Scientia Agricultura Sinica ›› 2014, Vol. 47 ›› Issue (19): 3791-3798.doi: 10.3864/j.issn.0578-1752.2014.19.007

• PLANT PROTECTION • Previous Articles     Next Articles

Cloning, Prokaryotic Expression and Polyclonal Antibody Preparation of Tropomyosin Gene in Laodelphax striatellus Fallén

XU Qiu-fang1, CHEN Qing-qing1,2, NI Hai-ping1, LI Shuo1, ZHANG Jin-feng1, ZHOU Yi-jun1   

  1. 1Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014
    2College of Life Science, Nanjing Normal University, Nanjing 210046
  • Received:2014-04-01 Revised:2014-06-17 Online:2014-10-01 Published:2014-10-01

Abstract: 【Objective】 By screening Laodelphax striatellus Fallén (small brown planthopper, SBPH) cDNA library, it was found that the tropomyosin (Tm) could interact with RBSDV P10 protein. To provide the antibody of Tm protein for analyzing the Tm gene’s function in the process of RBSDV and SBPH interaction, the Tm was cloned and expressed in prokaryotic system. Tm protein was purified and the polyclonal antibody was obtained by immunizing the rabbit. 【Method】 The complete open reading frame (ORF) of Tm from SBPH was determined by comparing the sequence to the highly homologous Tm in other species. The total RNA was extracted and the Tm ORF was cloned by reverse transcription polymerase chain reaction (RT-PCR). Tm was then inserted into the pMD-18T for sequencing. The nucleic acid sequence and the amino acids encoded by this ORF were analyzed by DNAstar software. Tm ORF was inserted into the expression vector pET-32a (+) by EcoR V and BamH I digestion. The recombinant plasmid was transformed into E. coli BL21 (DE3) and expressed under the induction with 0.4 mmol?L-1 IPTG for 4 h. Tm fusion protein was detected by SDS-PAGE. The E. coli BL21 (DE3) expressing the Tm fusion protein was collected and crushed by ultrasonic. The supernatant and the precipitate were collected, respectively, and SDS-PAGE was used to analyze the expression of the fusion protein. The soluble fusion protein was purified by Ni-NTA Agarose, washed with 100 mmol?L-1 imidazole and dialyzed with 0.01 mol?L-1 PBST. The purified fusion protein was used to produce polyclonal antibody via immunizing rabbit. The titer of rabbit anti-Tm antiserum was evaluated by indirect ELISA. To estimate the specificity of this antibody, the purified fusion Tm protein and the total proteins from SBPH were separated by SDS-PAGE and were tested by Western blotting assays using the prepared antibody.【Result】The sequence analysis results showed that the length of complete SBPH Tm ORF was 852 bp. It was amplified by RT-PCR and the sequencing result showed that the length of Tm ORF was 852 bp, encoded 283 amino acids, and its theoretical molecular weight was 32.6 kD. The blast results indicated that the encoded protein was conserved among different species. Tm was inserted into the vector pET-32a (+) and expressed in E. coli BL21 (DE3). The SDS-PAGE result showed that the Tm fusion protein of approximately 55 kD was mainly expressed as a soluble protein and there was also a small amount in inclusion body. The soluble fusion protein was purified and the polyclonal antibody was obtained by immunizing the rabbits using the purified fusion protein. The ELISA assay showed that the rabbit anti-Tm antiserum had a good sensitivity with the titer higher than 1﹕409 600. Using the polyclonal antibody to detect the purified Tm fusion protein and the total SBPH protein, specific band (55 and 37 kD, respectively) could be observed, suggesting that this antibody has strong specificity. 【Conclusion】 The ORF of SBPH Tm was cloned firstly. The Tm fusion protein was expressed in E. coli BL21 (DE3), purified successfully and used for preparing the antibody. The rabbit anti-Tm antiserum with high titer was obtained.

Key words: Laodelphax striatellus Fallén, tropomyosin, cloning, prokaryotic expression, polyclonal antibody

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