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Abstract Cry2Ab toxin of Bacillus thuringiensis is a toxic protein, which is wildly used in controlling lepidopteran pest in agricultural production. In this research, the cry2Ab gene (1 914 bp) was amplified from total DNA of B. thuringiensis WB9 strain by a pair of primer designed by the full-length sequence of published cry2Ab gene. Then, cry2Ab was ligated with linearized pGEX-KG vector by restriction enzyme BamHⅠand XhoⅠfor the construction of cry2Ab-pk expression vector. The soluble Cry2Ab-GST fusion protein (approximately 90 kD) was obtained after transferring Cry2Ab-PK expression vector into Escherichia coli BL21 (DE3) and then inducing by 0.8 mmol/L IPTG at 16℃ for 36 h. Total soluble protein was purified by batch purification and GST tag was removed by the use of prescission protease to obtain soluble Cry2Ab protein (approximately 65 kD). Polyclonal antibody against Cry2Ab was produced by immunizing the purified Cry2Ab to New Zealand white rabbit(Oryctolagus cuniculus) after three times of intramuscular injection and one time of intravenous injection. The titter of antibody was over 1∶150 000, measured by indirect ELISA. Specificity of the prepared antibody was determined by Western blot, showing that the polyclonal antibody against the Cry2Aa or Cry2Ab protein was positive and the antibody against Cry1Ab or Cry3Aa protein was negative. These results indicated that antibody against Cry2Ab protein can specifically identify Cry2A protein but cannot identify other three domains Cry protein including Cry1Ab and Cry3Aa. These results will provide technical support for further study of Cry2A toxins mechanism and the interaction between Cry2A toxins and its receptors.
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Received: 10 September 2012
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