Prokaryotic expression, purification and polyclonal antibody preparation of arginase in Agaricus bisporus / 国际药学研究杂志

ABSTRACT

Objective: To provide a method for preparing a specific polyclonal antibody of ARG. Methods: Firstly, the technology of polymerase chain reaction(PCR)was adopted to amplify the AbArg gene encoding arginase in Agaricus bisporus. Then the recombinant plasmid pET-28a(+)-Arg was constructed and transformed into E. coli BL21 for the expression of fusion protein. Afterwards, the purified protein was used to immunize New Zealand white rabbits, and the serum samples were collected. Results: The prokaryotic expression plasmid pET-28a(+)-Arg was constructed and the results of SDS-PAGE analysis showed that the fusion protein existing in form of inclusion body was successfully induced and expressed. After purification, the fusion protein with high purity (above 90%)was obtained. The immunity serum titer was 51 200 and Western blotting results showed that the polyclonal antibody could specifically recognize ARG protein in A. bisporus. Conclusion: Polyclonal antibody anti-ARG was prepared successfully, which lays a foundation for the further study on the mechanism of arginine metabolism.