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Mouse RANKL (Receptor Activator of Nuclear Factor Kappa B Ligand) mutant as well as establishment, expression and application of expression carrier of mutant

A technology for expressing vectors and mutants, which is applied in the fields of molecular biology, genetic engineering, and microbiology, and can solve the problems of unclear positioning and its impact mechanism

Active Publication Date: 2014-09-24
JIANGSU INST OF NUCLEAR MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, compared with the well-explained RANKL-RANK interaction signaling mechanism leading to osteoclast differentiation and maturation, the localization of RANKL protein in osteoblasts and its impact mechanism are still unclear.

Method used

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  • Mouse RANKL (Receptor Activator of Nuclear Factor Kappa B Ligand) mutant as well as establishment, expression and application of expression carrier of mutant
  • Mouse RANKL (Receptor Activator of Nuclear Factor Kappa B Ligand) mutant as well as establishment, expression and application of expression carrier of mutant
  • Mouse RANKL (Receptor Activator of Nuclear Factor Kappa B Ligand) mutant as well as establishment, expression and application of expression carrier of mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Mouse pEGFP-RANKL vector construction

[0048] Mouse osteoblast-like cells UAMS-32 were cultured, and the medium composition used was: 90% α-MEM, 10% FBS, 100U / ml penicillin, 0.1mg / ml streptomycin. Spread UAMS-32 cells on a 60mm culture dish, extract total RNA after the cells are full, and then reverse transcribe it into cDNA, use the cDNA as a template to carry out PCR reaction, the primers used are RANKLFor:

[0049] 5'-AT CTCGAG CTATGCGCCGGG-3' and RANKLRev:

[0050] 5'-TTCG AAGCTT GTCAGTCTATGTCCTG-3', the underlined bases above are the recognition sites of the introduced XhoI and HindIII restriction endonucleases, respectively.

[0051] The PCR reaction system is: 2×GCbuffer Ⅰ 25μL, cDNA template 1μL, primers (10μM) 1μL each, dNTP mix (2.5mM) 8μL, TaKaRa LA Taq 0.5μL, double-distilled H 2 O to 50 μL; the PCR reaction conditions are: 94 ° C pre-denaturation for 5 min, 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 2 min, 30 cycles, 72 ° C extension for ...

Embodiment 2

[0052] Embodiment 2 The acquisition of mouse RANKL gene and its expression vector with different Cys site mutations

[0053] Use the Agilent multi-site site-directed mutagenesis kit to perform site-directed mutation on different Cys sites of the mouse RANKL gene fragment, replace the codon sequence tgc of Cys in the RANKL gene fragment with the codon sequence ggc of Gly, and use the site-directed mutagenesis primers Using Agilent's site-directed mutagenesis primer design software (https: / / www.genomics.agilent.com / primerDesignProgram.jsp) to design (see Table 1):

[0054] Table 1 Primers for multi-site site-directed mutagenesis

[0055]

[0056]

[0057] Use the pEGFP-RANKL plasmid prepared in Example 1 above as a template to carry out PCR reaction. The PCR reaction system is: 10×QuikChange Lightning Multi reaction buffer 2.5μL, QuikSolution 0.5μL, ds-DNA template 100ng, mutagenic primers 50ng each, dNTP mix 1μL, QuikChange Lightning Multi enzyme blend 1μL, double-distil...

Embodiment 3

[0065] Embodiment 3 Contains the recombinant cell of described expression vector and the construction of expression system

[0066] The recombinant cells are constructed by the following method:

[0067] Transfection and screening: Take the pEGFP-C1 plasmid, the pEGFP-RANKL plasmid prepared in Example 1, and the mouse RANKL mutant expression vector prepared in Example 2, respectively, and digest the above plasmids with restriction endonuclease ApaLI to make them linearized. Then HEK293 cells were digested and plated in 6-well plates, with 500,000 cells per well, and cultured until the next day. Using Lipofectamine2000, 2.5 μg of linearized plasmids were used to transfect the HEK293 cells. After 24 hours of culture, the cells were digested to 100 mm culture After the cells adhere to the wall, change the medium, and add G418 with a final concentration of 400 μg / mL. The transfected HEK293 is the experimental group, and the control group is set as the screening control. This group...

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Abstract

The invention discloses a mouse RANKL (Receptor Activator of Nuclear Factor Kappa B Ligand) mutant as well as establishment, expression and application of an expression carrier of the mutant, provides the mouse RANKL mutant, an establishment method of the expression carrier of the mouse RANKL mutant, and a preparation method for transfecting the expression carrier of the mouse RANKL mutant into a cell, and further provides a cell and an expression system for expressing the expression carrier of the mouse RANKL mutant. The problem in the prior art that the mouse RANKL cannot be conveniently and efficiently studied is solved.

Description

technical field [0001] The invention belongs to the technical fields of microbiology, molecular biology and genetic engineering, and specifically relates to a mouse nuclear factor kappa B receptor activator ligand (RANKL) with different cysteine ​​site (Cys) mutations having biological activity. Construction, expression and application of mutants and their vectors. Background technique [0002] Bone has the characteristics of repeated formation, absorption and reconstruction for its own morphological changes and maintenance of blood calcium concentration. Normal bone is in a state of dynamic balance through bone formation mediated by osteoblasts and osteocytes and bone resorption mediated by osteoclasts. The maintenance of bone homeostasis is a multi-factor and multi-link complex regulatory process, and the signal coupling among osteoblasts, osteocytes and osteoclasts is the core. This signal coupling is regulated by a series of secreted proteins, the most important of whi...

Claims

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Application Information

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IPC IPC(8): C07K14/47C12N15/12C12N15/63C12N5/10C12N15/85G01N33/68
CPCC07K14/70575C12N15/85
Inventor 彭莹邓黎莉吴郁陈全成丁月娣束婷婷付强
Owner JIANGSU INST OF NUCLEAR MEDICINE
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