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| Expression of the open reading frame 27 of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus in Escherichia coli and cell localization |
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Abstract The open reading frame (ORF) 27 of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus is 768bp in length and potential to encode a polypeptide of 255 amino acids, with a molecular weight of about 29.5kD. The coding region of ORF27 was amplified from HaSNPV genome DNA by PCR, and cloned into pGEM-T easy vector, and then subcloned into the expression vector pGEX-4t-2 and the recombinant plasmid was named pGEXORF27. DNA sequencing was performed to confirm the correct amplification and orientation of the target sequence. pGEXORF27was transformed into competent cells of Escherichia coli BL21, and expression was induced for 3 hours in 1 mmol/L IPTG at 37℃. The molecular weight of fusion expression protein was 55.5kD, and it was produced to an amount about 9.2% of the total bacterial cellular protein. Western blot analysis showed the recombinant protein could react with GST antibody. Cutting gel and dialysis purified ORF27 protein. ORF27 gene was also inserted into pFastBacHTb and constructed pFastBacHTORF27. The recombinant donor plasmids were transformed to DH10 competent cells and constructed recombinant BacmidORF27. Mediating by Lipofectin, BacmidORF27 was transfected into Tn-5B1-4 cells of cabbage looper, Trichopolusia ni. SDS-PAGE analysis showed the expression product of ORF27 gene was 32kD that was identical to the predicted molecular weight. SDD-PAGE analysis demonstrated that the protein was produced to an amount about 2.9% of the total insect cellular protein. Western blotting analysis with anti-ORF27 further confirmed the preciseness of the expression product. Immunoelectron microscopy demonstrated ORF27-His fusion protein was localized in the cytoplasm and hardly present in the nucleus. The expression of ORF27 and its localization would lay the foundation for its function study.
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Received: 24 October 2006
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