Min Jie, Li Meng-Ying, Liu Yun-Hao, Liu Jun-Pin, Shuang Chao-Fan, Zhang Ling, Zhang Zhi-Bing
Department of Occupational and Environmental Health, School of Public Health, Wuhan University of Science and Technology, Wuhan, Hubei 430065, China.
Departments of Obstetrics and Gynecology and Biochemistry, Virginia Commonwealth University, Richmond, VA 23298, USA.
Zhonghua Nan Ke Xue. 2016 Dec;22(12):1059-1064.
To construct eukaryotic expression plasmids of the Tac1 gene and explore the interaction between TAC1 and sperm-associated antigen 6 (SPAG6).
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.
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.
The Tac1 recombinant plasmid was constructed successfully and the interaction between TAC1 and SPAG6 was confirmed with the plasmid.
构建Tac1基因的真核表达质粒，并探讨TAC1与精子相关抗原6（SPAG6）之间的相互作用。
从10只昆明雄性小鼠的心脏、肝脏、脾脏、肺脏、肾脏、大脑、肌肉和睾丸中提取RNA，逆转录成cDNA后，通过RT-PCR观察Tac1在上述组织中的表达。构建Tac1/pEGFP-N2和Tac1/pGADT7重组质粒，将Tac1/pEGFP-N2转染至CHO和COS-1细胞中，随后通过免疫荧光染色和蛋白质印迹法对TAC1进行定位和蛋白质表达检测。通过酵母双杂交实验和蛋白质印迹法确定TAC1与SPAG6之间的相互作用。
Tac1主要在睾丸、大脑和心脏中表达。限制性内切酶消化和测序结果表明重组质粒构建成功，限制性片段长度为390 bp。单独转染时，TAC1定位于CHO细胞的整个细胞体中，但与SPAG6共转染时，TAC1在细胞的微管中表达，分子量为40 000。酵母双杂交实验表明，在不含亮氨酸、色氨酸和组氨酸的培养基上培养的平板上，TAC1和SPAG6的菌落中均含有酵母融合蛋白。
成功构建了Tac1重组质粒，并证实了该质粒介导的TAC1与SPAG6之间的相互作用。
