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lgr4 protein fragment and its application in the preparation of medicine for treating osteoclast-induced bone disease

A protein fragment, osteoclast technology, applied in the field of medicine for bone diseases, can solve the problems of high mortality, no literature report, developmental delay and other problems

Active Publication Date: 2020-03-27
EAST CHINA NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the intrauterine growth retardation and high postnatal mortality of LGR4 gene knockout mice also indicate that LGR4 has a very important impact on mouse embryonic development (Mendive F, Laurent P, Van Schoore G, et al. Defective postnatal development of the male reproductive tract in LGR4 knockout mice.Dev Biol,2006,290(2):421-34.), but so far there is no literature report on the function of LGR4 in the embryonic development of specific organs

Method used

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  • lgr4 protein fragment and its application in the preparation of medicine for treating osteoclast-induced bone disease

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1: Purification and expression of LGR4 protein fragments

[0070] The LGR4 gene was amplified by PCR, and the amplified product was constructed on a PET-28a(+) prokaryotic expression vector, and PET-28a-ECD was transformed into Rosetta bacteria (full gold, cd801-03). The construction process refers to Table 1.

[0071] Table 1

[0072]

[0073]

[0074] According to the general prokaryotic expression method reported in "Acta Biophysica Sep. 2010, Vol. 26, No. 9: 790-798", the above vector was transformed into Rosetta bacteria to induce the expression of the LGR4 protein fragment. The experimental results are shown in Figure 1.

[0075] Figure 1a : LGR4 protein fragment (LGR4 ECD, h4) SDS-PAGE Coomassie Brilliant Blue result map, the arrow indicates the purified LGR protein fragment.

[0076] Figure 1b : Western blot results of LGR4 protein fragment (LGR4 ECD, h4), the target band in group h4 is the LGR4 protein fragment confirmed by His-tag antibody de...

Embodiment 2

[0077] Example 2: LGR4 protein fragment binding RANKL experiment

[0078] Use lipofectamine2000 to overexpress the plasmid containing the above LGR4 fragment (see below for the construction process) in 293T cells according to the manufacturer's protocol. After 24 hours of transfection, the cells were washed once with PBS, scraped off with a cell scraper, and centrifuged at 12,000rpm for one minute. Lyse the cell block with RIPA weak lysate on ice for 15 minutes, centrifuge at 12000rpm at 4 degrees for 10 minutes, take the supernatant into a new EP tube, add 500ng RANKL and 200ngRSPO-1 (positive control) respectively; set the EP tube in a silent mixer Incubate overnight at 4°C (<12 hours), add 5 microliters of FLAG-M2 beads to each tube the next day, continue to incubate at 4°C for 3 hours, then centrifuge at 3000rpm at 4°C for 1 minute, remove the supernatant, and wash 3 times with PBS The beads were then lysed with 1XSDS loading buffer, boiled at 100°C for 10 minutes, and the...

Embodiment 3

[0091] Example 3: LGR4 protein fragment inhibits RANKL-RANK binding experiment

[0092] Use lipofectamine2000 to overexpress 10ug LGR4 fragment plasmids (see Table 2), 6ugRank, 4ug FLAG-Vector, and 2ugEGFP-N3 in 293T cells respectively according to the manufacturer's protocol. After 24 hours of transfection, the cells were washed once with PBS and washed with cell Scrape off with a scraper, centrifuge at 12000rpm for one minute, then lyse the cell mass with RIPA weak lysate on ice for 15 minutes, centrifuge at 12000rpm at 4 degrees Celsius for 10 minutes, take the supernatant into a new EP tube, and mix the protein lysate according to the following design: 2ugEGFP -N3+2ugFLAG, 2ugRANK+2ugFLAG, 2ugRANK+2ugLGR4 fragments, 2ugRANK+8ugLGR4 fragments, and then add 100ngRANKL respectively; EP tubes were incubated overnight at 4 degrees Celsius on a silent mixer, and 4 microliters of RANK antibody (1: 50), incubate at 4°C for 3 hours, then add 20 microliters of proteinA / G beads, cont...

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Abstract

The invention provides a separated LGR4 protein fragment shown as SEQ ID NO.1. The protein has a protein sequence shown as SEQ ID NO.1. The invention further provides application of the protein fragment to preparation of a drug for treating the osteoclast induced bone disease. The protein fragment can inhibit differentiation of osteoclast in the body. Further, the invention provides application of the protein fragment to study of osteoclast differentiation inhibition in vitro or in vivo.

Description

technical field [0001] The present invention relates to the protein fragment of LGR4 (Leucine-rich repeat containing G-protein coupled receptor 4, leucine-rich repeating motif G-protein coupled receptor 4) in the field of biotechnology, specifically, the present invention relates to a class of The LGR4 protein fragment related to osteoporosis and giant cell tumor of bone, and its application in the preparation of medicine for treating osteoclast-induced bone disease. Background technique [0002] In recent years, the research on osteoclast regulation of human physiological functions has been increasing, and more and more studies have proved that osteoclasts not only participate in the homeostasis of bone metabolism in organisms, but also play a role in the regulation of the immune system (ImmunolRev.2005Dec; 208: 19-29.). More importantly, in many pathological conditions, osteoclast function loss or overactivation occurs from time to time, which increases the suffering of p...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/705C12N15/12A61K38/17A61P19/10A61P35/00A61P35/04
Inventor 罗剑杨正峰刘明耀
Owner EAST CHINA NORMAL UNIV
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