Welcome to Bulletin of Botanical Research! Today is Share:

Bulletin of Botanical Research ›› 2015, Vol. 35 ›› Issue (3): 333-339.doi: 10.7525/j.issn.1673-5102.2015.03.003

Previous Articles     Next Articles

Prokaryotic Expression of Caffeine Synthase Gene(TCS1),Its Polyclonal Antibody Preparation and Identification

DENG Wei-Wei;JIN Yang;LI Min;MA Lin-Long;ZHANG Zheng-Zhu*   

  1. State Key Laboratory of Tea Plant Biology and Utilization,Anhui Agricultural University,Hefei 230036
  • Online:2015-05-20 Published:2015-06-24
  • Supported by:

Abstract: Cloing and prokaryotic expressing of TCS1(Caffeine synthase 1) gene, preparing the antibody are the preliminary works for further research of the expression of TCS1 in tea plant. According to the cDNA fulllength sequence of TCS1 from GenBank, we found out the complete ORF(Open Reading Frame). The open reading frame(ORF) of TCS1 gene from the cDNA of leaves of tea plant was amplified. The ORF was then ligated into the expression vector pGEX-4T-2. The recombinant protein was expressed by induction with IPTG. Then, we tested in vitro enzyme activity, and purified the recombinant protein subsequently by affinity chromatography. The TCS1 antibody was further refined by immunizing white rabbits with the purified protein. The antibody titer and specificity of the polyclonal antibody were confirmed by ELISA and Western blot. Induced by optimizing the conditions, the best expression condition of the recombinant protein was at 30℃ for 4 h. After induction of total protein, soluble protein and inclusion body protein showed an obvious foreign proteins stripes. ELISA detection of the antibody recommended dilution ratio of 1∶2 000. We verified the favourable specific of the antibodies by western blot. The prokaryotic expression plasmid pGEX-4T-2-TCS1 was constructed, and the prepared polyclonal antibody had a high titer and good specificity.

Key words: Camellia sinensis, caffeine, tea caffeine synthase, prokaryotic expression, polyclonal antibody

CLC Number: