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


CONCLUSIONS
The yeast expression plasmids pGADT7-STAT4/6 are successfully constructed and can be applied in the detection of their interaction with other proteins.
Department of Respiratory, Children's Hospital, Chongqing Medical University, Chongqing, China. zhangmingxiang27@163.com
Ming-xiang
MX
Zhang
Zhou
Z
Fu
Dai-yin
DY
Tian
Li-jia
LJ
Wang
En-mei
EM
Liu
Zheng-xiu
ZX
Luo
Ji-hong
JH
Dai
D000818:Animals; D051379:Mice; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D050797:STAT4 Transcription Factor / Q000235:genetics*; D050800:STAT6 Transcription Factor / Q000235:genetics*; D017422:Sequence Analysis, DNA; D020798:Two-Hybrid System Techniques; D015003:Yeasts / Q000235:genetics
D011993:Recombinant Fusion Proteins; D050797:STAT4 Transcription Factor; D050800:STAT6 Transcription Factor; C494091:Stat4 protein, mouse; C494100:Stat6 protein, mouse