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目的分析重组H21G蛋白的化学修饰方法及化学修饰与免疫原性之间的相关性。方法应用琥珀酸酐和己二酸二酰肼(adipic acid dihydrazide,ADH)分别修饰重组H21G蛋白制备衍生物,赖氨酸单位减少率法测定重组H21G蛋白琥珀酰化物的琥珀酰化率;2,4,6-三硝基苯磺酸(2,4,6-trinitrobenzenesulfonic acid,TNBS)法测定重组H21G蛋白衍生物的-AH衍生率;SDS-PAGE及HPLC法分析重组H21G蛋白各衍生物的纯度及分子量分布。用各衍生物分别经皮下免疫小鼠,免疫浓度为25μg/ml,共免疫3次,于末次免疫后2周,经小鼠眼眶采血,分离血清,采用间接ELISA法检测血清IgG含量。采用非还原型SDS-PAGE分析重组H21G蛋白衍生物在制备初期、制备后4℃储存2周及6个月的稳定性。结果随着重组H21G蛋白与琥珀酸酐质量比(10∶1、10∶3、10∶4和10∶5)的提高,琥珀酰化率也逐渐升高,但质量比为10∶4和10∶5时无明显差异。重组H21G蛋白ADH衍生1和2 h的衍生率无明显差异。质量比为10∶4及10∶5的琥珀酰化物与原蛋白分子量分布差异较大;ADH衍生后,原蛋白的分子量分布也发生改变。重组H21G蛋白与琥珀酸酐质量比高于10∶3时,对蛋白的降解作用明显,当质量比达到10∶5时,重组H21G蛋白几乎不再以单体形式存在;而ADH衍生1和2 h对蛋白聚合作用无明显差异,均产生了二聚体和多聚体。重组H21G蛋白及其衍生物免疫小鼠后表现明显的剂次加强效应,且ADH衍生的产物的免疫原性高于琥珀酰化产物。琥珀酰化产物的稳定性较差,目标蛋白均明显降解,而ADH衍生的产物在4℃储存6个月后未见明显聚合或降解。结论重组H21G蛋白经ADH衍生的产物免疫原性及稳定性均优于琥珀酰化产物。
Objective To analyze the chemical modification of recombinant H21G protein and the correlation between chemical modification and immunogenicity. Methods The recombinant H21G protein was modified by succinic anhydride and adipic acid dihydrazide (ADH) respectively to prepare derivatives. The lysine unit reduction rate was used to determine the succinylation rate of recombinant H21G protein. 6-trinitrobenzenesulfonic acid (2,4,6-trinitrobenzenesulfonic acid, TNBS) was used to determine the -AH derivatization rate of the recombinant H21G protein derivative. The purity and molecular weight of each derivative of the recombinant H21G protein were analyzed by SDS-PAGE and HPLC distributed. The mice were immunized subcutaneously with each derivative at a concentration of 25μg / ml for 3 times. After 2 weeks after the last immunization, blood was collected from the orbit of mice and the serum was separated. The serum IgG level was measured by indirect ELISA. Non-reducing SDS-PAGE analysis of recombinant H21G protein derivatives in the preparation of the initial, after storage at 4 ℃ for 2 weeks and 6 months stability. Results As the mass ratio of recombinant H21G protein to succinic anhydride (10: 1, 10: 3, 10: 4 and 10: 5) increased, the rate of succinylation also increased gradually but the mass ratio was 10: 4 and 10: 5 no significant difference. There was no significant difference in the derivatization rates of recombinant H21G protein ADH derived 1 and 2 h. The molecular weight distributions of succinic compounds and protoplasts differ greatly between the mass ratio of 10: 4 and 10: 5. After ADH derivatization, the molecular weight distribution of the protoplasts also changes. When the mass ratio of recombinant H21G to succinic anhydride was higher than 10: 3, the degradation of protein was obvious. At the mass ratio of 10: 5, the recombinant H21G almost no longer existed as monomer; while ADH derived 1 and 2 h No significant difference in the polymerization of the protein, both produced dimer and multimer. Recombinant H21G protein and its derivatives showed obvious dose-boosting effect after immunized mice, and the immunogenicity of ADH-derived products was higher than that of succinylated products. The stability of succinylated products was poor, and the target proteins were significantly degraded. However, no obvious polymerization or degradation of ADH-derived products was observed after 6 months of storage at 4 ° C. Conclusion The immunogenicity and stability of recombinant H21G protein derived from ADH are better than that of succinylated product.