CAJ | 학술논문

目的：探讨致白内障基因热休克因子4b（Hsf4b） K65R位点突变对其调控下游热休克蛋白（HSP）表达的影响。方法：采用KOD－Plus－Mutagenesis－Kit试剂盒构建pWZL－blast－HA－Hsf4b／K65R赖氨酸突变质粒；通过慢病毒感染小鼠晶状体上皮细胞mLEC构建稳定表达Hsf4b／K65R突变质粒的细胞株；Western blotting 检测Hsf4b在mLEC K65R点突变细胞株和野生株中的表达；Western blotting 及real－timePCR检测K65R点突变后下游蛋白Hsp70、Hsp90、Hsp27和CryAB表达的变化。结果：阳性克隆PCR及基因测序证明慢病毒载体pWZL－blast－HA－Hsf4b／K65R构建成功。 K65R点突变后不影响Hsf4b在小鼠晶状体上皮细胞mLEC中的表达，但能影响下游蛋白CryAB、Hsp27、Hsp70i和Hsp90a的表达。结论：pWZL－blast－HA－Hsf4b／K65R载体可用于慢病毒感染稳定细胞株构建。 Hsf4b K65R位点突变能显著影响其对热休克蛋白的调控功能。
목적：탐토치백내장기인열휴극인자4b（Hsf4b） K65R위점돌변대기조공하유열휴극단백（HSP）표체적영향。방법：채용KOD－Plus－Mutagenesis－Kit시제합구건pWZL－blast－HA－Hsf4b／K65R뢰안산돌변질립；통과만병독감염소서정상체상피세포mLEC구건은정표체Hsf4b／K65R돌변질립적세포주；Western blotting 검측Hsf4b재mLEC K65R점돌변세포주화야생주중적표체；Western blotting 급real－timePCR검측K65R점돌변후하유단백Hsp70、Hsp90、Hsp27화CryAB표체적변화。결과：양성극륭PCR급기인측서증명만병독재체pWZL－blast－HA－Hsf4b／K65R구건성공。 K65R점돌변후불영향Hsf4b재소서정상체상피세포mLEC중적표체，단능영향하유단백CryAB、Hsp27、Hsp70i화Hsp90a적표체。결론：pWZL－blast－HA－Hsf4b／K65R재체가용우만병독감염은정세포주구건。 Hsf4b K65R위점돌변능현저영향기대열휴극단백적조공공능。
AIM:To clarify the impact of heart shock factor 4b (Hsf4b) K65R mutation on the regulation of downstream protein expression .METHODS:Non-functional Lys mutant plasmid pWZL-blast-HA-Hsf4b/K65R was genera-ted by replacing single , homologous amino acids using KOD-Plus-Mutagenesis-Kit.Mouse lens epithelial mLEC stable cell lines expressing Hsf4b or Hsf4b/K65R were constructed by lentivirus infection .The expression of Hsf4b in the mutant and the wildtype mLEC cells was confirmed by Western blotting .The expression of Hsf4b downstream proteins such as heat shock protein ( Hsp)70, Hsp90, Hsp27 and CryAB was examined by Western blotting and real-time PCR.RESULTS:The results of PCR and DNA sequencing confirmed the successful construction of mLEC Hsf 4b/K65R mutant.The K65R mutation didn’t influence Hsf4b expression in the mLEC cells.After K65R mutation in Hsf4b, the expression levels of Hsp27 and CryAB were down-regulated and the expression of Hsp 70i and Hsp90a upregulated.CONCLUSION: pWZL-blast-HA-Hsf4b/K65R can be used to construct a stable cell line by infecting with lentivirus .Hsf4b/K65R mutation influ-ences the regulation of downstream heat shock proteins .