Cloning and expression of the preS1 gene of hepatitis B virus in yeast cells.
OBJECTIVE
To investigate the complex functions of HBV preS1 protein, we constructed HBV preS1 gene expression vector and expressed it in yeast cells.


METHODS
Polymerase chain reaction (PCR) was performed to amplify the gene of HBV preS1 from the plasmid pCP10 containing the whole DNA fragment of HBV ayw subtype as template and the PCR product was cloned into the pGEM-T vector for sequencing. After being identified, the HBV preS1 gene was cut from the pGEM-T vector by EcoR I and Pst I restriction enzymes, and cloned into yeast expressive plasmid pGBKT7 to construct pGBKT7-preS1 recombinant expressive plasmid. This plasmid was transformed into yeast cell AH109 and expressed in it. The yeast protein was isolated and analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and Western blotting.


RESULTS
The HBV preS1 gene was amplified successfully and identified by DNA sequencing. The PCR products were coincided completely with the reported sequence. The digested fragments were cloned into the pGBKT7 vector and transformed into yeast cell AH109. The results of SDS-PAGE and Western blotting assay showed: (1) The HBV preS1 protein was expressed and existed in yeast cells; (2) The molecular weight of the expression product was about 30 000 D.


CONCLUSIONS
The HBV preS1 gene was successfully cloned and expressed in yeast cells.
Lu
Yin-Ying
YY
Gene Therapy Research Center, Institute of Infectious Diseases, Chinese PLA 302 Hospital, Beijing 100039, China.
Li
Ke
K
Cheng
Jun
J
Wang
Lin
L
Liu
Yan
Y
Zhang
Ling-Xia
LX
D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006514:Hepatitis B Surface Antigens / Q000235:genetics* / Q000378:metabolism*; D011498:Protein Precursors / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D015003:Yeasts / Q000166:cytology / Q000378:metabolism*
D006514:Hepatitis B Surface Antigens; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; C050806:presurface protein 1, hepatitis B surface antigen