Interaction between SPAG6 and TAC1 proteins

: 目的： 构建速激肽1（Tac1）基因真核表达载体并探讨其与精子相关抗原6（SPAG6）之间的相互作用。方法： 提取10只昆明雄性小鼠的心、肝、脾、肺、肾、脑、肌肉、睾丸8个组织的RNA，逆转录成cDNA后，用RT-PCR法观察Tac1在各组织中的表达。构建Tac1/pGADT7、Tac1/pEGFP-N2重组质粒，将Tac1/pEGFP-N2转染至CHO和COS-1细胞，采用免疫荧光染色及Western印迹检测TAC1在细胞中的定位和蛋白表达。将Tac1/pGADT7与Spag6/pGBKT7进行酵母双杂交实验，提取酵母蛋白用Western印迹检测TAC1与SPAG6之间是否存在相互作用。 结果： Tac1主要在睾丸、脑、心脏中表达。酶切和测序鉴定表明Tac1重组质粒构建成功，酶切鉴定片段390 bp。TAC1单独转染时，定位于CHO细胞的整个胞体，与SPAG6质粒共转染后，TAC1被募集至细胞微管中表达，用Western印迹检测到相对分子质量约为40 000的融合蛋白表达。与SPAG6的酵母双杂交实验显示TACl/SPAG6在缺少3种氨基酸（Leu、Trp、His）的培养板上有菌落生成，经Western印迹证实酵母融合蛋白中含有TAC1和SPAG6。结论： 成功构建Tac1重组质粒，并利用该质粒证实了TAC1与SPAG6之间存在相互作用关系。. METHODS: RNA was extracted from the heart, liver, spleen, lung, kidney, brain, muscle, and testis of 10 Kunming male mice and, after reverse transcription into cDNA, the expression of Tac1 in the above tissues was observed by RT-PCR. Tac1/pEGFP-N2 and Tac1/pGADT7 recombinant plasmids were constructed and Tac1/pEGFP-N2 was transfected into CHO and COS-1 cells, followed by localization and detection of the protein expression of TAC1 by immunofluorescence staining and Western blot. The interaction between TAC1 and SPAG6 was determined by yeast two-hybrid experiment and Western blot. RESULTS: Tac1 was expressed mainly in the testis, brain and heart. The results of restriction enzyme digestion and sequencing indicated successful construction of the recombinant plasmids, with the restriction fragment length of 390 bp. TAC1 was localized in the whole body of the CHO cells when transfected alone, but expressed in the microtubule of the cells when cotransfected with SPAG6, with the molecular weight of 40 000. Yeast two-hybrid experiment showed the colonies of TAC1 and SPAG6 on the culture plate without Leu, Trp and His, both contained in the yeast fusion protein. CONCLUSIONS: The Tac1 recombinant plasmid was constructed successfully and the interaction between TAC1 and SPAG6 was confirmed with the plasmid.