Preparation method and application of sclerostin single-chain antibody

A sclerostin and single-chain antibody technology, applied in biochemical equipment and methods, botanical equipment and methods, antibodies, etc., can solve the problem of increasing osteosarcoma, increasing bone density of postmenopausal women, unsatisfactory application of anabolic drugs, etc. question

Inactive Publication Date: 2018-06-01
GENERAL HOSPITAL OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Teriparatide is the most effective drug for preventing new non-vertebral fractures, and currently the only drug that can promote bone formation to treat osteoporosis, but there are many limitations in the clinical use of teriparatide: 1. Teriparatide Patients need to inject themselves every day and need to be frozen; 2. The United States restricts the use of teriparatide for no more than 2 years, and the EU recommends 18-24 months; 3. Teriparatide has an increased risk of osteosarcoma; 4. With the use of Over time, teriparatide will gradually increase the degree of bone resorption
At present, the clinical application of anabolic drugs to treat osteoporosis by promoting osteogenesis is not ideal
A humanized sclerostin monoclonal antibody (AMG785) was used in clinical randomized controlled trials and proved to be effective in increasing bone mineral density in postmenopausal women. However, the sclerostin monoclonal antibody has too large molecular weight and strong immunogenicity. and other shortcomings

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  • Preparation method and application of sclerostin single-chain antibody
  • Preparation method and application of sclerostin single-chain antibody
  • Preparation method and application of sclerostin single-chain antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1 Preparation of sclerostin single-chain antibody:

[0067] Sclerostin single-chain antibody needs to be prepared in advance, and then used in the treatment and experimental research of osteoporosis and osteoporosis-related fractures.

[0068] Preparation method of sclerostin single chain antibody:

[0069] Collect the total RNA of SOST monoclonal antibody 5H3D1 hybridoma cells in good growth state, break the cells with T R Izo1 reagent, extract with chloroform, precipitate with isopropanol, wash with 75% ethanol, dry and treat with diethyl pyrocarbonate Water dissolves the RNA. Using the extracted RNA as a template, cDNA was synthesized according to the operating procedures of the RT-PCR kit.

[0070] Design VL and VH primers based on the variable regions of rabbit monoclonal antibodies, and use degenerate codons to adapt to the diversity of light and heavy chains (S=G, C; M=A, C; R=A, G; W=A, T; Y = C, T);

[0071] The Linker is designed on the VL downstre...

Embodiment 2

[0084] Example 2 Analysis of the coding sequence of the sclerostin single-chain antibody:

[0085] The cells produced in Example 1 were purified from the plasmid in the cell line using a DNA extraction kit, and purified and separated by restriction endonuclease cutting and 2% agarose gel electrophoresis to obtain the coding sequence of the heavy chain variable region. The coding sequence of the light chain variable region was obtained by using the method, and the human antibody variable region universal primer was used for polymerase chain reaction amplification.

[0086] The light chain region of sclerostin scFv

[0087] SEQGACCATA TGCTGACCCA GAGTGCATCG CTCGTGTCTG CCGCTGTGCG AAGCATAGTC 60

[0088] ACCATCAATA GCCAGGCCAG TCAGAGTGTT TATAAGAACA ACTACTTAGC CTGGTTTCAG 120

[0089] CAGAAACCAG GGCAGCCTCC CAAGCGCCTG ATCTCATCTG CATCGACTCT GGCTTCTGCG 180

[0090] GTCTCGTCGG GGTTCAACGG CAGTCGATCC GGGACACAGA TCACTCTCAT CATCAGCGAC 240

[0091] GTGCAGTGTG ACGATGCTGG CACTTACTAC TGTCTGGACA ...

Embodiment 3

[0106] Example 3 Application of sclerostin single-chain antibody:

[0107] 1. Detection of the proliferation and differentiation ability of sclerostin single-chain antibody (Scl-scFv) on BMSCs; co-culture of sclerostin single-chain antibody (Scl-scFv) with mouse bone marrow mesenchymal stem cells, and the proliferation of cells was detected by MTT method Ability; BMSCs osteogenic induction 2 days after co-culture with sclerostin single-chain antibody, on the 7th day Real-Time PCR detection type I collagen (COL-1), alkaline phosphatase (ALP), RUNX2, osteopontin OPN, Osteocalcin (OCN) mRNA expression; on the 4th, 7th, and 10th day, Western blot was used to detect type I collagen, and ELISA was used to detect the expression of osteocalcin. Results The OD value of MTT in the Scl-scFv group was not significantly different from that in the blank control group; the expression of type I collagen, alkaline phosphatase, RUNX2, OPN, and OCN mRNA in the experimental group treated with Scl...

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Abstract

The invention provides a preparation method and application of a sclerostin single-chain antibody. The preparation method comprises the following steps: extracting anti-SOST monoclonal antibody 5H3D1hybrid tumor cell total RNA; amplifying the antibody light-chain variable region gene and heavy-chain variable region gene by an RT-PCR method; introducing Linker3 between genes VL and VH by an overlapping extending splicing PCR method; forming the single-chain antibody SOST-scFv through connection. The scFv gene with the correct sequencing is cloned to an expression vector PET-22b(+) and is thentransferred into E.coliBL21 for solubility expression; polyacrylamide gel electrophoresis SDS-PAGE is used for identifying expression products; ELISA and West-blot are used for detecting the reactionactivity of the expression protein. The preparation method and the application have the advantages that the molecular weight is small; the permeability is high; the antigen combination activity is high; the bone generation is obviously promoted; the bone loss is inhibited.

Description

technical field [0001] The patent of the invention belongs to the single-chain antibody of sclerostin, which is mainly used for the treatment of osteoporosis and osteoporosis-related fractures. Background technique [0002] Osteoporosis is a disorder of the skeletal system characterized by low bone mass and microstructural deterioration of bone tissue, leading to bone fragility and increased fracture risk. At present, osteoporosis has become a chronic common disease in the elderly worldwide, and the treatment of osteoporosis-related fractures is difficult. The average prevalence of osteoporosis among Chinese elderly is about 15.7%, and the prevalence will gradually increase with age. By the end of 2013, the number of elderly people over 60 years old is expected to reach 202.43 million. Osteoporosis bears a huge financial burden, and the risk of osteoporosis-related fractures in women and men is 40% and 13%, respectively. The prevalence of osteoporosis in Korean women and m...

Claims

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Application Information

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IPC IPC(8): C07K16/18C12N15/13C12N15/85A61K39/395A61P19/10A61P19/08
Inventor 姜华姚琦唐佩福
Owner GENERAL HOSPITAL OF PLA
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