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Preparation method for nacre protein N16 and application of nacre protein N16 in preparation of drugs for prevention and treatment of orthopedic diseases

The technology of pearl mother protein and N16 is applied in the field of preparation of pearl mother protein N16, which can solve the problem that the treatment mechanism of osteoporosis is very different, and achieve the effect of inhibiting the differentiation of osteoclasts and promoting the differentiation of osteoblasts.

Inactive Publication Date: 2013-06-05
SUN YAT SEN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

On the other hand, in China, due to the misleading propaganda of manufacturers and sellers, there are currently overwhelming calcium-containing products flooding the market, all of which are aimed at "supplementing calcium", which is far from the treatment mechanism of osteoporosis.
But there is no report about the application of nacre water-insoluble protein N16 in the treatment of orthopedic diseases

Method used

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  • Preparation method for nacre protein N16 and application of nacre protein N16 in preparation of drugs for prevention and treatment of orthopedic diseases
  • Preparation method for nacre protein N16 and application of nacre protein N16 in preparation of drugs for prevention and treatment of orthopedic diseases
  • Preparation method for nacre protein N16 and application of nacre protein N16 in preparation of drugs for prevention and treatment of orthopedic diseases

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1: Preparation of N16

[0036] Total mRNA was extracted from Pinctada fucata, and single-stranded cDNA was generated by reverse transcription. According to the gene sequence of NCBI database N16, design primer N16F (5'-GGAATTC CATATG GCTGTCCATTATAAGTGC-3’) and N16R (5’-CG GGATCC TTAATTGTCAAACCGTTC-3'), where the underlined bases are NdeI and BamHI restriction endonuclease sites. The N16 gene was amplified by PCR, and the reaction program was set at 95°C for 1 minute, 50°C for 20 seconds, and 72°C for 1 minute, a total of 25 cycles, and finally 72°C for 10 minutes. The amplified N16 gene was connected to the expression vector pET3α, and introduced into Escherichia coli BL21 (DE3) plysS to express the N16 protein. When Escherichia coli BL21 (DE3) plysS grew to the logarithmic phase, 0.4 mM isopropyl-β-D-thiogalactopyranoside was added to induce the expression of N16 at 37°C for 16 h, and the expression cells were collected.

[0037] Purification of N16. D...

Embodiment 2

[0041] Example 2: N16 inhibits the proliferation and differentiation of precursor osteoclasts.

[0042] 1. N16 inhibits the proliferation of precursor osteoclasts.

[0043] Mouse precursor osteoclast cell line RAW 264.7 was used to culture in DMEM medium with 100 U / mL penicillin and 100 g / mL streptomycin, and the cultured cells were placed at 37 °C and saturated humidity 5% C0 2 Under the lower concentration, change the medium once every 2 to 3 days. Cells in good growth state in the logarithmic growth phase were taken in 3-5×10 3 Each well was inoculated in a 96-well culture plate, 180 μl per well. 37℃, 5% CO 2 Incubate for 24 hours in the incubator. After the cells adhered to the wall, different concentrations of N16 were added to the experimental group, and 4 replicate wells were set for each concentration. At the same time, a negative control group (N16=0μM) was set up. Continue culturing for 24, 48, 72 hours. Cell proliferation was detected by MTT assay.

[0044] ...

Embodiment 3

[0057] Example 3: N16 promotes osteoblast differentiation experiment

[0058] 1. Effect of N16 on the proliferation of osteoblasts

[0059] Mouse precursor osteoblasts MC3T3-E1 were used to culture in α-MEM medium of 100 U / mL penicillin and 100 g / mL streptomycin, and the cultured cells were placed at 37°C and saturated humidity 5% C0 2 Under the lower concentration, change the medium once every 2 to 3 days. Cells in good growth state in the logarithmic growth phase were taken in 3-5×10 3 Each well was inoculated in a 96-well culture plate, 180 μl per well. 37℃, 5% CO 2 Incubate for 24 hours in the incubator. After the cells adhered to the wall, different concentrations of N16 were added to the experimental group, and 4 replicate wells were set for each concentration. Continue culturing for 48, 72, and 96 hours. Cell proliferation was detected by MTT assay.

[0060] see results Figure 7 . N16 had no significant effect on the proliferation of MC3T3-E1 cells after being...

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Abstract

The invention relates to a preparation method for nacre protein N16 and the application of the nacre protein N16 in preparation of drugs for prevention or treatment of orthopedic diseases such as osteoporosis and fracture. The nacre protein N16 is expressed in bacteria by using the method of genetic recombination, and a separation and purification scheme of the nacre protein N16 is established. The experiments of cells and animals prove that the nacre protein N16 can inhibit receptor activator of nuclear factor kappa B ligand (RANKL) from inducing the proliferation and the differentiation of osteoclast precursors RAW 264.7 cells, promotes the differentiation and the maturation of precursor osteoblast MC3T3-E1 cells, enhances the mineralization function of osteoblasts, treat the osteoporosis caused by ovariectomizing, and can be used in the preparation of drugs used for the prevention or the treatment of orthopedic diseases such as the osteoporosis and the fracture.

Description

technical field [0001] The invention relates to a preparation method of mother of pearl protein N16 and its use for preventing and treating orthopedic diseases such as osteoporosis and fractures. Background technique [0002] Osteoporosis refers to a systemic bone metabolic disorder characterized by low bone mass and tissue microarchitectural degeneration, increased bone fragility, and increased risk of fracture. The disease is especially common in middle-aged and elderly people. According to statistics from the International Osteoporosis Foundation, osteoporosis currently affects about one-third of women and one-fifth of men over the age of 50 in the world. At present, about 200 million people in the world suffer from osteoporosis, and its incidence rate has jumped to the seventh place among common diseases and frequently-occurring diseases. my country is the most populous country in the world, and the incidence of osteoporosis is as high as 90% among people over 65 years...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/70C07K14/435C07K1/18C07K1/16A61K38/17A61P19/10A61P19/08
Inventor 苏薇薇邵鹏柱马结仪王永刚彭维吴忠
Owner SUN YAT SEN UNIV
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