关键词: ALT1, 原核表达, 蛋白纯化, 多克隆抗体

Abstract: 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.

Key words: ALT1, Prokaryotic expression, Protein purification, Polyclonal antibody