MT-5细胞表达HBsAg的进一步研究

FURTHER STUDIES ON THE EXPRESSION OF HBsAg IN MT-5 CELLS

进一步观察了基因工程转化细胞株MT-5中HBsAg的表达,结果表明,HBsAg的表达分泌稳定,培养48小时收获,产率最佳;培养基中的小牛血清以10％为最适,通过建立多个单细胞亚克隆株证明,MT-5细胞中HBsAg的表达存在不均一性,亚株的HBsAg表达不受糖皮质激素的诱导,但受Cd^(2+)和Zn^(2+)的诱导,且细胞能够耐受较高浓度的ZnCl_2,Cd^(2+)和Zn^(2+)的最佳诱导浓度分别为1μM和100μM,最高诱导效率为350％,最高诱导产量达2.5mg/l。 MT-5培养上清中的HBsAg经亲和层析初步纯化后,SDS-PAGE呈现两条特异性多肽,分子量为23k和27k,它们代表HBsAg的主要多肽及其糖基化产品,DNA杂交结果不但确证重组S基因存在于MT-5细胞中,而且揭示重组DNA以游离体状态存在,似乎不进行整合和重排,不同亚系细胞中重组DNA的拷贝数估算为11～27个/细胞,拷贝数与HBsAg表达量有正相关关系。

The authors have investigated the experssion of HBsAg in the transformed mouse cell line MT-5. The results showed that the synthesis and secretion of HBsAg is stable. Comparison of different harvest time gave optimal interval of 48h. The effect of calf serum on the expression is obvious and the optimal concentration was 10%. Several subclones of MT-5 cells have been established. The expression of HBsAg in these subclones was not identical and was not induced by glucocorticoid. However, HBsAg expression was induced by Zn ++ and Cd++ , and the cells could stand the treatment of relatively high concentration of Zn++. The highest induction yield reached 2.5 mg/L and highest induction efficiency 350%. SDS-PA-GE analysis revealed two specific bands corresponding to 23k and 27k in the HBsAg samples purified from the MT-5 culture supernatant, which probably represent the HBsAg major po.lypeptide and its glycosylated form. An analysis of DNA from MT-5 cells revealed the existence of recombinant S gene in MT-5 cells and demonstrated that the cells retain the recom-binant plasmid in an extrachromosomal state with a copy number of 11 to 27 per cell in different subclones. There was no evidence indicative of integration or rearrangement. The yield of HBsAg was directly proportional to the copy number.