重组H21G蛋白衍生物的制备及其免疫原性

Preparation and immunogenicity of recombinant H21G protein derivative

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 method for recombinant H21 G protein as well as the relationship between chemical modification and immunogenicity. Methods Derivatives were prepared by modification of recombinant H21 G protein with succinic anhydride and adipic acid dihydrazide（ADH）respectively,and determined for succinylation rate by lysine unit reduction rate method,for AH derivation rate by 2,4,6-trinitreobenzenesulfonicacid（TNBS） method,and analyzed for purity and molecular weight distribution by SDS-PAGE and HPLC. Mice were immunized s. c. with the derivatives each at a dosage of 2. 5 μg / ml for 3 times,of which serum samples were collected2 weeks after the last immunization and determined for IgG content by indirect ELISA. The stabilities of derivatives at initial period of preparation as well as those after storage at 4 ℃ for 2 weeks and 6 months were analyzed by nonreduced SDS-PAGE. Results The succinylation rates increased gradually with the increasing mass ratio of recombinant H21 G to succinic anhydride,while showed no significant difference when the ratios were 10 ∶ 4 and 10 ∶ 5. No significant difference was observed between the derivation rates of H21 G derived with ADH for 1 and 2 h. However, the molecular weight distributions of succinyl chlorides at mass ratios of 10 ∶ 4 and 10 ∶ 5 showed significant differences with those of original H21 G proteins. The molecular mass distribution of H21 G also changed after derivation with ADH. The derivates in which the mass ratios of recombinant H21 G to succinic anhydride were more than 10 ∶ 3 showed significantly degradation effect on protein. When the ratio reached 10 ∶ 5,little recombinant H21 G existed in a form of monomer. Both dimer and polymer appeared when the H21 G was derived with ADH for 1 and 2 h,which showed no significant difference. The immuneeffects of recombinant H21 G and its derivates in mice were enhanced with the increasing doses,while the immunogenicity of derivates with ADH was higher than that with succinic anhydride. The derivates with succinic anhydride was less stable,of which all the target proteins were degraded significantly. However,no obvious polymerization or degradation was observed in the derivates with ADH after storage at 4 ℃ for 6 months. Conclusion Both immunogenicity and stability of derivatives of recombinant H21 G with ADH were superior to those with succinic anhydride.