Journal of East China Normal University(Natural Sc ›› 2010, Vol. 2010 ›› Issue (4): 77-84.

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Clone, expression of allantoicase gene from Dictyostelium disc-oideum and preparation of polyclonal antibody against ALC

CHEN Neng-xing, WEI Xiao-jing, LIU Wei, HOU Lian-sheng   

  1. School of Life Science, East China Normal University, Shanghai 200062, China
  • Received:2009-03-01 Revised:2009-07-01 Online:2010-07-25 Published:2010-07-25
  • Contact: HOU Lian-sheng

Abstract: The coding region of allCis obtained from total mRNA of Dictyostelium discoideumby RT-PCR. The results show that the length of allC is 1 100 bp with sequence analysis and coded protein is 42 kD.Becase it is a differentially expression fragments, indicating that the gene play an important role in multicellular development and allC was cloned into the fusion expression vector pET-32a(+) with 6 His tagged and expressed in E. coli BL21 (DE3) host cells. and then purified by Ni2+ affinity chromatography column and fusion protein purified was used to immune the New Zealand rabbits for preparing polyclonal antibody. The fusion protein was successfully expressed and polyclonal antibody was also successfully obtained. The potency of the antibody was as high as 1∶64 000. The specificity of antibody was proved by Western Blotting analysis of expression product of allC. These data suggest ALC fusion protein has the good antigenicity. The antibody with high titer and specificity was obtained in the satisfaction of Western blot and localization experiment request , which was the foundation to farther study the characters of the allantoicase protein and their function during Dictyostelium discoideumdevelopment.

Key words: allantoicase, prokaryotic expression, protein purification, polyclonal antibody, Dictyostelium discoideum, allantoicase, prokaryotic expression, protein purification, polyclonal antibody

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