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RANKL-TNF (Receptor Activator of Nuclear Factor Kappa-B Ligand-Tumour Necrosis Factor) sample region fusion protein and preparation method and application thereof

A kind of fusion protein, protein technology

Inactive Publication Date: 2011-11-16
CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In a randomized double-blind trial in Argentina, the application of humanized monoclonal antibody AMG-162 in the treatment of postmenopausal osteoporosis patients showed that RANKL monoclonal antibody can significantly increase bone mass and relieve bone pain and other symptoms. Obvious adverse reactions, but the disadvantage is that it is not effective for inflammatory synovitis and must be used in combination with other anti-TNF-α preparations
TNF-α antagonists are clinically effective preparations for inhibiting RA inflammatory response, but 1 / 3 of patients are ineffective against the three antagonists currently on the market, and because TNF-α antagonists are relatively expensive, it is difficult to obtain them widely. application

Method used

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  • RANKL-TNF (Receptor Activator of Nuclear Factor Kappa-B Ligand-Tumour Necrosis Factor) sample region fusion protein and preparation method and application thereof
  • RANKL-TNF (Receptor Activator of Nuclear Factor Kappa-B Ligand-Tumour Necrosis Factor) sample region fusion protein and preparation method and application thereof
  • RANKL-TNF (Receptor Activator of Nuclear Factor Kappa-B Ligand-Tumour Necrosis Factor) sample region fusion protein and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Construction of RANKL-TNF-like region fusion protein prokaryotic expression vector

[0041] Primers were designed according to the gene sequence of the RANKL-TNF-like region recombinant fragment (RANKL P171-234, P246-309, HIV-1 BRU strain reverse transcriptase RT (414-424aa)) and the characteristics of the pET-28a(+) expression vector ( See the table below), using the full-length cDNA of RANKL as a template (Gen Bank NO: BC117286, 1097bp; pDONR223, 5004bp), retaining the His tag of pET-28a, and connecting the three gene fragments by recombinant PCR method, wherein The gene sequence of the BRU strain reverse transcriptase RT (414-424aa) of HIV-1 is inserted in the middle of the two RANKL-TNF sample region gene fragments, and the expression vector obtained by constructing is pET-28a(+)-RTFP-1; The gene sequence of the BRU strain reverse transcriptase RT (414-424aa) of HIV-1 is inserted at the end of the two RANKL-TNF sample region gene fragments, and the express...

Embodiment 2

[0045] Example 2 Induced expression of RANKL-TNF-like region fusion protein in Escherichia coli

[0046] Transform Escherichia coli BL21 (DE3) competent cells with the correct sequenced recombinant cloned plasmid, pick a single colony and inoculate it in 10 mL LB medium (containing 30 mg / L kanamycin), and culture overnight at 37°C with shaking at 250 rpm. The next day, take 30 mL of the overnight culture and transfer it to 1 L of LB medium (containing 30 mg / L of kanamycin), and culture it to OD at 37°C and 250 rpm. 600 to 0.5, add IPTG to make the final concentration 0.1mM, continue shaking culture at 37°C, 250rpm for 4.5h, and collect the bacteria by centrifugation. SDS-PAGE and Western-blot analysis show that a large amount of fusion protein can be obtained after being induced by 0.4mMIPTG, see image 3 , 4.

Embodiment 3

[0047] Example 3 Purification and renaturation of RANKL-TNF-like region fusion protein

[0048] The positive clones were cultured at 0.4mM IPTG at 37°C for 4.5h, then centrifuged at 6000rpm at 4°C for 15min to collect the bacteria, washed with pre-cooled PBS for 6 times, and ultrasonically resuspended for 10min (over 4s and stop for 6s, power: 40%). Add 10 times the volume of bacterial cell protein lysate per gram of wet bacteria to resuspend, add lysozyme to 1mg / mL, and add protease inhibitor PMSF to 1mM at the same time, place on ice for 30min, then ultrasonically lyse the bacteria in an ice bath for 10min, The precipitate was collected by centrifugation at 12,000 rpm for 10 min. Wash once with TE buffer and 1% trixton-100 respectively, dissolve the precipitate in PBS and resuspend the bacteria by ultrasonic (5mL / g wet bacteria), at the same time slowly add an equal volume of 8M urea solution dropwise, magnetic stirrer Stir for 30 min, centrifuge and wash the precipitate o...

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Abstract

The invention provides an RANKL-TNF (Receptor Activator of Nuclear Factor Kappa-B Ligand-Tumour Necrosis Factor) sample region fusion protein, and in particular relates to a fusion protein of an RANKL-TNF sample region protein and a Th2 cell epiposition segment with broad host range property. An experiment shows that an antibody produced by an RANKL-TNF sample region fusion protein immune mouse has the effect of combining RANKL with TNF-alpha, can be used for treating diseases such as rheumatoid arthritis and the like and can play effects of antagonistic bone destruction and inflammation inhibition. The RANKL-TNF sample region fusion protein disclosed by the invention can be used as a therapeutic vaccine and provides a new therapeutic approach and thinking for the diseases such as rheumatoid arthritis and the like. According to the invention, the RANKL-TNF sample region fusion protein is expressed in vitro, and therefore the method has the advantages of high expression yield, strong controllability and relatively low production cost;, and the product has good immunogenicity and is easy to realize mass production.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a fusion protein of RANKL-TNF-like region, and also relates to a preparation method of the fusion protein and an application in a therapeutic vaccine for rheumatoid arthritis. technical background [0002] Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by destructive arthritis, lesions characterized by erosive synovitis, and the resulting destruction of articular cartilage and bone, and eventually lead to joint deformity and disability. 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. Aiming at the two key pathological...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N1/21A61K39/00A61P19/02A61P19/04A61P29/00C12R1/19
Inventor 袁慧慧赵文明余丹
Owner CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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