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Recombinant antibody of anti-human cardiac troponin I

A troponin and antibody technology, applied in the field of immunity, can solve the problems of poor affinity and low activity, and achieve the effect of high affinity and strong binding protein activity

Active Publication Date: 2020-04-17
DONGGUAN PENGZHI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Due to low activity and poor affinity, the existing cTnI antibodies cannot be well applied to the detection of cTnI protein. Therefore, there is a strong demand in this field for antibodies that can effectively and specifically bind to and detect cTnI

Method used

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  • Recombinant antibody of anti-human cardiac troponin I
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  • Recombinant antibody of anti-human cardiac troponin I

Examples

Experimental program
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Effect test

Embodiment 1

[0120] This example provides an exemplary preparation method of a recombinant antibody against human cardiac troponin I.

[0121] S10, construction of expression plasmids:

[0122] In this embodiment, restriction endonuclease and Prime Star DNA polymerase were purchased from Takara Company;

[0123] MagExtractor-RNA extraction kit was purchased from TOYOBO;

[0124] BD SMART TM The RACE cDNA Amplification Kit was purchased from Takara;

[0125] The pMD-18T vector was purchased from Takara;

[0126] The plasmid extraction kit was purchased from Tiangen Company;

[0127] Primer synthesis and gene sequencing were completed by Invitrogen;

[0128] Secreting Anti-cTnI 12D2 monoclonal antibody is an existing hybridoma cell line, which is recovered for use.

[0129] S11, design and synthesis of primers:

[0130] 5' RACE upstream primers for amplification of heavy and light chains:

[0131] SMARTER II A Oligonucleotide:

[0132] 5'>AAGCAGTGGTATCAACGCAGAGGTACXXXXX<3';

[013...

Embodiment 2

[0152] Transient Transfection of Recombinant Antibody Expression Plasmids into CHO Cells and Identification of Antibody Activity in Expression Supernatant

[0153] The plasmid was diluted to 400ng / ml with ultrapure water, and the CHO cells were adjusted to 1.43×10 7 cells / ml in a centrifuge tube, mix 100ul plasmid with 700ul cells, transfer to electroporation cup, electroporation, transfer to 10ml CD CHO AGT medium, culture in 37 degree shaker (8% CO 2 , amplitude 150); sampling every day to detect cell viability, when the cell viability is lower than 50%, centrifugal cell culture supernatant, the antibody obtained (has sequence such as light chain and heavy chain shown in SEQ ID NO:11 and 12 ). Take 4 μg of purified antibody for reducing SDS-PAGE.

[0154] After analysis, the complementarity determining region (WT) of the heavy chain:

[0155] CDR-VH1 is G-F(X1)-T-F-T-E(X2)-Y-N-V(X3)-H;

[0156] CDR-VH2 is Y-I(X1)-Y-P-N(X2)-N-G-I-S(X3)-G-Y-N-Q;

[0157] CDR-VH3 is R-D(X1...

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Abstract

The invention relates to a recombinant antibody of an anti-human cardiac troponin I, particularly to a novel isolated binding protein containing a cTnI antigen binding domain, and researches of the preparation, the applications and the like of the binding protein. The binding protein is high in activity, has high affinity with human cTnI protein, and can be widely applied to the field of cTnI protein detection.

Description

technical field [0001] The invention relates to the technical field of immunization, in particular to a recombinant antibody against human cardiac troponin I. Background technique [0002] Before the 1980s, the World Health Organization (WHO) has always regarded myocardial enzyme activity as one of the diagnostic criteria for acute myocardial infarction (AMI). In the late 1980s, researchers discovered that the sensitivity and specificity of troponin (Tn) was higher than that of phosphocreatine kinase (CK), phosphocreatine kinase isoenzyme (CK-MB), lactate dehydrogenation enzymes and biomarkers such as aspartate aminotransferase. Cardiac troponin I (cTnI) exists only in the myocardium and is a marker of cardiomyocytes. Its abnormal changes can affect the diastolic and systolic functions of the heart, and can be used to diagnose myocardial necrosis and judge myocardial injury. One of the most specific markers is recognized as the main biochemical marker for the rapid diagnos...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C12N15/13G01N33/68
CPCC07K16/18G01N33/6893C07K2317/565C07K2317/92G01N2333/4712G01N2800/324G01N2800/12
Inventor 孟媛钟冬梅游辉范凌云
Owner DONGGUAN PENGZHI BIOTECH CO LTD
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