神经元特异核蛋白单克隆抗体的构建

Construction of the monoclonal antibody of neuron-specific nuclear protein

目的 根据Pubmed报道的FOX3cDNA序列,构建神经元特异核蛋白（Neun）单克隆抗体。方法 获得FOX3cDNA序列,设计引物,PCR扩增,利用pcDNATM3.3-TOPO TA将纯化后的PCR产物插入真核表达载体中,得到FOX3蛋白,然后利用该蛋白免疫小鼠,免疫后小鼠的脾细胞与骨髓瘤细胞（SP2/0）进行融合,筛选后获得的杂交瘤细胞小鼠腹腔接种,收获腹腔积液后进行相关检测。结果 所获得的腹腔积液经免疫印迹检测,在（46~48）×103处显示两条带,免疫荧光检测后细胞核显示阳性信号。结论 根据FOX3cDNA序列构建的抗体与商业化的抗Neun抗体在免疫荧光和免疫印迹上表现一致,Neun与FOX3为同一基因。

Objective To construct the monoclonal antibody of anti-Neun basis on the sequence of FOX3 eDNA published on Pubmed. Methods The specific primers were designed basis on the sequence of FOX3 cDNA which was amplified with RT-PCR, then the PCR product wag inserted into peDNATM 3.3-TOPO TA to get the eukaryotic expression vector FOX3. The splenocyte from mice which were immunized with the protein of FOX3 was merged with SP2/0 cell line to gain hybridoma cell. The hybridoma cell was transplanted into the abdominal cavity of mice and the abdominal dropsy through selecting was collected and used into im- munofluorescence staining arid immunohlotting. Results There were two specific bands from （46-48）×10^3 through immunoblot- ting and positive cells through immunofluorescence staining. Conclusion The antibody anti-Neun from the FOX3 cDNA sequence was showed the same information from the commercialized Neun antibody,through checking with immunofluorescence staining and immunoblotting. The gene Neun was as same as to gene FOX3.