关键词: 铁氧还蛋白Ⅰ, 原核表达, 抗体, 番茄花叶病毒

Abstract:

【Objective】 The objective of the study is to construct the prokaryotic expression vector of Fdn-I and express the soluble Fdn-I in E. coil BL21 for preparation of rabbit anti-Fdn-I antibody for the analysis of expression of Fdn-I in tobacco leaves infected by ToMV. 【Method】 Fdn-I was amplified from the full-length cDNA of Fdn-I by PCR and cloned into pEGX-6p-1, an expression vector fused with GST, to construct the pEGX-Fdn-I. The recombinant plasmid was transformed into E. coil BL21 to express the soluble GST-Fdn-I protein in the optimized condition. GST-Fdn-I protein was purified with high-affinity GST resin to immunize rabbit for preparing anti-Fdn-I antibody. The title was determined by enzyme-linked immunosorbant assay (ELISA). The specificity of anti-Fdn-I antibody and the expression of Fdn-I in tobacco leaves infected by ToMV were detected by Western blot method. 【Result】 The soluble GST-Fdn-I protein with molecular weight 41.3 kD was successfully expressed in E. coil BL21 induced with 0.3 mmol?L-1 IPTG at 28℃. About 4 mg fusion protein was purified and used to immunized rabbit. Anti-Fdn-I antibody with the title of 1/6 400 was obtained. Western blotting analysis showed that it could bind with Fdn-I specially expressed in E. coil and Yeast. In ToMV infected tobacco leaves, the expression of Fdn-I was lower than that in health tobacco leaves. 【Conclusion】 The recombinant GST-Fdn-I protein in E. coli and its polyclonal antibody with high title and specificity were successfully achieved. The result of Western blot with the polyclonal antibody showed that the expression of Fdn-I in tobacco leaves was decreased by the infection of ToMV.

Key words: ferredoxin I, prokaryotic expression, antibody, Tomato mosaic virus