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Method for obtaining RANKL recombinant protein with biological activity and application of RANKL recombinant protein with biological activity

A biologically active and recombinant protein technology, applied in chemical instruments and methods, animal/human proteins, medical preparations containing active ingredients, etc., can solve the problems of inhibition of osteoclast bone resorption and poor treatment effect of patients , to achieve good biological activity

Pending Publication Date: 2020-07-14
NANTONG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] At present, traditional medicine or microsurgery have achieved certain effects in the treatment of osteosclerosis and periapical granuloma, but the treatment effect is not good for patients with severe osteosclerosis caused by autosomal abnormalities, which is mainly due to the process of bone metabolism. The bone resorption function of osteoclasts is inhibited in

Method used

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  • Method for obtaining RANKL recombinant protein with biological activity and application of RANKL recombinant protein with biological activity
  • Method for obtaining RANKL recombinant protein with biological activity and application of RANKL recombinant protein with biological activity
  • Method for obtaining RANKL recombinant protein with biological activity and application of RANKL recombinant protein with biological activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1. Investigate the induced expression of RANKL recombinant protein 1. The induced expression of RANKL recombinant protein

[0044] Resuscitate BL21 competent cells containing pET-32a-mRANKL recombinant plasmid, inoculate in 0.5ml LB medium containing Amp resistance; 37°C, 150rpm shaking culture overnight, take 10ul bacterial liquid evenly plated in LB solid state culture Plates were incubated overnight at 37°C. Select monoclonal colonies for plate picking, inoculate in 5ml LB medium containing Amp resistance at 37°C and shake at 150rpm / min, culture overnight, inoculate 500ml LB medium containing Amp resistance at a ratio of 1:100, 37 ℃, shake the bacteria at 250rpm until the OD is between 0.4-0.6, add IPTG for induction for 5h, and extract the protein of the bacterial fluid.

[0045] 2. Extraction of RANKL recombinant protein

[0046] Centrifuge at 3000rpm for 5min to collect the bacteria, discard the supernatant, add PBS to resuspend, centrifuge at 3000rpm fo...

Embodiment 2

[0049] Example 2, Purification and Identification of RANKL Recombinant Protein

[0050] 1. Purification of RANKL recombinant protein

[0051] The extracted RANKL recombinant protein was purified through the His tag, that is, a peptide segment containing 6 histidines was added to the RANKL recombinant protein during design. Recombinant protein was purified.

[0052] 2. Identification of purified RANKL recombinant protein by SDS-PAGE

[0053] Electrophoresis was performed on 10% SDS-PAGE gel, and samples were loaded in the order of purified control supernatant protein sample, purified control precipitated protein sample, purified RANKL recombinant protein supernatant sample and purified RANKL recombinant protein precipitated sample, each Lane 40ug protein. After 120V for 90min, carry out Coomassie Brilliant Blue G-250 staining, the result is as follows figure 2 As shown in A, by Coomassie Brilliant Blue staining, it was found that the RANKL recombinant protein was mainly ex...

Embodiment 3

[0056] Example 3, Purification and Identification of RANKL Recombinant Protein

[0057] 1. RANKL recombinant protein induces osteoclast precursor Raw264.7 cells to differentiate into osteoclasts

[0058] Raw264.7 cells were seeded in 96-well cell culture plates, 10,000 cells / well, cultured in α-MEM medium with 10% fetal bovine serum (FBS), added 50ng / ml RANKL recombinant protein, 37°C 5% Osteoclasts were induced for 5-6 days under CO2 saturated humidity. Stained by tartrate-resistant acid phosphatase (TRAP) staining kit, photographed, and the photographed results were as follows: image 3 As shown in A, the cell staining is wine red. The irregular shaped cells containing multiple nuclei are osteoclasts, which contain filopodia and lamellipodia structures. Mature osteoclasts also contain folded border structures. The black cut-off head refers to the osteoclasts induced and differentiated by Raw264.7 cells.

[0059] 2. RANKL recombinant protein induces bone marrow macrophages...

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Abstract

The invention discloses a method for obtaining RANKL recombinant protein with biological activity. The method is characterized by comprising the following steps: (1) expressing the RANKL recombinant protein in a large quantity through induction in vitro; (2) obtaining RANKL recombinant protein with biological activity by purification; and (3) identifying the biological activity of the RANKL recombinant protein through promotion of differentiation of osteoclast of the RANKL recombinant protein in vitro. The invention further discloses application of the RANKL recombinant protein with biologicalactivity in preparation of a drug for treating bone metabolic disorder diseases. The application is characterized in that the RANKL recombinant protein with biological activity is obtained by in-vitro induction expression, purification and identification of the RANKL recombinant protein. A large quantity of the RANKL recombinant protein with biological activity can be obtained through in-vitro induction expression and purification of the RANKL recombinant protein, and has potentials to become a new way for treating and improving bone sclerosis and periapical granuloma.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and specifically relates to a method for obtaining RANKL recombinant protein with biological activity and its application. Background technique [0002] At present, traditional medicine or microsurgery have achieved certain effects in the treatment of osteosclerosis and periapical granuloma, but the treatment effect is not good for patients with severe osteosclerosis caused by autosomal abnormalities, which is mainly due to the process of bone metabolism. The bone resorption function of osteoclasts was inhibited. Studies have shown that RANKL can promote osteoclast differentiation and enhance bone resorption. The occurrence of bone metabolic diseases is mostly due to the abnormal close relationship between osteoblast-mediated bone formation process and osteoclast-mediated bone resorption process. Therefore, how to obtain biologically active RANKL recombinant protein is the problem to be solv...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C07K14/705A61K38/17A61P19/08
CPCC12N15/70C07K14/70575A61P19/08A61K38/00
Inventor 王东闵雯嫣
Owner NANTONG UNIVERSITY
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