汉坦病毒核蛋白原核表达纯化及其单抗制备
Expression and purification of nucleocapsid protein of hantaan virus and creation of anti-NP monoclonal antibody
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摘要:目的 制备稳定、特异的汉坦病毒核蛋白重组抗原和单克隆抗体。方法 构建汉坦病毒(HTNV)-76118株核蛋白原核表达载体(pBV)220-S1.3和(pET)28a-S1.3, 大肠埃希菌诱导表达。十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)和Western-blot分析显示, 在48kD附近有目的蛋白表达, 且有较好的抗原活性。用纯化的包涵体抗原免疫Balb/c小鼠, 取脾细胞与SP2/0骨髓瘤细胞融合, 经镍合氨基三乙酸(Ni-NAT)亲和纯化的核蛋白包被酶标板, 进行ELISA筛选阳性克隆株, 并建立细胞系, 选2株高效分泌抗核蛋白单克隆抗体(McAb)的阳性杂交瘤细胞, 常规制备腹水, 进行单抗鉴定。结果 成功构建了重组核蛋白原核表达载体pBV220-S1.3和pET28a-S1.3, 获得了高纯度重组核蛋白(rNP)及其高效价单克隆抗体2E6和4D8。结论 重组核蛋白抗原及其单克隆抗体在流行性出血热的监测、诊断和科研中有重要价值。Abstract:Objective To prepare stable and unique recombined nucleocapsid protein(rNP)and monoclonal antibody (McAb)of Hantaan virus(HTNV).Method To establish prokaryotic expression carriers of HTNV strain 76118 which are pBV2202S113 and pET28a2S113 through inducing in E.coli.The bacterial products were analyzed by using SDS2PA GE and Western-Blot.The results of analysis showed that there was a special antigen about 48kD, whick was rNP of HTNV.Myeloma cell SP2/0 were hybridized with lymphocytes B which taken from Immunized Balb/C.The positive hybridoma were screened with rNP antigen derived from pET28a2S113 purified by DEAE-Sephadex A50 and Ni-NAT chromatography.To establish cell line and select two lines for producing McAb of rNP routinely.Finally, McAb was identified.Results Rceombination prokaryotic expression carriers pBV2202S113 and pET28a2S113 were constructed for producing nucleoprotein of HTNV successfully.High purity rNP and high titer Mc/Ab of anti-NP which were 2 E6 and 4D8 were obtained.Conclusion The rNP and McAb of HTNV have great value in epidemiological sdurveillance, diagnosis and scientific research of epidemic hemorrhagic fever.
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