孙明颢，叶林伯**，郜金荣，贺石汉，吴正辉

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 HBV