重组汉逊酵母表达的HBsAgP24亚基间二硫键桥联程度等生化性状分析

Analysis of biochemical properties including cross-linking degree of disulfide bond between P24 subunits of HBs Ag expressed in recombinant Hansenula polymorpha

目的分析重组汉逊酵母(Hansenula polymorpha,HP)表达的HBs Ag(HP-HBs Ag)P24亚基间二硫键桥联程度等生化性状。方法采用蔗糖垫层离心法检测HBs Ag的浮力密度,SDS-PAGE分析HBs Ag P24亚基间结合程度的变化,高效液相色谱法(high performance liquid chromatography,HPLC)分析HBs Ag VLP在硫氰酸盐处理前后百分含量的变化。以酿酒酵母(Saccharomyces cervesiae,SC)表达的HBs Ag(SC-HBs Ag)作为对照。结果 HP-HBs Ag的浮力密度为(1.085±0.014)g/ml,与SC-HBs Ag的浮力密度[(1.090±0.009)g/ml]接近。SDS-PAGE分析表明,未经硫氰酸盐处理时,HP-HBs Ag不能进入分离胶,而SC-HBs Ag能进入分离胶;经硫氰酸盐处理后,HP-HBs Ag和SCHBs Ag均不能进入分离胶。HPLC分析表明,流动相中含SDS时,HP-HBs Ag VLP的百分含量在经硫氰酸盐处理前后变化不明显,SC-HBs Ag VLP的百分含量在经硫氰酸盐处理后较处理前升高;在经硫氰酸盐处理前后,HP-HBs Ag VLP的百分含量均高于SC-HBs Ag。结论 HP-HBs Ag的浮力密度与SC-HBs Ag相近,HP-HBs Ag P24亚基间二硫键桥联程度较高,结合紧密,不需要经硫氰酸盐处理。

Objective To analyze the biochemical properties including the cross-linking degree of disulfide bond between P24 subunits of HBsAg expressed in recombinant Hansenula polymorpha（HP-HBsAg）. Methods The buoyant density of HBsAg was determined by sucrose cushion centrifugation, while the cross-linking degree betweens P24 subunits of HBsAg by SDS-PAGE, and the percents of HBsAg VLPs before and after treatment with thiocyanate by HPLC. The HBsAg expressed in recombinant Saccharomyces cervesiae（SC-HBsAg） was used as control. Results The buoyant density of HP-HBsAg was（1. 085 ± 0. 014）g / ml, which was similar to that of SC-HBsAg [（1. 090 ± 0. 009）g / ml]. SDS-PAGE demonstrated that the HP-HBsAg untreated with thiocyanate could not enter to the separation gel illustrate, while the untreated SC-HBsAg could. However, after treatment with thiocyanate, neither HP-HBsAg nor SC-HBsAg entered to the separation gel. HPLC demonstrated that, when the mobile phase contained SDS, the percentage of HP-HBsAg VLPs showed no obvious change before and after treatment with thiocyanate, while that of SC-HBsAg VLPs after treatment was higher than that before treatment. However, both the percentages of HP-HBsAg VLPs before and after treatment with thiocyanate were higher than those of SC-HBsAg VLPs. Conclusion The buoyant density of HP-HBsAg were similar to that of SC-HBsAg. However, the cross-linking degree of disulfide bond between the P24 subunits of HP-HBsAg was higher, while the conjugation was tighter, which needed no treatment with thiocyanate.