adw与adr亚型乙型肝炎表面抗原基因在哺乳动物细胞中的表达

EXPRESSION OF HEPATITIS B SURFACE ANTIGEN ( SUBTYPE adw, adr) IN MAMMALIAN CELLS

以pSV2-dhfr DNA为载体,在其单一酶切点Bg1Ⅱ处分别插入adw或adr亚型的HBVDNA Bg1 ⅡA片段(包括完整的pre-s及s基因),构成PSDHB_(r-1)和PSDHBw重组质粒。将其分别与pX1 DNA用磷酸钙沉淀法共转化LTK-细胞,在HAT培基选择压力下,挑选LTK^+细胞,这些细胞均能有效表达HBsAg。再改用MTX选择培基,并逐渐增加其药量,从MTX抗性细胞中获得稳定表达HBsAg的Mwc-1和Mrm细胞系。

Using pSV2-dhfr DNA as a plasmid vector which has a single Bgl Ⅱ site, we have constructed two new recombinant plasmlds by inserting Bgl Ⅱ A fragment of hepatitis B ( subtype adw, adr)virus DNA into plasmid vector pSV2-dhfr.The 2.8 kb DNA of Bgl Ⅱ A fragment contains complete pre-S and S genes. Ltk- cells were cotransformed with recombinant plasmid pSDHBw or pSDHBr-1 DNA and pX1 DNA. Under the selective pressure of HAT medium,Ltk+ cells were selected. These Ltk+ cells expressed HBsAg efficiently.The Mwc-1 and Mrm cell lines expressing HBsAg at high level were established by progressively increasing the amount of methotrexate ( MTX ) in the culture medium.The amount of HBsAg excreted by MWC-1 was 10μg/107 cellsXday, i.e. 1.0×10-12 g/cell/day, by Mrm was 15μg/107 cells/day,i.e. 1.5×10-12g/cell/day.These cell lines have stable HBsAg excretion.HBsAg excreted into the culture medium was shown by immuno-electronmtcroscopy to be composed of spherical and tube-like particles of about 22nm diameter, being similar to those found In human serum.