小鼠DNAJB13与HK1的相互作用

Interaction of DNAJB13 with HK1 in mouse

目的探讨小鼠DNAJB13与HK1是否具有相互作用。方法应用双酶切连接方法构建p GEX-4T-1/Dnajb13原核表达载体,测序验证;重组质粒转化感受态细胞DH5α,用IPTG诱导融合蛋白GST-DNAJB13表达,采用SDS-PAGE考马斯亮蓝染色和Western blotting分析蛋白和鉴定;提取小鼠睾丸蛋白,采用GST pull down检测DNAJB13与HK1是否具有相互作用。结果成功构建p GEX-4T-1-Dnajb13重组质粒,测序结果与标准序列一致;转化重组质粒的大肠杆菌在37℃,IPTG浓度1 mmol/L诱导下高效表达融合蛋白;GST pull down检测结果阳性,显示DNAJB13与HK1存在相互作用。结论在小鼠睾丸中,DNAJB13与HK1存在相互作用,可能参与精子形成和精子运动。

Objective To investigate the presence of interactions between DNAJB13 and HK1. Method The open reading frame of Dnajb13 gene was amplified from mouse testis c DNA by PCR. The PCR products were then inserted into p GEX-4T-1 vector after double digestion and identified by sequencing. The recombinant plasmids were transformated into competent DH5 a cells, and the fusion protein was expressed with IPTG induction. SDS-PAGE Coomassie brilliant blue staining and Western blot analysis were used to detect the fusion protein expression. The protein precipitated by GST-DNAJB13 in GST pull down assay was detected by Western blotting. Results The recombinant plasmid p GEX-4T-1-Dnajb13 was successfully constructed and verified. E.coli transformed with the recombinant plasmid expressed abundant fusion protein. GST pull down assay showed interactions between DNAJB13 and HK1. Conclusion DNAJB13 interacts with HK1 in mouse testis and probably participates in spermatogenesis and the regulation of sperm motility.