Preparation and use of OPG-HSP70 fusion protein

A technology of fusion proteins and proteins, which can be applied in the fields of peptide/protein components, chemical instruments and methods, medical preparations containing active ingredients, etc., and can solve the problems of ineffectiveness of synovial inflammation.

Inactive Publication Date: 2009-05-20
CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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  • Summary
  • 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 d...

Method used

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  • Preparation and use of OPG-HSP70 fusion protein
  • Preparation and use of OPG-HSP70 fusion protein
  • Preparation and use of OPG-HSP70 fusion protein

Examples

Experimental program
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Effect test

Embodiment 1

[0050] Cloning of embodiment 1 OPG 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 are...

Embodiment 2

[0052] Example 2 Construction of 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 digest...

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, and select a single colony to inoculate in 5 mL 2×YT medium (containing 25 mg / L kanamycin), and shake overnight at 37°C. After the transfer at 1:50 the next day, 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 bacteria by centrifugation.

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Abstract

The invention relates to 'a preparation method of OPG-HSP70 fusion protein and an application thereof', pertaining to the new technology field of biological medicaments. The preparation method provides an OPG-HSP70 fusion protein (osteoprotegerin-heat shock protein 70) medicament. According to the most significant pathological features of the rheumatoid arthritis (RA) i.e. the arthrosynovitis with cartilage and bone destruction, the medicament uses the osteoprotegerin as the decoy receptor to combine with the receptor activator of nuclear factor-kB ligand (RANKL) of the nuclear factor kB expressed by osteoblast and other cells, and to interrupt the combination between the RANKL and the RANK expressed by the osteoclast so as to inhibit the bone resorption involved by the osteoclast; and the medicament uses the protective polypeptide segments of the heat shock protein (HSP) to inhibit the inflammatory arthritis. The method contains the DNA sequence which encodes the fusion protein, the method for generating the fusing protein by the recombinant technology, the biological activity of the fusion protein and the like.

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 pathological problems of bone injury and ...

Claims

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Application Information

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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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