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Virologica Sinica, 18 (2) : 99-103, 2003
Instiute of virology,Wuhan University,Wuhan 430072,China
Hepatitf B virus(HBV1 genome DNA was directly extracted from human serum infected
with HBV.The full length preS gene DNA was obtained by PCR using the HBV genome DNA as
template.Then it w0s cloned into the pUCm—T vector and sequenced using the M 1 3 Primers.The
sequencing data shows that it contains 1 203bp an d it length with 24 different nucleotides compared
with the standard sequence of HBV (subtype adr)in China,it was confirmed to be the preS gene with
three ATGs which corresponded to the initiation codon of PrsS1,PreS2 an d S proteins respectively.Th is
DNA fragment was cloned into the SnaB 1-Avr II sites of expressing vector pPIC9K,in framed with the
AOXI promotor and then the pPIC9K—PreS recombined plasmid DNA linearized by Sal 1 was introduc‘
ed to Pichia pastoris GSll5 by electroporation device.GSll5一pPIC9K—PreS was got by selecting with
MD-G418 plates an d identifying with PCR.Th e GSll5-pPIC9K,PreS Can grow in the media with
methan ol and can produce the PreS protein in secreted form with molecular weight 48KD as detected by
SDS—PAGE.ELISA experiment proved that the protein Can react with the positive human serum against
Key Words: preS gene;  Pichia pastoris;  ELISA
  Published online: 15 Apr 2003
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