乙型肝炎病毒表面抗原S和前S1融合基因转基因细胞系的建立与细胞性质的研究

Study on a Transgenic Cell Line Excreting High Level of S and Pre S1 Fusion Protein of HBsAg

构建了 pCHBSSIG质粒 ,其特点是CMV立即早期启动子调控乙型肝炎 (乙肝 )表面抗原S +前S1融合基因在前 ,SV40早期启动子调控GS扩增基因在后 ,此质粒转化到CHO dhfr-细胞中 ,经克隆加MSX及MTX筛选、扩增 ,建立了 7个高效表达乙肝表面抗原S及前S1融合蛋白细胞系GdSS1,并检测了其中GdSS1 18细胞系的生物学特性 ,结果表明 :未发现微生物污染 ,无致瘤性 ,遗传稳定 ,电镜下可观察到 2 2nm的颗粒 ,纯化后的蛋白在SDS PAGE及Westernblot中显示出特异性的 2 7kD、30kD乙肝表面抗原S和前S1的融合蛋白带。主蛋白的反向被动血凝(RPHA)滴度为 1∶2 5 6～ 1∶5 12 ,前S1蛋白的ELISA滴度为 1∶12 8。

The plasmid pCHBSS1G was constructed with HBsAg S+PreS1 fusion gene and glutamine systhetase (GS) gene separately regulated by CMV IE promoter and early promoter of SV40. CHO dhfr - cells were transfected with this recombinant plasmid DNA and the cell lines were cloned. Seven CHO cell lines secreting S+PreS1 fusion protein of HBsAg at high level were establilshed by selection under increasing concentration of methotrexate (MTX) and methionine sulphoximine (MSX) in the culture medium. Biological characteristics of one of the 7 cell lines, namely, the GdSS1 18 cell line was studies. The results showed that 22nm particles of HBsAg were visualized under the electron microscope. GdSS1 18 cell line was not contaminated by microorganisms including bacteria, fungi, mycoplasma and exogenous or endogenous viruses. It showed no tumorigenesis in nude mice. The genetic stability was confirmed. Analysis of HBsAg polypeptides by SDS PAGE and Western blot revealed special bands of 27kD, 30kD fusion protein of HBsAg. Reverse passive haemagglutination (RPHA) titer for major protein was 1∶256 1∶512, the ELISA titer for PreS1 protein was 1∶128.