| 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220 r/min振摇5 h，当IPTG终浓度为0.1 mmol/L时，GST-Atac2在E.coli BL21(DE3)中高效表达，GST-Atac2融合蛋白大小约为30 ku，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。 |
| 关键词: 果蝇 Atac2 原核表达 单克隆抗体 |
| DOI： |
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| 基金项目:西北农林科技大学引进人才启动项目(Z111021005) |
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| Preparation of monoclonal antibody against the Drosophila Atac2 gene |
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| Abstract: |
| 【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． |
| Key words: Drosophila Atac2 prokaryotic expression monoclonal antibody |
