Construction And Verification Of Yeast Bait Plasmids Carrying Hpv18e6 Cdna Fragme

Objective To construct and identify the bait expression vector pGBKT7- HPV18E6 in yeast two-hybrid system and examine whether the recombinant bait plasmid has self-activating and toxicity effect. To establish an experimental basis of utilizing yeast two-hybrid system to screen the objective proteins which interact with HPV18 E6.Methods Cultured cervical adenocarcinoma cell line Hela, conventional extraction of total cellular RNA for reverse transcription after synthesis of cDNA, Full fragment of open reading frame of HPV18 E6 cDNA was amplified using PCR, HPV18E6 and the yeast expression vector pGBKT7 were digested by the restriction endonucleases EcoRI and BamHⅠafter purificated, products were recoveryed and the vector was inserted into the bait expression vector pGBKT7, the recombinant plasmid was named pGBKT7- HPV18 E6. Recombinant plasmid by restriction enzyme digestion to verify the correctness and through the re-sequencing of plasmid sequences to check whether the changes The recombinant plasmid bait vector pGBKT7- HPV18E6 with correct sequence was transformed into yeast cell AHl09 by using Lithium acetate method (Li- Ac). And its toxicity and transcriptional activation was tested by both the phenotype and the color assay.Results The amplified product of 650 bp was insert into pGBKT7 vector and proven correctly by double restriction enzyme digestion. Sequence analysis revealed that the fragment was correctly inserted into pGBKT7 with a right reading frame. And then its expression in yeast was verified. HPV18E6 was amplified and cloned into pGBKT7 successfully. The recombinant pGBKT7-HPV18E6 plasmids and empty pGBKT7 vector could grow white colonies on SD/-Trp/X-α-gal plates and none could survive on SD/-His /-Trp /X-α-gal, SD/-Ade/-Trp/X-α-gal plates. After being cultured in SD/-Trp liquid medium for 16h, the OD600 of them were 0.98 and 0.99. The bait vector was transformed into AHl09 as well and no toxicity and self-activation were found.Conclusion The bait expression vector of HPV18E6 was constructed successfully, and can not activate the transcription of reporter gene alone.the vector can be used as yeast two-hybrid systerm the"bait", which layed the foundations for screening target proteins interacting with the bait protein using the yeast two-hybrid technique.