乙型肝炎病毒表面抗原中蛋白基因在中国地鼠卵巢细胞中的高效表达及其产物活性的初步检测

构建了pSV2DHWS2S质粒,使dhfr扩增基因及乙型肝炎病毒的Pre-S_2+S基因分别在两个SV40早期启动子的调控之下。此质粒转化到CHO-dhfr~-细胞,经克隆、加氨甲喋呤(MTX)筛选、扩增,建立了3个高效分泌HBsAg中蛋白及主蛋白的克隆细胞系。检测了其中的M6细胞系生物学特性。结果表明,免疫电镜下可观察到22nm的颗粒;该细胞用转瓶连续培养60天,每2天收、换液1次,每升HBsAg平均产量为2.9mg。经初步纯化,在SDS-PAGE中显示23k、27k主蛋白带及33k、36k中蛋白带。主蛋白及中蛋白的反相血凝(RPHA)滴度分别为64和128;中蛋白的ELISA滴度为320。部分品系小鼠免疫后能产生滴度为8的抗Pre-S_2抗体。3只家免中仅有1只在免疫后第1、2周可测出Pre-S_2抗体,而3只兔的S抗体滴度都较高,持续时间也较长。

HIGH LEVEL EXPRESSION OF MIDDLE PROTEIN OF HEPATITIS B SURFACE ANTIGEN(HBsAg) IN CHINESE HAMSTER OVARY(CHO) CELL LINE AND THE BIOLO-GIC PROPERTIES OF PRODUCTS

The plasmid pSV2DHBWS2S was constructed so that the dhfr gene and Pre-S2+S gene of HBsAg were separately regulated by the early promoter of SV40. CHO-dhfr celis were transformed with this recom-binant plasmid DNA and cell lines cloned. Three cell lines secreting the middle and major proteins of HBsAg at high Ievel were established by progressively increasing the amoun of methotrexate in the culture me-dium.One of the transformed cell line M6 showed 22nm particles of HBs Ag under the electron microscope. Expressiorr of HBsAg was 2.9mg/L in roller bottle cultures. Analysis of HBsAg polypeptides by SDS-PAGE after silver staining revealed bands of P23, GP27 major proteins and GP33, GP36 middle proteins of HBsAg. Reverse passive haemagglutina-tion ( RPHA ) titer for major protein was 64-128, then ELISA titer for middle protein was 320.Studies of immunogenicity of the middle protein in various mouse strains showed that anti-Pre-S2 antibody titer in some strains of mice was 8 by ELISA. Of the three rabbits, only one produced anti-Pre-S antibody after immunization. High Ievel of anti-S antibody was detec ted in the serum of all rabbits.