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目的采用高效液相色谱法(high performance liquid chromatography,HPLC)分析重组酿酒酵母表达的乙型肝炎表面抗原(hepatitis B surface antigen,HBsAg)病毒样颗粒(virus-like particle,VLP)的结构性状。方法采用HPLC法,分别以磷酸盐缓冲液和含去垢剂SDS的磷酸盐缓冲液为流动相,分析3批重组酿酒酵母表达的纯化HBsAg VLP的出峰规律。结果 3批纯化的HBsAg样品在不含SDS的流动相条件下,均只在(15.39±0.01)min处出现单一的相对分子质量2 000 000的HBsAg VLP峰,峰面积积分百分含量为(100.00±0.0)%。3批纯化的HBsAg样品在含SDS的流动相条件下,在(15.07±0.02)min处出现相对分子质量2 000 000的HBsAg VLP峰,峰面积积分百分含量为(55.07±0.05)%;在(28.62±0.04)min处出现1个相对分子质量24 000的P24亚基多肽峰,峰面积积分百分含量为(44.93±0.04)%。结论 HBsAg在常规保存条件下为P24亚基通过非共价键或(和)共价键聚合形成的相对分子质量为2 000 000的大分子蛋白颗粒,在含SDS的条件下,HBsAg颗粒可部分解聚为P24亚基。
Objective To analyze the structural characteristics of hepatitis B surface antigen (HBsAg) virus-like particle (VLP) expressed in Saccharomyces cerevisiae by high performance liquid chromatography (HPLC). Methods The peaks of purified HBsAg VLPs expressed by three batches of recombinant Saccharomyces cerevisiae were analyzed by HPLC using phosphate buffer and phosphate buffer containing detergents SDS respectively. Results A single HBsAg VLP peak with a relative molecular mass of 2 000 000 appeared at (15.39 ± 0.01) min in the SDS-free mobile phase of 3 batches of purified HBsAg samples. The peak area integral percentage was 100.00 ± 0.0)%. Three batches of HBsAg VLPs with relative molecular mass of 2 000 000 appeared at (15.07 ± 0.02) min in SDS-containing mobile phase with the peak area integral percentage of (55.07 ± 0.05)%; At 28.62 ± 0.04 min, one P24 subunit polypeptide peak with a molecular weight of 24 000 appeared, with a peak area integral percentage of (44.93 ± 0.04)%. Conclusions Under the routine storage conditions, HBsAg is a macromolecular protein particle with a relative molecular mass of 2 000 000 formed by non-covalent or covalent bond polymerization of P24 subunit. Under the condition of containing SDS, the HBsAg particles may partially Disaggregated to P24 subunit.