatic sequencing, identification of inserts and reading frames, and construction of prokaryotic expression plasmid pGEX-Latexin fusion protein.

[0096] 2. Induction, expression and purification of GST-Latexin recombinant protein

[0097] 1. Aseptically draw 5ml of pGEX-4T-3-Latex...

Embodiment 3

[0116] Example 3. Preparation, purification and identification of monoclonal antibody against human carboxypeptidase A inhibitor Latexin

[0117] 1. Animals: female, 4 Balb / c mice aged 6-8W (purchased from Institute of Experimental Animals, Chinese Academy of Medical Sciences), 18-20g / mouse.

[0118] 2. Antigen: GST-Latexin fusion protein is expressed by prokaryotic, purified by SDS-PAGE gel, the purity reaches the electrophoresis grade, 50μg / animal / time.

[0119] 3. Immunization procedures and approaches:

[0120] First immunization: Latexin antigen was fully mixed with an equal volume of complete Freund's adjuvant to form water-in-oil, and injected intradermally at multiple points on the back of the mouse. Four weeks later, the first booster immunization was performed. Latexin antigen was mixed with incomplete Freund's adjuvant in equal volumes, and injected intradermally at multiple points on the back. Afterwards, immunization was boosted once every other month, and 2 μl ...

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Abstract

The invention relates to a monoclonal antibody of a carboxypeptidase A inhibiting factor Latexin as well as a preparation method of the monoclonal antibody and a hybridoma cell strain for generating the monoclonal antibody. The invention also relates to a reagent kit containing the monoclonal antibody, an application of the monoclonal antibody in detecting the Latexin gene as well as the level of the encoded protein thereof and an application of the monoclonal antibody in preparing a composition for detecting and / or diagnosing the tumor for assistance.

Description

technical field [0001] The present invention relates to a monoclonal antibody of human carboxypeptidase A inhibitor Latexin, a preparation method of the monoclonal antibody, a hybridoma cell line producing the monoclonal antibody, and a kit containing the monoclonal antibody , the application of the monoclonal antibody in detecting the level of latexin gene and its encoded protein, and the application of the monoclonal antibody in the preparation of a composition capable of detecting or assisting in the diagnosis of tumors. Background technique [0002] The study of oncogenes and tumor suppressor genes enables people to gradually understand the molecular process of carcinogenesis, promotes the accumulation of basic theoretical knowledge of cell biology, and provides molecular targets for tumor prevention and treatment. [0003] Carboxypeptidase A has dual functions of proteolysis and protein protection, and can improve the stability of elastin-binding protein (EBP), which is...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C12N5/18G01N33/577
CPCC07K16/18
Inventor 柯杨李勇陆哲明宁涛巴桑卓玛丁慧荣魏浩然
Owner BEIJING CANCER HOSPITAL PEKING UNIV CANCER HOSPITAL
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