【摘要】 从人胎肝cDNA文库中钓取得到SLC38A3全长cDNA序列.利用生物信息学方法对SLC38A3蛋白序列进行分析,根据亲水性、抗原性、柔韧性及表面性等指标选择一段多肽序列作为抗原用于抗体制备.将该片段克隆到pET-DsbA融合蛋白表达载体上,在异丙基硫代半乳糖苷(IPTG)诱导下产生SLC38A3抗原肽.纯化目的蛋白并制备兔抗SLC38A3蛋白的多克隆抗体.利用Western blot鉴定抗体特异性,并初步分析该蛋白在人肺癌细胞A549内的定位.结果显示,成功构建了SLC38A3片段的原核表达载体,在大肠杆菌中实现可溶性表达,制备了特异性较高的人SLC38A3蛋白多克隆抗体,并证实该蛋白表达于细胞质内.为进一步研究SLC38A3的生物学功能奠定了基础.更多还原

【Abstract】 Solute carrier family 38 member 3(SLC38A3) cDNA was cloned from a human fetal liver cDNA library.The structure of SLC38A3 protein was analyzed with bioinformatics methods.A peptide segment was chosen as the antigen for antibody production according to hydrophilia,antigenicity,flexibility and surface.The cDNA fragment was cloned into pET-DsbA.The fusion protein was induced by IPTG in E.coli BL21(DE3).The target protein was purified by Ni2+-NTA chromatography and used to immunize the rabbit.The specificity of antibody was confirmed by Western blot.The protein localization was analysed in lung cancer cell line A549.The recombinant vector pET-DsbA-SLC38A3p was successfully constructed and expressed.The obtained antibody was specific.The protein was confirmed expressed in the cytoplasm of A549 cells.Our work plays a basis for the further study on SLC38A3.更多还原