抗Luman募集因子N端单克隆抗体的制备及鉴定

Preparation and identification of monoclonal antibodies against Luman-recruiting factor N-terminal

【目的】制备抗Luman募集因子N端(Luman-recruiting factor N terminal,LRF-N)蛋白的单克隆抗体。【方法】PCR扩增LRF-N端的194bp序列,构建LRF-N重组质粒pGEX-LRF-N,转染BL21(DE3)菌,诱导表达并纯化GST-LRF-N融合蛋白,以其作为抗原免疫6～8周龄雌性BALB/c小鼠,免疫4次,每次间隔10d,第4次加强免疫3d后采取小鼠脾脏,分离脾细胞,与SP2/0细胞融合,用ELISA和Western blot进行阳性细胞株的筛选和鉴定,对获得的杂交瘤细胞的细胞核型、分泌稳定性以及所分泌的单克隆抗体进行鉴定。【结果】pGEX-LRF-N在大肠杆菌BL21(DE3)中得到高效表达,GST-LRF-N蛋白分子质量约为33ku,与预期结果一致。试验共获得3株稳定分泌抗LRF-N抗体的杂交瘤细胞株,分别命名为2AC9、2AG10和3G7。单抗2AC9、2AG10、3G7能够特异性地识别GST-LRF-N蛋白,亚型鉴定结果显示,其重链分别为IgG2a、IgM和IgG1。【结论】获得的抗LRF-N抗体的杂交瘤细胞株2AC9、2AG10、3G7单抗特异性良好,能够稳定分泌抗体,可用于小鼠LRF蛋白生物学功能及活性的进一步研究。

[Objective] This study was to develop and characterize monoclonal antibodies against Lu- man-recruiting factor N terminal（LRF-N）. [Method] PCR was used to amplified 194 bp fragment of LRF gene N-terminal and a recombinant plasmid pGEX-LRF-N was constructed. Then,the GST-LRF-N recom- binant protein was expressed and purified before being used to immunize the female BALB/c Mus musculus （with age of 6--8 weeks） once every 10 days for a total of 4 times. The spleens were extracted 3 days after the fourth immunization,and splenocytes were fused with $P2/0 cells. The selection and identification of positive samples and the identification of karyotypes, secretion stabilities and the obtained 3 monoclonal an- tibodies against GST-LRF-N （MAb,named as 2AC9,2AG10 ,and 3G7 ,respectively） were finally conducted by indirect ELISA and Western blot assays. [Result] Recombinant plasmid pGEX-LRF-N was expressed efficiently in the Escherichia coli BL21 （DE3）,and the molecular weight of its expressive GST-LRF-N pro-tein was 33 ku,Mouse positive antiserum could recognize GST-LRF-N protein. The MAbs 2AC9,2AG10, and 3G7 could specifically recognize pGEX-LRF-N,from pGEX-Luman protein,pGEX-Atac2 protein and pGEX-4T-1 protein. The results of isotype analysis showed that MAbs 2AC9,2AG10, and 3G7 could be categorized to IgG2a, IgM, and IgG1 subclasses, respectively. [Conclusion] The obtained MAbs from 2AC9,2AG10, and 3G7 hybridoma cells have good monospecificities and can stably secrete antibodies,thus can be used for further research of LRF protein in mouse tissues.