[Construction and identification of yeast expression plasmids containing mouse signal transducers and activators of transcription-4/6].

ABSTRACT

AIM: To construct yeast expression plasmids containing mouse STAT4/6 gene for further study of their interaction with other proteins in yeast two-hybrid system. METHODS: Mouse STAT4/6 genes were amplified by PCR and T-A was cloned with pMD19-T simple vector and then was cloned into yeast expression vector pGADT7 cut with incision enzymes and treated with CIAP. The yeast expression vector pGADT7 was identified by enzyme cutting and sequencing. The yeast expression plasmids pGADT7-STAT4/6 were transformed into AH109 yeast cells and the expression of the STAT4/6 fusion proteins was detected by Western blot. Their toxicity and self-activation were also detected. RESULTS: Mouse STAT4/6 genes were successfully amplified and cloned into pMD19-T simple vector and pGADT7. Sequencing analysis revealed that both plasmids met the design of the study. The yeast expression plasmids pGADT7-STAT4/6 were successfully transformed into AH109 yeast cells, without toxicity or self-activation. The expression of STAT4/6 fusion proteins was confirmed by Western blot. CONCLUSION: The yeast expression plasmids pGADT7-STAT4/6 are successfully constructed and can be applied in the detection of their interaction with other proteins.