腺病毒载体携带全长人乙肝病毒表面前S2蛋白抗体基因在体外的表达

Expression of adenoviral vector encoding the whole humanized antibody gene of Pres2 protein of hepatitis B virus in vitro

目的 构建携带全长人乙肝病毒表面PRES2 蛋白抗体基因的腺病毒载体 ,观察其在体外的表达。方法 将全长 2 5 70bp人乙肝病毒表面PRES2 蛋白抗体基因克隆入 5型腺病毒穿梭质粒载体 ,在 2 93细胞中重组产生重组腺病毒 ,体外感染CHO细胞 ,感染复数 (MultiplicityofInfec tionVirus/Cell)为 2 0 ,获得表达产物 ,并测定其亲和力。结果 TCID5 0法测定重组腺病毒滴度为(2 .1× 10 10 )pfu/ml。病毒感染CHO细胞后 ,表达蛋白经蛋白质印迹分析证实为全长人源化抗体。抗体亲和力常数为 (2 .16± 0 .2 0 )× 10 8L/mol。结论 全长人乙肝病毒表面PRES2 蛋白抗体基因腺病毒载体成功构建和表达全长高亲和力人源化抗体 ,为乙型肝炎治疗提供了一个全新的途径。

Objective To construct the adenoviral vector encoding the whole humanized antibody gene of pres2 protein of hepatitis B virus and detect its expression in CHO cells.Methods The whole 2570bp humanized antibody gene of pres2 protein was cloned into 5 type adenoviral shuttle plasmid pDC315.The corresponding recombinant viruses were obtained by homologous recombination in 293 packaging cells.The viruses containing the whole humanized antibody gene of pres2 were transfected into CHO cells.MOI was 20.Results The viral titer was 2.1× 10 10 pfu/ml determined by TCID50 analysis.Supernatant of CHO cells culture transfected with recombined adenoviruses was collected and the recombinant protein was purified.Western blot revealed the product was whole humanized antibody.The affinity for pres2 antigen was 2.16± 0.20× 10 8 L/mol.Conclusion The adenoviral vector encoding the whole humanized antibody gene of pres2 protein was constructed successfully and the whole humanized antibody was expressed.