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

作　　者：余鹏博[1] 王敬军[1] 张家驹[1] 郝志明[2] 刘中华[2] 董建华[1] 唐青[3] 许文波[3] 

机构地区：[1]陕西省疾病预防控制中心,西安710054 [2]西安交通大学 [3]中国疾病预防控制中心病毒病研究所

出　　处：《中国公共卫生》2005年第12期1445-1447,共3页Chinese Journal of Public Health

基　　金：陕西省首届医药卫生重点科研基金项目(99zh-001)

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。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 pBV220-S1.3 and pET28a-S1.3 through inducing in E. coll. The bacterial products were analyzed by using SDS-PAGE and Western-Blot. The results of analysis showed that there was a special antigen about 48kD, whick was rNP of HTNV. Myelo- ma cell SP2/0 were hybridized with lymphocytes B which taken from Immunized Balb/C. The positive hybridoma were screened with rNP antigen derived from pET28a-S1.3 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 Rceombi- nation prokaryotic expression carriers pBV220-S1.3 and pET28a-S1.3 were constructed for producing nucleoprotein of HT- NV successfully. High purity rNP and high titer Mc/Ab of anti-NP which were 2E6 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.

关 键 词：汉坦病毒 原核表达 重组核蛋白 单克隆抗体 

分 类 号：R373.9[医药卫生—病原生物学]