毕赤酵母表达的含前S1、前S2和S表位乙肝表面抗原疫苗的制备和免疫原性研究

Preparation and Immunogenicity of a Pichia pastoris-derived Hepatitis B Vaccine Containing PreS1, PreS2 and S Epitopes

摘要:

整合了乙肝表面抗原嵌合基因SS1和SS2的毕赤酵母工程菌株GS115-SS1S2经高密度发酵培养，甲醇诱导，抗原表达量达到300～600mg/L发酵液。SS1S2抗原经细胞破碎、硅胶吸附、疏水层析和凝胶过滤纯化，纯度达99%以上，每升培养物可收获纯化抗原82mg。纯化的SS1S2抗原经Al(OH)₃吸附，在NIH小鼠中进行免疫效果评价。三组NIH雌性小鼠，分别腹腔接种2.5μg、0.625μg和0.156μg SS1S2疫苗或商品化的单S疫苗。部分小鼠在30天时采血，测定各疫苗组的ED₅₀值。在SS1S2疫苗组，前S1、前S2和S抗原的ED₅₀值分别为0.46、0.29和0.84μg，而S疫苗组S抗原的ED₅₀为0.99μg。另一部分小鼠分别在7天和14天时采血，考察抗体阳转率与时间的关系。SS1S2疫苗前S1、前S2抗体阳转率在7d和14d时比S抗体的阳转率为高，提示前S抗体出现的时间较早。上述结果显示SS1S2疫苗比单S疫苗具有更好的免疫原性。

Abstract:

The preparation process and immunogenicity of a novel hepatitis B vaccine containing preS1, preS2 and S epitopes were investigated in this study. APichia pastoris stain GS115-SS1S2 harbouring two chimeric HBsAg gene constructs, SS1 and SS2 was cultivated by high-density fermentation. 300～600mg/L of the expression level was achieved through 48～72h methanol induction. SS1S2 antigen was extracted and purified by silica adsorption, HIC and SEC to 99% purity from the harvested cells. 82mg purified antigen could be achieved from one liter of fermentation culture. The immunogenicity of the purified antigen was evaluated in NIH mice. Three groups of female NIH mice, 14～16g in weight, were injected once intraperitoneally with 2.5, 0.625, 0.156μg of each of the two vaccines: SS1S2 or a commercially available S vaccine. Part of the mice were bled in 30 days after injection to compare the ED₅₀ of the two vaccines. For the SS1S2 vaccine, the ED₅₀ is 0.46, 0.29 and 0.84μg respectively for the preS1, preS2 and S antigens. For the S vaccine, the ED₅₀ is 0.99μg for the S antigen. Another part of the mice were bleed in 7 or 14 days to detect preS1, preS2 and S antibodies. Higher ratios of mice were seroconverted for preS1 and preS2 antibodies as compared to the S antibody in these two time points. These results suggest that the SS1S2 vaccine may be more immunogenic than the conventional S vaccines.