分泌抗丙型肝炎病毒NS_5蛋白单克隆抗体杂交瘤细胞株的建立及初步鉴定

ESTABLISHMENT AND PRELIMINARY IDENTIFICATION OF ANTI HCV NS 5 McAb HYBRIDOMA CELL LINES

目的建立能稳定分泌抗丙型肝炎病毒（ＨＣＶ）ＮＳ５重组蛋白单克隆抗体（ＭｃＡｂ），并对其生物学活性进行实验室鉴定。方法用重组ＨＣＶＮＳ５区蛋白为抗原免疫ＢＡＬＢ／ｃ小鼠，取其脾细胞与骨髓瘤细胞ＳＰ２／０进行融合，经有限稀释克隆３次后制备分泌ＭｃＡｂ的杂交瘤细胞株，并用酶免疫（ＥＩＡ）法对其分泌抗体进行初步鉴定。结果融合后的阳性克隆中筛选出６株能稳定分泌ＭｃＡｂ的杂交瘤细胞株，命名为２Ｃ８，２Ｄ２，１Ｄ６，２Ｇ２，１Ｆ５和１Ｆ１０。这６株ＭｃＡｂ与ＮＳ５重组抗原均有良好的反应性，杂交瘤培养上清的ＥＩＡ抗体滴度为１∶４００～１∶１６００，其中１株诱生的同系小鼠腹水滴度为１∶８００００。这６株ＭｃＡｂ均为ＩｇＧ１亚型。结论该ＭｃＡｂ的制备，可为ＨＣＶ感染者血清和肝组织中抗原检测方法的建立和生物工程药物的研制创造条件。

ObjectiveThe goal is to establish hybridoma cell line which can secrete monoclonal antibodies against HCV recombinant NS 5 protein and to determine biological activity of the monoclonal antibodies. MethodsThe BALB/c mice were immunized through intraperitoneal injection of the NS 5 region protein of hepatitis C virus. After three times injection, the splenocytide positive for the NS 5 region antigen was fused with SP 2/0 mouse myeloma cells. To prepare secretable McAb byhridoma cell lines, three times subcloning method, limited dilution method, were used. The antibody strains were analyzed by EIA. ResultsSix hybridoma cell lines of positive for HCV NS 5 protein were selected designed 2C 8,2D 2, 1D 6, 2G 2,1F 5 and 1F 10 from cloning strains. The titer of EIA of the culture supernatant were 1∶400～1∶ 1 600 . The titer of EIA in one animal of ascites was 1∶ 80 000 . All the monoclonal 1 antibody strain was IgG 1 subtype. ConclusionThe McAb can be used in detection of HCV antigen in sera and hepatocyte and used in study of new bioengineering medicine.