Preparation method and application of opg-hsp70 fusion protein

A fusion protein and protein technology, applied in the direction of peptide/protein components, chemical instruments and methods, medical preparations containing active ingredients, etc., can solve the problem of no effect of synovial inflammation

Inactive Publication Date: 2011-12-14
CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

In the collagen-induced arthritis (CIA) model, OPG can also reduce or prevent bone destruction, but has no effect on synovial inflammation (Am J Pathol.2002, 161(4): 1419-27)

Method used

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  • Preparation method and application of opg-hsp70 fusion protein
  • Preparation method and application of opg-hsp70 fusion protein
  • Preparation method and application of opg-hsp70 fusion protein

Examples

Experimental program
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Effect test

Embodiment 1

[0050] The cloning of embodiment 1OPG full-length gene

[0051] According to the sequence of the human OPG coding region cDNA reported in the literature (GenBank No. NM_002546), two oligonucleotide primers for the OPG coding region cDNA were designed, and the human osteosarcoma cell line MG63 was cultivated to the early logarithmic growth stage, and rhBMP-2 (in The final concentration in the culture medium reaches 100ug / L), and after continuing to cultivate to the logarithmic growth phase, the cells are counted and centrifuged to collect the cells. Total cellular RNA was extracted according to the instructions of the Trizol Reagent total RNA extraction reagent. According to the operating steps of the MMLV first-strand cDNA synthesis kit, the first-strand cDNA was synthesized using oligo(dT) as a primer. Take 8uL of the reverse transcription product as a template, under the action of TaqDNA polymerase, carry out PCR with P1 and P2 primers respectively, the reaction conditions ...

Embodiment 2

[0052] The construction of embodiment 2 OPG-HSP70 fusion protein prokaryotic expression vector

[0053] The recombinant plasmid pGEMT-Easy-OPG with correct sequencing was used as a template, and P3 and P4 were used as primers for the first PCR amplification, and then the PCR product was used as a template, and P3 and P5 were used as primers for the second PCR amplification. The OPG-HSP70 fusion gene was obtained. After purification, the PCR product was subjected to a double-enzyme digestion reaction with the prokaryotic expression vector pET-28a using Nco I and Xho I restriction endonucleases. After digestion, the carrier and the target DNA were purified and connected, transformed into E. coli DH5a competent cells, and a single colony was selected and inoculated in a 5 mL LB medium (containing 25 mg / L kanamycin) Erlenmeyer flask, 37 °C shaking overnight. The bacteria were collected, a small amount of plasmid was extracted, and then identified by Nco I and Xho I double enzyme...

Embodiment 3

[0054] Example 3 Induced expression of OPG-HSP70 fusion protein in Escherichia coli

[0055] Transform Escherichia coli E.coli BL21 (DE3) competent cells with the recombinant clone plasmids identified by restriction enzyme digestion, pick a single colony and inoculate in 5 mL 2×YT medium (containing 25 mg / L kanamycin), and shake overnight at 37°C. The next day, after the 1:50 transfer, culture at 37°C for 2 hours, add IPTG to make the final concentration 0.1mmol / L, continue shaking culture at 30°C for 5 hours, and collect the cells by centrifugation.

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Abstract

The invention is a "preparation method and application of OPG-HSP70 fusion protein", which belongs to the field of biomedical new technology. The invention provides an osteoprotegerin-heat shock protein 70 (OPG-HSP70) fusion protein drug. The drug targets synovitis, the most important pathological feature of rheumatoid arthritis (RA), accompanied by destruction of cartilage and bone. Through osteoprotegerin (OPG) as a decoy receptor, it can interact with cells expressed by osteoblasts and other cells. Receptor Activator of Nuclear Factor κB Ligand (RANKL) binds, blocks the binding of RANKL to RANK expressed by osteoclasts, thereby inhibiting bone resorption involving osteoclasts; uses the protective polypeptide fragment of heat shock protein (HSP) to inhibit joint inflammation. The invention includes the DNA sequence encoding the fusion protein; the method of producing the fusion protein through recombinant technology; and the biological activity of the fusion protein, etc.

Description

technical field [0001] The invention relates to a method for expressing and preparing recombinant osteoprotegerin-heat shock protein 70 (OPG-HSP70) fusion protein by using Escherichia coli, and a pharmaceutical preparation containing recombinant osteoprotegerin-heat shock protein 70 (OPG-HSP70) prepared by the method And the application of the pharmaceutical preparation in the preparation of medicines for preventing or treating rheumatoid arthritis. technical background [0002] Rheumatoid arthritis (RA) is an autoimmune disease mediated by T cells, characterized by synovial inflammation, accompanied by articular cartilage and bone damage. The prevalence of RA in China is 0.32% to 0.38%, and the disability rate is high. RA has caused huge losses to the labor force and social economy. And RA is a kind of refractory disease. Although the existing treatment methods are various, they are not ideal. The present invention aims at the key pathologica...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/09A61K38/16A61P19/02
Inventor 王炜赵文明马静李慎涛
Owner CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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