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Preparation and detection kit for human ARMET protein monoclonal antibody

A monoclonal antibody and kit technology, applied in the field of human ARMET protein monoclonal antibody preparation and detection kits, can solve problems such as lack of detection, and achieve the effect of wide application value

Inactive Publication Date: 2009-05-20
ANHUI MEDICAL UNIV
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AI Technical Summary

Problems solved by technology

The reason why its research is not widely carried out is related to the lack of methods to detect it, because so far there is no commercially available commercial ARMET protein and related antibodies

Method used

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  • Preparation and detection kit for human ARMET protein monoclonal antibody
  • Preparation and detection kit for human ARMET protein monoclonal antibody
  • Preparation and detection kit for human ARMET protein monoclonal antibody

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Embodiment Construction

[0044] (1) Cloning of ARMET gene, induced expression and purification of protein

[0045] The cDNA coding sequence of human ARMET was cloned into the pET28a(+) vector with restriction enzymes (NcoI / XhoI) to express the recombinant ARMET protein fused with 6 histidines at the C-terminal. The pET28a(+)-ARMET plasmid was transformed into competent cell BL21, and placed in a 37°C incubator overnight. Inoculate in fresh LB medium containing kanamycin at a ratio of 1:100 (V:V) for expanded culture, shake culture at 37°C until bacterial solution A 600 0.6-0.8, adding IPTG to induce expression. Bacteria were collected and lysed, the supernatant was taken, added to the prepacked Ni-beads column, non-specifically bound proteins were washed away with a buffer containing 50mmol / L imidazole, and then washed with an elution buffer (50mmol / L NaH 2 PO 4 pH8.0, 300mmol / L NaCl, 250mmol / L imidazole) to elute the target protein, and store the eluted protein in a -80°C refrigerator.

[0046] ...

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Abstract

The invention relates to a preparation method of a monoclonal antibody of human ARMET protein and a testing reagent kit. Restriction enzyme NcoI / XhoI is used for cloning the cDNA coding sequence of the human ARMET protein to the carrier pET28a (+), which expresses that the C-terminal fuses together with the recombination ARMET protein of 6 histidines; the plasmid of pET28a(+)-ARMET is converted into a competent cell BL21, which induces and expresses the ARMET protein, collects the thalli and purifies the human ARMET protein; meanwhile, the monoclone of anti-ARMET is prepared and the kit for testing ARMET is established. The method can be applied to the study on the functions of the ARMET, the basic studies on the physiological mechanism and pathomechanism of a human body and can also be used in the auxiliary diagnosis to the clinical diseases.

Description

Technical field: [0001] The present invention relates to a method for expression and purification of human protein ARMET (Arginine rich, mutated in early stage of tumors, also known as ARP, MANF), monoclonal antibody preparation and detection. Background technique: [0002] ARMET (Arginine rich, mutated in early stage of tumors) is also called ARP (arginine-rich protein) or MANF (mesencephalic astrocyte-derived neurotrophic factor) in public databases, and its amino acid sequence is found in figure 1 . Initial studies found a single nucleotide mutation in this gene in early tumors (Shridhar et al, 1996a and 1996b), but subsequent studies found that this mutation in the ARMET gene was a polymorphism of a normal gene , rather than tumor-specific (Evrone et al., 1997; Tanaka et al., 2000). The results of this study caused people to lose interest in the study of ARMET. However, the neurotrophic factor MANF isolated from the culture medium of rat mesencephalic astrocytes by Pe...

Claims

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Application Information

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IPC IPC(8): C07K16/18C12N15/12C12N15/70C07K14/47G01N33/577
Inventor 沈玉先方圣云王法财余永强王海萍孙爱民沈玉君黄学桂张晶晶
Owner ANHUI MEDICAL UNIV
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