致白内障基因Hsf4b K65R位点突变对下游热休克蛋白表达的影响

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-time PCR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-HAHsf4b/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。

Eeffect of K65R site-mutagenesis of cataracts-related gene Hsf4b on downstream heat shock proteins expression

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 generated 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 Hsf4b/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 Hsp70i 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 influences the regulation of downstream heat shock proteins.