[Interaction between SPAG6 and TAC1 proteins].

OBJECTIVE: To construct eukaryotic expression plasmids of the Tac1 gene and explore the interaction between TAC1 and sperm-associated antigen 6 (SPAG6). 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.

[Construction of a GFP-fused mouse PACRG baculovirus recombinant vector and expression of the fusion protein in Sf9 inset cells].

OBJECTIVE: To construct a GFP-fused mouse Parkin co-regulated gene (PACRG) baculovirus recombinant PACRG/GFP-pFastBac1 vector and express the fusion protein in Sf9 insect cells. METHODS: Full-length mouse PACRG cDNA was amplified by PCR and cloned in frame to the vector pFastBac1 with eGFP (rpFBac-PACRG-GFP recombinant vector). The plasmid was transformed into DH10Bac cells to obtain the recombinant bacmid plasmid, the bacmid was transfected into Sf9 insect cells, and the expressed PACRG/GFP fusion protein was analyzed by Western blot and fluorescence microscopy. RESULTS: The construction of the PACRG/GFP-pFastBac1 baculovirus plasmid was confirmed by sequencing and restriction enzyme digestion. Western blot showed the expression of the fusion protein carrying a green fluorescence in the Sf9 insect cells. CONCLUSIONS: Conclusion: A PACRG/GFP-pFastBac1 recombinant baculovirus vector was successfully constructed and the fusion protein was highly expressed in the Sf9 insect cells. Our findings have provided a basis for further studies on the structure of the PACRG protein and regulation of spermatogenesis.