Objective: To construct a gene plasmid vector carrying NLRP3 and NLRP12 mutations in Wistar rats according to the NLRP3 (p.V72M, c.214G＞A) and NLRP12 (p.R754H, c.2261G＞A) gene mutation sites of the familial cold autoinflammatory syndrome (FCAS) family. Methods: According to the amino acid homology, the NLRP3 and NLRP12 gene mutation plasmids of rats were designed. The target genes were amplified by polymerase chain reaction (PCR) and recovered. The double enzyme digestion and connection were performed on the pCMV-mCherry-MCS-Neo vector. The connected products were transformed into competent cells. The positive transformants were identified by colony PCR, plasmid extraction electrophoresis and sequencing. Results: Agarose gel electrophoresis showed that the NLRP3 and NLRP12 mutant genes were successfully amplified. The gene mutation plasmid was confirmed by enzyme digestion electrophoresis and DNA sequencing. The gene sequence was completely correct and the recombinant plasmid vector was successfully constructed. Conclusion: The plasmid vectors of NLRP3 and NLRP12 gene mutations is successfully constructed, which provides a biological basis for further exploring the pathogenesis of FCAS caused by NLRP3 and NLRP12 gene mutations and the functional study of NLRP3 and NLRP12 genes.
HUANG Yuxian
,
HAO Jinqi
,
YU Yanqin
,
JIA Ximei
,
ZHANG Hongjia
,
WANG Liquan
,
QIU Ruize
,
WANG Xiuchun
,
SHI Jihai
. Construction and application of NLRP3 and NLRP12 gene mutation plasmid vectors[J]. Journal of Baotou Medical College, 2025
, 41(1)
: 14
-19
.
DOI: 10.16833/j.cnki.jbmc.2025.01.003
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