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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 carrying the antibody against human PRES2 protein on the surface of full-length human hepatitis B virus and observe its expression in vitro. METHODS: The PRES2 protein gene of 25 575 bp human hepatitis B virus was cloned into shuttle plasmid vector of adenovirus type 5, recombinant adenovirus was produced in 293 cells, and CHO cells were infected in vitro. The multiplicity of infection virus 20, to obtain the expression product, and determine its affinity. Results The titer of recombinant adenovirus was (2. 1 × 10 10) pfu / ml by TCID50 assay. After the virus was infected in CHO cells, the expressed protein was confirmed to be a full-length humanized antibody by Western blot analysis. The antibody affinity constant was (2.16 ± 0.20) × 10 8 L / mol. Conclusion The recombinant adenoviral vector carrying human PRES2 protein on the surface of full-length human hepatitis B virus successfully constructed and expressed human full-length high affinity humanized antibody, which provided a new approach for the treatment of hepatitis B virus.