Human wnt5a-nl nucleic acid recombinant and its preparation method and application

A recombinant and nucleic acid technology, applied in the field of biomedicine, can solve problems such as bone destruction

Inactive Publication Date: 2021-06-22
CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The results of Wnt5a heterozygous mice and co-culture experiments show that too much or too little Wnt5a will lead to bone destruction, too much Wnt5a will promote the formation of osteoclasts through the Wnt5a-Ror2 pathway, and too little Wnt5a will inhibit the formation of osteoblasts Function, osteoblast and osteoclast function imbalance will cause bone destruction

Method used

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  • Human wnt5a-nl nucleic acid recombinant and its preparation method and application
  • Human wnt5a-nl nucleic acid recombinant and its preparation method and application
  • Human wnt5a-nl nucleic acid recombinant and its preparation method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Example 1 Construction of Human Wnt5a-NL Nucleic Acid Recombinant

[0034] According to the characteristics of Wnt family protein structure, construct phWnt5a-NL nucleic acid recombinant, such as figure 1 As shown, the selected target expression vector is pCMV6-XL5, and the target fragment used is a fragment of human Wnt5a N-terminal linker (NTD-Linker, NL) (125-273aa, SEQ ID NO: 1). First, the amino acid and cDNA full-length sequences of hWnt5a were obtained from the BLAST database, and the amino acid sequence of hWnt5a was input into the SignalP online signal peptide prediction server to obtain the signal peptide sequence of hWnt5a (SEQ ID NO:5). At both ends of the signal peptide and the cDNA sequence of the selected hWnt5a-NL region, artificially design primers according to the conventional primer design principles; add XbaI and EcoRI restriction internals to the 5' end of the upstream primer of the signal peptide and the downstream primer of the hWnt5a-NL fragment,...

Embodiment 2

[0040] Embodiment 2 Amplification and extraction of human Wnt5a-NL nucleic acid recombinant in Escherichia coli

[0041] The Escherichia coli transformed with the recombinant plasmid was inoculated in 10 mL of LB medium (containing 100 mg / L of Amp + antibiotics), cultured overnight at 37°C with shaking at 250rpm. The next day, 10 mL of the overnight culture was transferred to 1 L of LB medium (containing 100 mg / L of Amp + Antibiotics), 37°C, 250rpm culture to OD 600 Value around 2.0. Bacteria were collected by centrifugation. The recombinant plasmid was extracted according to the instruction manual of plasmid extraction. The concentration and purity of the DNA were measured by an ultraviolet spectrophotometer to ensure that the ratio of the extracted plasmid A260 / A280 was between 1.8-2.0.

Embodiment 3

[0042] Example 3 In vivo and in vitro expression identification of human Wnt5a-NL nucleic acid recombinant

[0043] The recombinant plasmid prepared in Example 2 was transiently transfected into human 293T cells, and the cells were collected 48 hours later; the total protein was extracted according to the instructions of the protein extraction kit; the expression product was identified in vitro by Western blot. Ensure that the confluence of the cells is 70%-90% during transfection; dilute 1 μg of plasmid and 3 μL of Lipofectamine 2000 transfection reagent (purchased from Invitrogen, catalog number: 11668-027) with 250 mL of Opti-MEM respectively, and then mix them and incubate at room temperature 20min. After taking out the cells, discard the supernatant, wash with PBS once, add the mixed plasmid and transfection reagent mixture, and drop gently into the culture dish. After 4-5 hours, add DMEM complete medium to culture overnight, replace the medium once with complete medium,...

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Abstract

The present invention provides a novel human Wnt5a-NL nucleic acid recombinant and its preparation method and application. The human Wnt5a-NL nucleic acid recombinant of the present invention is a recombinant plasmid obtained by constructing the human Wnt5a protein truncated gene encoding gene shown in SEQ ID NO: 1 on a eukaryotic expression vector. Experiments have shown that human Wnt5a-NL nucleic acid recombinant injection mice can inhibit inflammation and bone destruction, and can be developed for the treatment of rheumatoid arthritis and other diseases. The nucleic acid recombinant provided by the invention belongs to the therapeutic plasmid, which can provide a new treatment approach and idea for rheumatoid arthritis and other diseases. The nucleic acid recombinant has good therapeutic effect, simple preparation, low cost, good stability and convenient storage, and is suitable for large-scale production.

Description

technical field [0001] The present invention relates to the field of biomedicine, in particular to a human Wnt5a-NL nucleic acid recombinant and its preparation method and application. Background technique [0002] Rheumatoid arthritis (RA) is a chronic inflammatory disease that can lead to systemic bone loss and increased fracture risk. The bone imaging can show periarticular osteoporosis and marginal erosion. Erosive synovitis, erosive destruction of articular cartilage and bone tissue can be seen in pathological examination. Periarticular bone loss in the early stages of the disease and later development of marginal erosions can lead to periarticular osteoporosis, which in turn leads to a generalized decrease in bone mineral density. Bone loss, a complication of RA, occurs both locally and systemically, which increases fracture risk. Recent reports suggest that activation of the wingless (WNT) signaling pathway can accelerate bone resorption by stimulating the activati...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/47C07K19/00C12N15/12C12N15/62A61K38/17A61K48/00A61P19/02A61P29/00
CPCA61K38/00A61K48/00C07K14/47C07K2319/02C12N15/63
Inventor 曹伟袁慧慧王映霏赵文明
Owner CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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