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Recombinant ADAM15h fusion protein, preparation method and applications thereof

A fusion protein, psct-adam15h technology, applied in the field of ADAM15h and its preparation, can solve the problem that the expression of recombinant ADAM15 has not been reported, etc.

Inactive Publication Date: 2014-07-23
北京神州细胞生物技术集团股份公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, the extracellular segment of recombinant ADAM15 has been expressed in sf21 insect cells, and its disintegrin domain protein fragments have also been successfully expressed in Escherichia coli and Pichia pastoris expression systems. The expression of recombinant ADAM15 in mammalian cells has not yet been seen. Reported, there is no relevant literature involved in the construction of stable strains and the production of complete ADAM15 extracellular segment recombinant proteins

Method used

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  • Recombinant ADAM15h fusion protein, preparation method and applications thereof
  • Recombinant ADAM15h fusion protein, preparation method and applications thereof
  • Recombinant ADAM15h fusion protein, preparation method and applications thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0022] Embodiment 1, the construction of the CHO cell line of recombinant ADAM15h

[0023] (1) Construction of expression vector

[0024] ADAM15 cDNA was prepared by PCR method using human spleen cDNA library, and it could be cloned into pMD18-T vector to obtain pMD-ADAM15. Nucleotide sequences were verified using sequences deposited in GenBank (Gene Bank NM_207191.1). Using this pMD-ADAM15 DNA as a template, 2130 bp ADAM15 cDNA for ADAM15 expression was prepared by PCR method with forward primer (CTGAAGCTTACCATGCGGCTGGCGCT) and reverse primer (GGATCTAGATTTAATGGTGATGATGGTGGTGATGGTGGTGATGAGCTGTGGTCAGGGAGCTGGTT). The upstream primer includes a HindIII restriction site sequence for cloning, and the downstream primer contains a 10His tag and an XbaI site sequence. The cDNA was cloned into the pSCT vector (the vector carries the dhfr and neo genes) to prepare pSCT-ADAM15h, and the sequence of ADAM15h was verified again by gene bank sequence.

[0025] (2) Transfection of pSCT-ADA...

Embodiment 2

[0037] Example 2, ELISA detection of recombinant human ADAM15

[0038] The protein concentration of recombinant human ADAM15 was detected by ELISA method. The protein and antibody required for this experiment were all from Sino Biological Inc. The specific operation steps are as follows:

[0039] (1) Dilute the rabbit anti-human ADAM15 monoclonal antibody to 2ug / ml with the coating solution and coat on the microtiter plate, 100ul / well, overnight at 4°C.

[0040] (2) Discard the liquid in the well, spin dry, add washing solution at 300ul / well, and wash the plate twice.

[0041] (3) Add blocking solution at 300ul / well and react at room temperature for 1 hour.

[0042] (4) Repeat step (2).

[0043] (5) Dilute the recombinant human ADAM15 standard to 1ng / ml, 0.5ng / ml, 0.25ng / ml, 0.125ng / ml, 0.0625ng / ml and 0.03125ng / ml, and dilute the ADAM15 sample accordingly. The final samples were spotted at 100ul / well and reacted at room temperature for 2 hours.

[0044] (6) Dilute the HRP...

Embodiment 3

[0050] Embodiment 3, the purification of recombinant ADAM15h

[0051] Recombinant ADAM15h protein was affinity purified with Ni column, the specific method is:

[0052] (1). Balance column: 5 column volume balance buffer (PH7.0 20mM PBS 500mM Nacl 20mM imidazole 0.4mM PMSF 10% glycerol) balance Ni column.

[0053] (2). Load the sample, wash the Ni column with 5 column volume equilibration buffer.

[0054] (3). Elution: 5-10 column volumes of Elution Buffer (B1: pH7.0 20mM PBS 500mM Nacl 50mM imidazole 0.4mM PMSF 10% glycerol; B2: pH7.0 20mM PBS 500mM Nacl 500mM imidazole 0.4mM PMSF 10% glycerol) to elute the target protein.

[0055] (4). After all the samples have completed the above operations, take the samples for electrophoresis. For electrophoresis results, see figure 1 .

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Abstract

The invention discloses a complete extracellular fragment of a disintegrin-metalloproteinase ADAM15 obtained through recombinant expression of a eukaryotic cell, a preparation method and applications thereof, and aims to provide an ADAM15 protein capable of being applied to production and research. The recombinant ADAM15h protein provided by the invention is an ADAM15h fusion protein which is obtained by converting and expressing a host cell line by pSCT-ADAM15h. The invention provides a preparation method of the recombinant ADAM15h fusion protein. The preparation method comprises the following steps: converting a host cell line by using pSTC-ADAM15h, culturing the converted host cell line, and screening the recombinant ADAM15h high expression clone so as to obtain the recombinant ADAM15h fusion protein. The invention also provides a method for purifying ADAM15h fusion protein in cell culture supernatant and a method for detecting the enzyme activity of recombinant ADAM15h by cutting a fluorescent substrate.

Description

technical field [0001] The present invention relates to the field of biotechnology. Specifically, the present invention relates to a disintegrin metalloprotease ADAM15 fragment expressed through recombinant fusion and its preparation method and application, in particular to a kind of protein that can be soluble in Chinese hamster ovary cells (CHO). , stably expressed ADAM15h and its preparation method and application. Background technique [0002] A Disintegrin And Metalloproteinase (ADAM), also known as MDC (metalloproteinase / disintergrin / cystein-rich), is a newly discovered family of transmembrane glycoproteins containing multiple functional domains in recent years. This family protein has a high homology with the snake venom metallop proteinase (SVMP) family, and its structure is similar, usually consisting of 800-1200 amino acids, including the leader domain, metalloprotease functional domain, disintegrin functional domain, cysteine-rich functional domain, epidermal gro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/64C12N15/85G01N21/31C12Q1/37
CPCC12N9/6489C12Q1/37
Inventor 罗春霞杨茜孙春昀李成红胡萍黄序
Owner 北京神州细胞生物技术集团股份公司
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