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B cell epitope peptide segment of amino-terminal pro-brain natriuretic peptide and applications thereof

An amino-terminal, brain natriuretic peptide technology, applied in the medical field, can solve the problems of difficult patient treatment, insufficient specificity and timeliness, and inability to assess the long-term impact of myocardial damage, and achieve high titer, good specificity and high purity. Effect

Active Publication Date: 2013-01-23
重庆业为基生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, both inpatients and outpatients, clinical routine methods are not only difficult to diagnose HF, but also uncertain when analyzing the results of treatment.
For example, the traditional electrocardiogram, echocardiography and other examination methods are not specific and timely enough, and the traditional examination methods cannot evaluate the long-term impact of myocardial injury, so it is difficult to guide the treatment of patients in a timely and convenient manner

Method used

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  • B cell epitope peptide segment of amino-terminal pro-brain natriuretic peptide and applications thereof
  • B cell epitope peptide segment of amino-terminal pro-brain natriuretic peptide and applications thereof
  • B cell epitope peptide segment of amino-terminal pro-brain natriuretic peptide and applications thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1 recombinant NT-proBNP gene cloning

[0029] The human NT-proBNP coding gene sequence was obtained from GenBank (accession number NCBI Reference Sequence: NM_002521.2), and submitted to the biological online analysis software GCUA (Graphical codon usage analyzer, http: / / guca.schoedl.del) for analysis and evaluation, And carry out codon bias tropism transformation and optimization. The optimization method is to synonymously replace codons whose expression activity of NT-proBNP protein is less than 20% in E. coli with codons preferred by E. coli (the same function can also be achieved without codon replacement). SEQ ID No: 1 is the nucleotide sequence of the gene encoding human NT-proBNP obtained from GenBank, and the underlined part is the codon whose expression activity is lower than 20% in Escherichia coli according to the results of GCUA analysis.

[0030] cacccgctgggcagc ccc ggt tca gcc tcg gac ttg gaaacg tccgggtta caggagcagcgcaaccat ttg cagg...

Embodiment 2

[0041] Example 2 Recombinant pET42a-NT-proBNP plasmid construction

[0042] The pET42a vector and the whole gene synthesis sequence subcloning vector (see Example 1 for details) were digested by Sal I and BamH I for 4 hours, recovered by agarose gel electrophoresis, and washed with T 4 DNA ligase was ligated overnight at 4°C. Put 200 μL of the prepared competent DH5α bacteria into the ice bath, pipette 1 μL of the ligation product into the tube, transform the DH5α bacteria, pat and mix, ice bath for 30 minutes, 42°C water bath for 90 seconds, take out the centrifuge tube and ice bath for 2 minutes, Add 800 μL of 2×YT culture medium at room temperature and mix well, shake and culture at 37°C at 220 rpm for 1 hour, and apply 50 μL, 200 μL and all remaining transformed bacteria to three 2×YT cultures containing kanamycin resistance Cultivate overnight on the plate in a constant temperature incubator at 37°C. The next day, the albino-free colonies were picked and inoculated in LB...

Embodiment 3

[0043] Example 3 Induced expression of recombinant NT-proBNP protein

[0044] Transform BL21 codon plus (DE3) engineering bacteria by conventional methods, take the recombinant pET42a-NT-proBNP plasmid to transform BL21 codon plus (DE3) bacteria, spread on LB solid medium containing chloramphenicol resistance and kanamycin resistance , cultivated overnight in a 37°C incubator, picked white colonies and inoculated them in LB medium for expanded culture the next day, cultured at 30°C, and added IPTG with a final concentration of 0.6mmol / L to induce expression for 4 hours when the measured bacterial OD value reached 0.6-0.8 . After cultivation, each gram of wet bacteria was resuspended with 10 times the volume of PBS buffer (pH 7.3), mixed evenly, and ultrasonically destructed. After the bacterium was completely broken, it was centrifuged at 10,000 rpm at 4°C for 15 minutes, and the supernatant was filtered with a 0.45 μm microporous membrane. . Take the supernatant and precipi...

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Abstract

The invention belongs to the field of medicine, particularly relates to a diagnostic technique of heart failure, and specifically relates to a B cell epitope peptide segment of amino-terminal pro-brain natriuretic peptide. An amino acid sequence is shown in SEQ ID NO:4 or SEQ ID NO:5. The prepared specific antibody can be used as a diagnostic reagent of heart failure. The amino-terminal pro-brain natriuretic peptide prepared by the invention is high-purity monomer recombinant protein of the amino-terminal pro-brain natriuretic peptide. The protein can be used for immunogen and screening collagen prepared from an antibody, and can be simultaneously used as a calibrator when the amino-terminal pro-brain natriuretic peptide is built to carry out quantitative detection. A monoclonal antibody prepared by immune mice at the B cell epitope peptide segment of the amino-terminal pro-brain natriuretic peptide has the advantages of high purity (SDS-PAGE detection purity greater than 96%), high valence (the valence of ELISA up to 1:512000), good specificity, mass preparation, and the like.

Description

technical field [0001] The invention belongs to the field of medicine, in particular to a diagnosis technique for heart failure. Background technique [0002] Cardiovascular disease is an important disease that seriously threatens human life and health. Among them, heart failure (Heart Failure, HF), as a pathological syndrome in cardiovascular diseases that seriously endangers human health, is no longer simply regarded as an independent clinical disease, but as a cardiovascular disease that develops to a later stage. A stage, that is, from the initial risk factors (hypertension, hypercholesterolemia, diabetes) to left ventricular hypertrophy, myocardial cell dysfunction, coronary heart disease, and finally to heart failure. According to statistics by some scholars, once typical symptoms appear in patients with heart failure, the 5-year survival rate is 25% for men and 38% for women, which is similar to that of patients with malignant tumors. In 2003, the China Cardiovascul...

Claims

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Application Information

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IPC IPC(8): C07K7/08C07K19/00C07K16/26C12N5/20G01N33/577C12R1/91
Inventor 黄洪涛石延宾姚静张宪胡伟魏勇
Owner 重庆业为基生物科技有限公司
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