基因工程乙肝疫苗的研制——Ⅲ.MT-5细胞的HBsAg纯化与鉴定

DEVELOPMENT OF GENETIC ENGINEERING HEPATITIS B VACCINE Ⅲ. PURIFICATION AND DETERMINATION OF HBsAG PRODUCED BY MT-5 CELLS

采用超过滤、PEG沉淀和超速离心综合步骤对MT-5细胞培养上清中的HBsAg进行纯化。所得纯化HBsAg经SDS-PAGE银染色结果显示两条多肽,分子量分别为23k和27k,与血源HBsAg的多肽成分相同,为重组HBsAg的两条特异性多肽,且在凝胶中无其它杂蛋白带存在,纯度高。经PAGE银染色结果证实纯HBsAg中的杂蛋白含量符合疫苗生产的要求。经此综合方法提纯的HBsAg回收率可达44.1％以上。

The purification of HBsAg in MT-5 cells culture supernatant was carried out by three different steps including ultrafiltration, precipitation by PEG 6000 and three-step ultracentrifugations. The purified HBsAg was composed of two specific bands found in the result of SBS-PACE silver stain. The molecular weights were 23k and 27k respectively, similar to those of human plasma HBsAg. There was no other protein band in the gel. The quantity of other protein in purified HBsAg accorded with the demands of va-cine confirmed by the result of PAGE silver stain. Up to 44.1% of the HBsAg was recovered at the end of purification process.