title: [Construction and expression of DNA-binding domain plasmid with hepatitis B virus e antigen in yeast double hybrid system].
abstract: BACKGROUND
  Using hepatitis B virus e antigen (HBeAg) gene to construct the DNA-binding domain vector, which can express HBeAg in yeast cell, and can be used in yeast double hybrid as "bait plasmid" to look for the gene from the cDNA library, which expresses the protein that can interact with HBeAg.
  
  
  METHODS
  PCR was performed to amplify the HBeAg gene from a sera of hepatitis B patient. The product of the amplification was inserted into T-vector and was verified by sequencing. Then it was inserted into the "bait" plasmid pGBKT7 after the digestion with the restricted endonuclease of EcoR I and Sal I. The plasmid was transformed into the yeast cell. PCR was used to verify whether the plasmid was transformed into yeast. The HBeAg protein expressed in the cell was confirmed by Western blot. Using nutrition selection assay to verify the constructed plasmid alone could not activate the reporter gene in the yeast cell.
  
  
  RESULTS
  Sequenced and digested by two endonucleases, the recombined vectors pGBKT7-eAg produced anticipated fragment. PCR verified that there was HBeAg fragment in the yeast. Having assayed by Western blotting, it was shown that the yeast cell transformed with pGBKT7-eAg vector had positive signal which could not be seen in the control. Tested by the nutrition selection assay, the recombined vectors pGBKT7-eAg could not activate LacZ reporter gene in the yeast.
  
  
  CONCLUSIONS
  DNA-binding domain plasmid was successfully constructed and could express HBeAg proteins in the yeast cell but could not activate transcription of LacZ reporter gene alone. The recombined plasmid can be used in yeast double hybrid.
authors.affiliation: Department of Immunology, The No. 302 Hospital of PLA, Beijing 100039, China.
authors.forename: Bo-an
authors.initials: BA
authors.lastname: Li
authors.forename: Yang
authors.initials: Y
authors.lastname: Qi
authors.forename: Cui-li
authors.initials: CL
authors.lastname: Shu
authors.forename: Yan
authors.initials: Y
authors.lastname: Liu
authors.forename: Hao
authors.initials: H
authors.lastname: Cheng
authors.forename: Jing
authors.initials: J
authors.lastname: Li
authors.forename: Rong
authors.initials: R
authors.lastname: Gao
authors.forename: Jun
authors.initials: J
authors.lastname: Hou
authors.forename: Yun
authors.initials: Y
authors.lastname: Cheng
mesh_terms: D005822:Genetic Vectors; D006513:Hepatitis B e Antigens / Q000096:biosynthesis* / Q000235:genetics; D019694:Hepatitis B, Chronic / Q000097:blood; D006801:Humans; D008297:Male; D008875:Middle Aged; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D020798:Two-Hybrid System Techniques; D015003:Yeasts / Q000235:genetics
chemical_list: D006513:Hepatitis B e Antigens; D011993:Recombinant Fusion Proteins