抗果蝇组蛋白乙酰转移酶Atac2单克隆抗体的制备  

Preparation of monoclonal antibody against the Drosophila Atac2 gene

作　　者：马延森[1] 李晓[1] 陈志[1] 兰向丽[1] 武小椿[1] 靳亚平[1] 刘伟 

机构地区：[1]西北农林科技大学动物医学院,农业部动物生物技术重点实验室,陕西杨凌712100

出　　处：《西北农林科技大学学报（自然科学版）》2013年第3期33-38,共6页Journal of Northwest A&F University(Natural Science Edition)

基　　金：西北农林科技大学引进人才启动项目(Z111021005)

摘　　要：【目的】制备抗果蝇组蛋白乙酰转移酶Atac2的单克隆抗体,为进一步研究Atac2基因的功能提供重要的试验工具。【方法】PCR扩增果蝇Atac2蛋白N端前26个氨基酸的编码基因,构建GST-Atac2融合蛋白表达载体pGEX-Atac2。转化大肠杆菌E.coli BL21(DE3),进行诱导表达,通过GST亲和层析法纯化GST-Atac2融合蛋白。以GST-Atac2为抗原免疫8周龄的Balb/c小鼠,取脾细胞,与SP2/0骨髓瘤细胞融合,间接ELISA法检测阳性孔,经有限稀释法亚克隆,筛选单克隆杂交瘤细胞株,检测抗体的特异性、效价和亚型,并检测其亲和常数。【结果】PCR扩增获得了目的片段。成功构建了GST-Atac2融合蛋白表达载体,于37℃下220r/min振摇5h,当IPTG终浓度为0.1mmol/L时,GST-Atac2在E.coli BL21(DE3)中高效表达,GST-Atac2融合蛋白大小约为30ku,与预期结果一致。获得1株稳定分泌抗Atac2抗体的杂交瘤细胞株,命名为1C7。单抗1C7能够特异地识别GST-Atac2蛋白,效价为1∶2×105;亚型鉴定表明其重链为IgG1,所得单克隆抗体的亲和常数为2×105。【结论】获得了1株特异性好、能够稳定分泌抗果蝇Atac2蛋白的单克隆抗体杂交瘤细胞株。【Objective】 This work was to prepare monoclonal antibodies against the Drosophila acetyltransferase Atac2 gene,so as to provide an important test tool for further research on Atac2 gene.【Method】 Atac2 gene N-terminal 78 bp fragment was amplified by PCR to construct a recombinant plasmid pGEX-Atac2.Inducible expression and affinity chromatography purified protein GST-Atac2 was used to immunize 8-week-old female Balb/c Mus musculuson.Spleen cells were collected on the fourth day after four times of immunization.After fusion between SP2/0 myeloma cell and the stimulated splenocytes,one hybridoma cell strain against Atac2 named 1C7 was obtained.It was checked by indirect ELISA and Western blot.【Result】 Recombinant plasmid pGEX-Atac2 was expressed efficiently in the E.coli BL21（DE3）,and its expressive GST-Atac2 protein was about 30 ku.The MAb 1C7 could specifically recognize pGEX-Atac2 from pGEX-Zhangfei protein and pGEX-4T-1 protein.Isotype analysis showed that MAb 1C7 belonged to IgG1 subclass with κ light chain.【Conclusion】 These results suggested that we got the MAb against Atac2,which could serve as a useful tool for further functional study of Atac2.

关 键 词：果蝇 Atac2 原核表达 单克隆抗体 

分 类 号：Q291[生物学—细胞生物学] Q785