Expression of the melittin gene of Apis cerana cerana ( Hymenoptera : Apidae ) in insect cells

Abstract

A fragment encoding melittin cDNA from Apis cerana cerana fused with glutathione S-transferase gene was inserted into the multiple cloning site of the pBacFastHTb to construct a recombinant donor plasmid, pBacHT-GSTAccM, which was transposed to the target bacmid in E. coli (DH10) by Tn7 transposition function. Then the recombinant baculovirus Bacmid-GSTAccM was transfected into Tn-5B1-4 cells of the cabbage looper, Trichoplusia ni, mediated by lipofectin. The expressed protein of about 34 kDa was detected by Western blotting and triple antibody sandwich ELISA, indicating that the recombinant protein is the fusion protein of GSTAccM. Thin layer scanning showed that the expression level of GSTAccM was about 7% of the total cell protein. Purified and recovered recombinant melittin of A. c. cerana showed bioactivity in activating rabbit platelets to aggregate. 867 * Corresponding authors. Generation of recombinant baculovirus The recombinant plasmid pGEM®-GSTAccM was digested with XbaI/HindIII endonucleases and ligated into a pBacFastHTb vector cut with XbaI/HindIII. This final recombinant baculovirus expression vector pBacHT-GSTAccM was identified by PCR with primers FP1-GST and RP2-AccM. After being transformed into E. coli DH10Bac cells, the recombinant baculovirus Bacmid-GSTAccM was checked by PCR with PUC/M13 primers (Bacmid-FP1 primer: 5’-GTAAAACGACG GCCAGT; Bacmid-RP2 primer: 5’-AACAGCTATGACCATG) designed from the left and right arms sequences of the bacterial transposon Tn7 (mini-att Tn7) and GST-AccM primers (FP1-GST and RP2-AccM), respectively. Transfection of Tn-5B1-4 cells with recombinant bacmid

Cite this paper

@inproceedings{Shi2006ExpressionOT, title={Expression of the melittin gene of Apis cerana cerana ( Hymenoptera : Apidae ) in insect cells}, author={Wan Shi and Jia Cheng and Chuan Xi Zhang}, year={2006} }