截短ALT1蛋白的获得及其多克隆抗体的制备

 目的 原核表达、纯化截短ALT1蛋白，制备ALT1多克隆抗体。方法 利用pCold TF载体原核表达系统，IPTG诱导，表达经生物信息学分析含有两个编码ALT1抗原表位的ALT1 N端1~115个氨基酸序列的基因片段，依次经镍(Ni)离子亲和柱纯化、HRV 3C蛋白酶酶切、二次镍离子亲和柱纯化和分子筛柱层析得到不带标签的截短ALT1纯蛋白，用纯蛋白免疫新西兰大白兔制备多克隆抗体。结果 经测序证实，成功构建了截短pCold TF-ALT1表达载体。获得纯度达90%的不带标签的截短ALT1重组蛋白，制备了效价达4.0×106的ALT1多克隆抗体，经western blot鉴定，能特异性地识别ALT1抗原和肝细胞裂解液。结论 成功获得高质量的截短ALT1无标签蛋白及其特异的多克隆抗体，为ALT1的免疫学检测试剂的研发提供了依据。

Expression of truncated ALT1 protein and preparation of its polyclonal antibody

Objective To express and purify tag free truncated ALT1 protein by prokaryotic expression system, and prepare its polyclonal antibody through immunizing rabbits with this protein. Methods The gene coding sequence of ALT1 N-terminal 1~115 amino acids was inserted in pCold TF vector, and this vector was transformed to E.coli BL21(DE3) for expression under induction of IPTG at low temperature. The recombinant protein was purified successively by Ni-Sepharose 6FF affinity chromatography, HRV 3C protease digested, Ni-Sepharose 6FF affinity chromatography and size-exclusion chromatography. New Zealand rabbits were immunized with the truncated protein and the antiserum was obtained. Results The truncated ALT1 expression vector pCold TF-ALT1 was constructed and identified by sequencing. The truncated ALT1 protein were prepared and it reached a purity of 90% after purification, The polyclonal antibody to this protein was also prepared and the titers of antiserum reached at 4.0×106. Western blot identified that the antiserum can specially recognize lysate of HepG2 and human serum with hepatitis B. Conclusion High qualified ALT1 protein and its specific polyclonal antibody were prepared, which laid a foundation for the development of immunological reagent of ALT1.