目的:根据家族性寒冷性自身炎症综合征(familial cold autoinflammatory syndrome, FCAS)家族的NLRP3(p.V72M,c.214G＞A)、NLRP12(p.R754H,c.2261G＞A)基因突变位点,构建携带Wistar大鼠NLRP3、NLRP12突变的基因质粒载体。方法:依据氨基酸同源性,设计大鼠NLRP3、NLRP12基因突变质粒,利用聚合酶链式反应(polymerase chain reaction, PCR)方法扩增目的基因并回收,双酶切并连接在pCMV-mCherry-MCS-Neo载体上,连接产物转化感受态细胞,菌落PCR鉴定阳性转化子,质粒小提电泳并测序鉴定。结果:琼脂糖凝胶电泳显示,成功扩增出NLRP3、NLRP12突变基因,基因突变质粒经酶切电泳及DNA测序证实,基因序列完全正确,重组质粒载体构建成功。结论:成功构建NLRP3、NLRP12基因突变的质粒载体,为进一步探讨NLRP3、NLRP12基因突变导致FCAS发病的机制研究以及NLRP3、NLRP12基因的功能性研究提供生物学基础。
黄毓娴
,
郝金奇
,
余艳琴
,
贾喜梅
,
张宏佳
,
王利全
,
裘瑞泽
,
王秀春
,
石继海
. NLRP3、NLRP12基因突变质粒载体构建及应用*[J]. 包头医学院学报, 2025
, 41(1)
: 14
-19
.
DOI: 10.16833/j.cnki.jbmc.2025.01.003
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.
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