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Preparation of monoclonal antibody 7H8 capable of resisting epitope at N terminal of human procalcitonin protein

A monoclonal antibody and protein technology, applied in the biological field, can solve the problems of high price and achieve the effect of improving specificity, ensuring integrity and reducing false positive rate

Active Publication Date: 2017-02-15
HUNAN NORMAL UNIVERSITY
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Problems solved by technology

Moreover, the antibodies used in the current detection system are all from large foreign companies, which are expensive

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  • Preparation of monoclonal antibody 7H8 capable of resisting epitope at N terminal of human procalcitonin protein
  • Preparation of monoclonal antibody 7H8 capable of resisting epitope at N terminal of human procalcitonin protein
  • Preparation of monoclonal antibody 7H8 capable of resisting epitope at N terminal of human procalcitonin protein

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Embodiment Construction

[0035] The present invention will be further clarified through the detailed description of specific embodiments below, but it is not intended to limit the present invention, but only for illustration.

[0036] Materials used in this embodiment are as follows:

[0037] (1) Bacterial species, sequence and cell strain: Escherichia coli E. coli The top 10 strains, ER2566, were preserved by our laboratory, and the codon-optimized PCT gene sequence and SP 2 / 0 mouse myeloma cells were donated by Professor Xia Ningshao, School of Public Health, Xiamen University.

[0038] (2) Main reagents: HRP-goat anti-rabbit was donated by Professor Xia Ningshao of Xiamen University; standard PCT polyclonal antibody was purchased from Proteintech Company; protein relative molecular mass standards were purchased from Thermo Company; Freund’s adjuvant was purchased from American Sigma Company; HT and HAT medium additives were purchased from Sigma Company of the United States; 1640 medium was purch...

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Abstract

The invention discloses a hybridoma cell capable of secreting monoclonal antibodies 7H8 capable of resisting amino acid sequences at the N terminal of human PCT (procalcitonin) protein. Through the prokaryotic expression of a human PCT whole-genome sequence with codon optimization in escherichia coli, the purified human PCT protein is taken as an antigen for immunizing BALB / c mice. Through cell fusion and screening, the hybridoma cell capable of stably secreting the monoclonal antibodies aiming at epitope at the N terminal of the human PCT protein is obtained, and the hybridoma cell is named as 7H8. The 7H8 monoclonal antibody can be used for testing PCT concentration of serum, and has high sensitivity and specificity. The 7H8 antibody identifies the amino acid sequences at the N terminal of the human PCT protein, the cross reaction with the calcitonin in the body is avoided, the specificity in identifying the PCT protein is improved, a very good novel material is provided for developing a diagnostic reagent corresponding to the PCT, and the technical scheme can be widely applied to the cytobiology and immunology experiments, and has a very good application value.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to a monoclonal antibody, in particular to a hybridoma cell secreting a monoclonal antibody against the N-terminal epitope of human PCT protein and the preparation of the specific monoclonal antibody. Background technique [0002] Procalcitonin (PCT) is derived from the calcitonin I gene on human chromosome 11. After the gene is translated and expressed in thyroid C cells, it is converted by endogenous polypeptide enzyme The signal peptide of 25 amino acids at the N-terminus is cut off to form a 116 amino acid non-hormonally active calcitonin propeptide substance, namely procalcitonin (PCT). PCT consists of three parts: N-terminal (1-57aa)-calcitonin (60-91aa)-calcistatin (96-116aa), which are composed of 2 peptides (-Lys-Arg-) and 4 peptides (-Gly-Lys -Lys-Arg-) connection, the relative molecular mass is about 13 KDa, PCT is processed and hydrolyzed by a series of intracellular proteases...

Claims

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Application Information

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IPC IPC(8): C12N15/06C12N5/20C07K16/18G01N33/68G01N33/577
Inventor 刘如石郑姣李晓丹廖旻晶吴意邱义兰何赛
Owner HUNAN NORMAL UNIVERSITY
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