Construction, expression and application of a mouse rankl mutant and its expression vector
A technology of expressing vectors and mutants, which is applied in the fields of microbiology, molecular biology and genetic engineering, and can solve the problems of unclear positioning and its influencing mechanism
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Embodiment 1
[0047] Example 1 Construction of mouse pEGFP-RANKL vector
[0048] The mouse osteoblast-like cells UAMS-32 were cultured in the following medium: 90% α-MEM, 10% FBS, 100 U / ml penicillin, 0.1 mg / ml streptomycin. The UAMS-32 cells were placed in a 60mm petri dish, and the total RNA was extracted after the cells were confluent, and then reverse transcribed into cDNA. The cDNA was used as a template for PCR reaction. The primers used were RANKLFor:
[0049] 5'-AT CTCGAG CTATGCGCCGGG-3' and RANKLRev:
[0050] 5'-TTCG AAGCTT GTCAGTCTATGTCCTG-3', the above underlined bases are the recognition sites of the introduced XhoI and HindIII restriction enzymes, respectively.
[0051] The PCR reaction system was: 2×GCbuffer Ⅰ 25 μL, cDNA template 1 μL, primers (10 μM) 1 μL each, dNTP mix (2.5 mM) 8 μL, TaKaRa LA Taq 0.5 μL, double-distilled H 2 O supplemented to 50 μL; the PCR reaction conditions were: pre-denaturation at 94 °C for 5 min, 94 °C for 30 s, 55 °C for 30 s, 72 °C for 2 min,...
Embodiment 2
[0052] Example 2 Acquisition of mouse RANKL gene with different Cys site mutations and its expression vector
[0053] The Agilent multi-site-directed mutagenesis kit was used to perform site-directed mutagenesis of different Cys sites in the mouse RANKL gene fragment, and the codon sequence tgc of Cys in the RANKL gene fragment was replaced with the codon sequence ggc of Gly. The site-directed mutagenesis primers used Use Agilent's site-directed mutagenesis primer design software (https: / / www.genomics.agilent.com / primerDesignProgram.jsp) to design and obtain (see Table 1):
[0054] Table 1 Multisite site-directed mutagenesis primers
[0055]
[0056]
[0057] The pEGFP-RANKL plasmid prepared in Example 1 was used as a template for PCR reaction. The PCR reaction system was: 10×QuikChange Lightning Multi reaction buffer 2.5 μL, QuikSolution 0.5 μL, ds-DNA template 100 ng, mutagenic primers 50 ng each, dNTPs mix 1μL, QuikChangeLightning Multi enzyme blend 1μL, double-disti...
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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