HK11的相互作用.PDFVIP

J South Med Univ, 2016, 36(12): 1684-1688 doi 10.3969/j.issn. 1673-4254.2016.12.16 ·1684 · 基础研究 小鼠小鼠DNAJBDNAJB1133 与与HKHK11 的相互作用的相互作用 1 2 1 1 1 杨梦月 ，熊紫薇 ，李维娜 ，贾苗苗 ，刘 刚 1 2 中南大学生殖与干细胞工程研究所，湖南 长沙 410078 ；益阳市中心医院，湖南 益阳 413000 摘要：目的 探讨小鼠DNAJB 13与HK 1是否具有相互作用。方法 应用双酶切连接方法构建pGEX-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DNAJB 13与HK 1是否具有相互作用。结果 成 功构建pGEX-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DNAJB 13与HK 1存在相互作用。结论 在小鼠睾丸中，DNAJB 13与 HK 1存在相互作用，可能参与精子形成和精子运动。 关键词：DNAJB 13；重组质粒；融合蛋白；HK 1；GST pull down ；精子发生；精子运动 Interaction of DNAJB13 with HK1 in mouse 1 2 1 1 1 YANG Mengyue , XIONG Ziwei , LI Weina , JIA Miaomiao , LIU Gang 1Institute of Reproductive and Stem Cell Engineering, Central South University, Changsha 410078, China; 2 Yiyang Central Hospital, Yiyang 413000, China Abstract: Objective To investigate the presence of interactions between DNAJB13 and HK1. Method The open reading frame of Dnajb13 gene was amplified from mouse testis cDNA by PCR. The PCR products were then inserted into pGEX-4T-1 vector after double digestion and identified by sequencing. The recombinant plasmids were transformated into competent DH5a cells, and the fusion protein was expressed with IPTG induction. SDSCoomassie 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 pGEX-4T-1-Dnajb13 was successfully constructed and verified. E.coli transformed with the recombin