Cloning and expression and purification of hepatitis B e-antigen precursor in Escherichia coli.
OBJECTIVE
To investigate the role of the 25 kD hepatitis B e antigen (HBeAg) precursor that only exist inside hepatocytes and study its effect on the pathopoiesis of hepatitis B and QIAGEN expression and purification system.


METHODS
Hepatitis B virus (HBV) preC/C gene for the 25 kD HBeAg precursor was cloned into the expression vector pQE30 and the 25 kD HBeAg precursor was expressed in Escherichia coli (E. coli) and purified. Its antigenicity for 21 kD mature HBeAg was tested by western blot analysis.


RESULTS
Cloned fragments in the expression vector were sequenced and verified to be homogeneous with that of HBV (ayw subtype). Expression of the HBeAg precursor in E. coli under the transcriptional regulation of T5 promoter yielded a soluble cytosolic protein with an apparent molecular mass of 25 kD. Recombinant HBeAg precursor exhibited identical potencies with 21 kD mature HBeAg that reacted with anti-HBeAg antibodies. The purification rate of the expressed HBeAg precursor was up to 89.6% and the yield of purified HBeAg precursor from this procedure was 2.4 mg/L.


CONCLUSIONS
25 kD HBeAg precursor exhibited biological activity and might play an important role in pathopoiesis of hepatitis B.
Zhou
Fuyuan
F
Department of Infectious Diseases, Nanfang Hospital, Guangzhou 510515, China. fuyuan@fimmu.edu.cn
Sui
Lili
L
Luo
Kangxian
K
Hou
Jinlin
J
D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006513:Hepatitis B e Antigens / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011498:Protein Precursors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D006513:Hepatitis B e Antigens; D011498:Protein Precursors; D011994:Recombinant Proteins