重组酵母表达HBsAg P24亚基结合程度对抗原性的影响

目的观察HBsAg P24亚基结合紧密程度对其抗原性的影响。方法3批重组酵母表达纯化的P24亚基松弛结合型HBsAg经半转型和转型处理后，成为混合和紧密结合型HBsAg，用HPLC法检测P24亚基结合紧密程度，松弛型和紧密型HBsAg免疫小鼠后，检测NK细胞杀伤活性及抗原特异性脾淋巴细胞增殖活性。将HBsAg吸附于佐剂Al（OH）3凝胶，免疫小鼠后用半数有效剂量法（ED50）检测所诱导的体液免疫应答，用体外相对效力法（RP）检测抗原的反应原性。结果3批松弛型、混合型和紧密型共9组HBsAg P24亚基紧密结合型比率分别在23．00％～32．00％、38．43％～50．98％和39．65％～52．76％之间，混合型组P24亚基紧密结合程度接近紧密型组；3批紧密型HBsAg免疫小鼠后可比松弛型HBsAg诱导更强的NK细胞活性，松弛型HBsAg免后4周内诱导抗原特异性脾淋巴细胞活性高于紧密型HBsAg，但总体上HBsAg仅诱导较弱的特异性脾淋巴细胞应答；紧密型HBsAg组比松弛型组诱导较强的体液免疫应答，1批混合型抗原体液免疫应答低于同批松弛型和紧密型组，2批松弛型组和其同批紧密型组的体液免疫应答程度接近；混合型HBsAg RP平均值显着高于松弛型组（P〈0．01），紧密型组RP平均值小于混合型组。结论重组酵母表达P24亚基结合程度不同的HBsAg均保持一定程度的抗原性。紧密型HBsAg免疫可诱导较强的非特异性NK细胞和体液免疫应答，混合型HBsAg的免疫反应原性较高。

Impacts of HBsAg P24 conjugating extent on the antigenicity of the recombinant yeast expressed HBsAg

Objective To identify the impact of the HBsAg P24 unit conjugating extent on the antigeni- city of the recombinant yeast expressed HBsAg. Methods Three lots of purified HBsAg derived from recombinant yeast were treated with or without thiocyanate to prepare non-transformed, semi-transformed and transformed HBsAg, the P24 unit is theoretically loose-conjugated, semi-tight-conjugated and tight-conjugated. The tight-conjugation of polypeptide P24 unit for HBsAg was detected by HPLC. The BALB/c mice NK activities and HBsAg specific spleen lymphocytes proliferation immunized with the loose-conjugated and tight-conjugated HBsAg were tested. Results The average tight-conjugated P24 percentages of the three lots loose-conjugated, semi-tight-conjugated and tight-conjugated HBsAg are 23.00%-32.00%, 38.43%-50.98% and 39.65%-52.76%, respectively. The extent of tight-conjugated P24 semi-transformed HBsAg is similar to transformed ones. The NK activities induced by tight-conjugated HBsAg were higher than that induced by loose-conjugated antigen, the HBsAg specific spleen lymphocytes proliferation induced by loose-conjugated antigen was higher than that of tight-conjugated antigen in 4 weeks. All loose-conjugated, semi-tight-conjugated and tight-conjugated HBsAg effectively induced humoral immunoresponse. Although all vaccines showed effective immunogenicity, the semi-tight-conjugate vaccines are significantly more effective than that of loose-conjugated (P<0.05). Both the median effective dose (ED_ 50 ) and in vitro relative potencies (RP) of the loose-conjugated and the semi-tight-conjugated trial hepatitis B vaccines meet the requirement of the recombinant yeast derived hepatitis B vaccine. Conclusion Various P24 unit conjugated degree HBsAg maintains antigenicity. The tight-conjugated HBsAg could induce stronger NK activities than loosed HBsAg. The semi-transform treatment may improve the immunogenicity of HBsAg.