Prokaryotic expression of human ⅠAlanine aminotransferase and prepara tion of its antiserum
AIM: To construct a prokaryotic expression vector of ALT1 (Alanine aminotransferasel), to purify ALT1 protein produced by the expression system and to prepare its antiserum. METHODS: Total RNA was isolated from HepG2 cells and human alt gene was amplified with RT-PCR. The code fragment was cloned into pMD19-T rector and was confirmed by sequencing. The enzyme-digested target fragment was cloned into PET-32a ( + ) expression vector and transfected into E. coli. ALT1 expression was induced in BL21 ( DE3 ). After the inclusion body protein was purified through Ni 2+ affinity chromatography, processed by dialysis and identified by Western Blotting, rabbits were immunized with the fusion protein and the antiserum was obtained. RESULTS: The result of DNA sequence analysis showed that the cloned alt gene sequence was completely corresponding to that in Gene Bank data. SDS-PAGE and Western Blotting showed that the expressed ALT1 fusion protein was about 75 × 103, existing in the inclusion body of E. coli, which could be purified through Ni2+ affinity chromatography. The liter of the antiserum to the purified protein was 1:100 000 by ELISA and Western Blotting confirmed that the antiserum reacted specifically to the ALT protein. CONCLUSION: A recombinant ALT1 protein and the specific polyclonal antibody have been obtained, which provides a basis for the establishment of immunoassays of human ALT1.
NETL
NSTL
万方数据
维普
