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Preparation method and application of osteoporotegerin-heat-shock protein 65-fused protein

A technology of fusion protein and protein, which is applied in the direction of hybrid peptide, bone disease, drug combination, etc., can solve the problem of no effect of synovial inflammation

Inactive Publication Date: 2010-08-25
李慎涛 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 osteoporotegerin-heat-shock protein 65-fused protein
  • Preparation method and application of osteoporotegerin-heat-shock protein 65-fused protein
  • Preparation method and application of osteoporotegerin-heat-shock protein 65-fused protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Construction of OPG-HSP65 fusion protein prokaryotic expression vector

[0048] According to the gene sequence of the OPG-HSP65 recombinant fragment (OPG P22-194, HSP65P256-270) and the characteristics of the pET-28a expression vector, three primers were designed: the upstream primer P1 and the downstream primers P2 and P3, which retained the His of pET-28a Tag, with the recombinant plasmid pGEM-TEasy-OPG as a template, the MTB256-270aa gene sequence was designed into the primers, and the gene fragments of the two were connected by two PCR methods, and the TCC flexible fragment was introduced into the downstream primer P2, so that The spatial conformation of OPG-HSP65 recombinant protein was formed correctly. PCR reaction conditions: denaturation at 95°C for 5 min, cycled 30 times according to the following parameters: denaturation at 94°C for 40 s, annealing at 53°C for 35 s, extension at 68°C for 1 min, and finally extension at 72°C for 7 min. PCR products ...

Embodiment 2

[0049] Example 2 Induced expression of OPG-HSP65 fusion protein in Escherichia coli

[0050] 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 25 mL LB medium (containing 25 mg / L kanamycin), and culture overnight at 37°C with shaking. The next day, take 50ml of the overnight culture and transfer it to 1L LB medium (containing 25mg / L kanamycin), culture at 37°C until OD 600 To 0.5, add IPTG to make the final concentration 0.5mmol / L, continue shaking culture at 30°C for 5h, and then centrifuge to collect the bacteria.

Embodiment 3

[0051] Example 3 Purification and renaturation of OPG-HSP65 fusion protein in Escherichia coli

[0052] Positive clones were cultured at 0.5mmol / L IPTG at 30°C for 5h, centrifuged at 6000r / min for 15min at 4°C, collected the cells, and washed with pre-cooled 20mmol / L Tris-HCL (pH=7.9) buffer , Wash the cells with pre-cooled 20mmol / LTris-HCL (pH 7.9) buffer. Add 10 times the volume of bacterial cell protein lysis solution per gram of wet bacteria to resuspend, add lysozyme to 1mg / ml, and add protease inhibitor PMSF to 1mmol / L at the same time, place on ice for 30min, then ultrasonically lyse the bacteria in an ice bath After 10 min, the precipitated inclusion bodies were collected by centrifugation. Wash twice with 2mol / L and 3mol / L urea inclusion body washing solution respectively, then dissolve the inclusion body with 4mol / L urea denaturing solution, centrifuge at 12000r / min for 45min at 4°C, and take the supernatant through a 0.22μm micropore The denatured solution of the ...

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Abstract

The invention relates to a preparation method and application of an osteoporotegerin-heat-shock protein 65-fused protein, belonging to the new technical field of biomedicine. The invention provides an osteoporotegerin-heat-shock protein 65 (OPG-HSP 65)-fused recombinant protein medicament. Aiming at the most important pathological feature of rheumatoid arthritis (RA) that articular synovitis is accompanied with the destroy of cartilages and bones, the medicament can be combined with a receptor activator of nuclear factor kappa B ligand (RANKL) expressed by cells comprising osteoblasts and the like through using osteoporotegerin (OPG) as a decoy receptor to block the combination of the RANKL and a RANK (Receptor Activator of NF-kB) expressed by osteoclasts, thereby inhibiting the bone absorption that the osteoclasts participate; and the medicament inhibits articular inflammations by utilizing the protective polypeptide segment of the heat-shock protein (HSP). The invention comprises a DNA (Deoxyribonucleic Acid) sequence for encoding recombinant protein, a method for generating the recombinant protein through a recomposition technique, the biologic activity of the recombinant protein and the like.

Description

technical field [0001] The invention relates to a method for expressing and preparing a recombinant osteoprotegerin-heat shock protein 65 (OPG-HSP65) fusion protein in Escherichia coli using DNA recombination technology, a pharmaceutical preparation containing OPG-HSP65 prepared by the method and the preparation of the pharmaceutical preparation Use in a medicament for preventing or treating rheumatoid arthritis. technical background [0002] Rheumatoid arthritis (RA) is a T cell-mediated chronic systemic autoimmune disease characterized by joint synovial inflammation and bone damage. Due to the high prevalence and disability rate of RA, the etiology has not yet been fully elucidated, especially RA has caused serious losses to the social economy and labor force, so the research on the treatment of RA has important medical and socioeconomic significance. And RA is a kind of refractory disease. Although the existing treatment methods are various, they are not ideal. The pres...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12P21/02C07K19/00A61P19/04C12R1/19
Inventor 李慎涛赵文明张月
Owner 李慎涛
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