Construction and identification of a yeast two-hybrid bait vector and its effect on the growth of yeast cells and the self-activating function of reporter genes for screening of HPV18 E6-interacting protein.
By using a yeast two-hybrid system, a yeast two-hybrid bait vector was constructed and identified for screening of the HPV18 E6-interacting proteins, and its effects on the growth of yeast cells and the activation of reporter genes were investigated. Total mRNA extracted from Hela cells was reversely transcribed into cDNA. Fragment of HPV18 E6 cDNA was amplified using RT-PCR and directly ligated to the pGBKT7 vector. The recombinant plasmid was confirmed by restriction endonuclease analysis and DNA sequencing. The recombinant pGBKT7-HPV18 E6 plasmid and empty pGBKT7 vector were transformed into the yeast cell AH109, respectively. After they were cultured respectively in YPDA liquid medium and nutrition-deficient culture medium, their toxicity and transcriptional activation were tested by both the phenotype assay and the color assay. The bait plasmid HPV18 E6 was successfully obtained. After being cultured in YPDA liquid medium for 16h, the A (600 nm) values of two yeast fluids were 0.98+/-0.03 and 0.99+/-0.02, respectively. The recombinant pGBKT7-HPV18 E6 plasmid and empty pGBKT7 vector could grow to white colonies on SD/-Trp/X-alpha-gal plates, while no colony could survive on SD/-His/-Trp/X-alpha-gal, SD/-Ade/-Trp/X-alpha-gal plates, indicating that the bait plasmid pGBKT7-HPV18 E6 was constructed successfully and expressed correctly, and could not activate the transcription of reporter gene alone. The yeast two-hybrid GAL4 system 3 can be utilized to find HPV18 E6 interacting proteins.
Cancer Biology Research Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China. merchant688@yahoo.com.cn
Quan
Q
Mei
Shuang
S
Li
Ping
P
Liu
Ling
L
Xi
Shixuan
S
Wang
Yuhan
Y
Meng
Jie
J
Liu
Xinwei
X
Yang
Yunping
Y
Lu
Hui
H
Wang
D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D009856:Oncogene Proteins, Viral / Q000096:biosynthesis* / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D025941:Protein Interaction Mapping; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D020798:Two-Hybrid System Techniques; D015003:Yeasts / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism
D004268:DNA-Binding Proteins; C052603:E6 protein, Human papillomavirus type 18; D009856:Oncogene Proteins, Viral; D012333:RNA, Messenger; D011994:Recombinant Proteins