兔抗Red蛋白抗血清的纯化及MC3T3细胞中Red蛋白表达时相的分析

Purification of Rabit Anti-Red Polyclonal Antibody and Determination of the Time Course of Red Proteins Expression in MC3T3 cells

为了纯化兔抗Red蛋白的3种多克隆抗体,并用其研究Red蛋白在真核细胞中的表达时相,首先于30℃培养了不含目的基因的空载体(pDH2)大肠菌,经彻底裂菌后用丙酮沉淀全菌蛋白,用其在不同时间吸附3种待纯化的Red抗体,然后用间接 ELISA法及Western-blot法检测了3种抗体的效价和特异性。最后用纯化后的3种Red抗体检测三顺反子表达载体pCMV-gIbIx的3 种蛋白表达产物在MC3T3细胞中的表达时相。ELISA及Western-blot检测结果表明:3种抗血清经纯化后特异性好且效价没有受到影响,随后用纯化的抗体成功地研究了Red基因在真核细胞中的表达时相,此结果为Red重组系统在真核生物基因功能的研究中提供了必要的条件。

To obtain purificate of rabit anti-red polyelonal antibody and determinate of the time course of red protein expression in subcellular, firstly, all pDH2 protein in Escherichia coli with acetone at 30℃ is deposited. Anti-red antiserum with all deposited protein in different time is adsorbed. Secondly, the titer and specificity of polyclonal antibody were detected by ELISA and Western-blot. Finally, the time course of expression of three red proteins in eukaryotic cells have been detected with the purified antibodies. Experimental result indicated that the purified antibodies had steady antibody titer by ELISA analysis, and Western blot analysis indicated that the three purified antibodies had the high specificity to the corresponding proteinsteady, which set up a technique foundation for studying the homogenous recombination of red proteins.