OBJECTIVETo explore use of retroviral vector in gene therapy of hepatitis B.

METHODSThe recombinant vector Plxsn-HBs was constructed by inserting HBV S gene into pLXSN. The pseudovirus, which was produced from PA317 after transferring with pLXSN-HBs by electroporation, were frozen at different temperature. The activities of the pseudovirus to infect eukaryotic cells and express antigen were determined by comparing the numbers of G418-resistant clones and assaying HBsAg in the supernatant of the cells with ELISA after infection HepG2, NIH3T3 and 293 cells.

RESULTSIt was hard to find changes in HBsAg amount at different intervals and different temperatures. G418 resistant clones, however, were variable. When frozen at -20 degrees C, the numbers of clones were half less than that of the beginning after 6 months, few clones were formed after 12 months, and no clone was found after 24 months. When frozen at -40 degrees C, the numbers of clones were 121, 332 and 89 42, 137 and 43 for HepG2, NIH3T3 and 293 cell lines at 12 and 24 months, respectively. When frozen at -70 degrees C, the numbers of clones were 159 463 and 112 for HepG2, NIH3T3 and 293 cell lines at 24 months, respectively. There was no statistical difference compared to that of zero months.

CONCLUSIONSThe activity of the peseudovirus to infect eukaryotic cells and expressed antigen was not changed after 2 years frozen at -70 degrees C.