Scientia Agricultura Sinica ›› 2013, Vol. 46 ›› Issue (16): 3369-3376.doi: 10.3864/j.issn.0578-1752.2013.16.007

• PLANT PROTECTION • Previous Articles     Next Articles

Prokaryotic Expression and Polyclonal Antibody Preparation of Trichothecene 3-O-Acetyltransferase Gene Tri101

 DONG  Fei, JI  Fang, WU  Ji-Rong, YIN  Xian-Chao, XU  Jian-Hong, SHI  Jian-Rong   

  1. Institute of Food Safety and Detection, Jiangsu Academy of Agricultural Sciences/Jiangsu Center for GMO Evaluation and Detection, Nanjing 210014
  • Received:2013-01-22 Online:2013-08-15 Published:2013-04-15

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Abstract: 【Objective】Tri101 was cloned and the purified protein was obtained by prokaryotic expression. Polyclonal antibody of high titer and high sensitivity was obtained by SPF rabbits immunization, thus providing a basis for the establishment of the transgenic plants and products.【Method】Tri101 was obtained from Fusarium graminearium Schwabe F3210 by PCR and connected to the expression vector pET29a (+), the recombinant vector was transformed into E. coli BL21(DE3) and induced by IPTG. The results indicated that the recombination expression plasmid was constructed and transformed into E. coli BL21 (DE3). The Tri101 protein was induced by IPTG and used as the antigen to immune the SPF rabbits. 【Result】The result of SDS-PAGE showed that the molecular weight of expressed protein was about 50 kD, existing in the form of inclusion body. The expressed protein was purified by HisTrap, and the best imidazole concentration was 200 mmol•L-1. His-tag present in recombinant protein was removed by TEV protease and the digestion product was purified by HisTrap. The purified protein was used to prepare polyclonal antibody. The result of ELISA showed that the titer of rabbit anti-Tri101 antiserum was 1﹕12 000，the detection sensitivity was 0.05×10-6 µg•mL-1. The result of Western blotting demonstrated that polyclonal antibody had a very good specificity.【Conclusion】Through Tri101 cloning and prokaryotic expression, the purified fusion protein was prepared by immunizing SPF rabbits. Polyclonal antibody was obtained and used for the establishment for the transgenic wheat and products.

Key words: trichothecene 3-O-acetyltransferase , Tri101 , prokaryotic expression , polyclonal antibody , Fusarium head blight (FHB)

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