| Melittin, a peptide constituting about 40%~50% of dry venom, is the main toxin of Apis mellifera venom. It has important biological activities. It causes the lysis of erythrocytes and the release of marker ions from liposomes and of histamine from mast cells. Its amphophilic structure-a large apolar portion and a very basic polar end-is ideal for promoting interactions of melittin with lipid membranes. Extensive studies on melittin have been made in recent years and more and more attentions have been paid to its basic and clinical iatrology, biological engineering, and plant protection in agriculture.The main results in this study are shown as following:Six cDNA fragments encoding prepromelittin were amplified by RT-PCR from the total RNA of venom gland of female Polistes hebraeus, Vespula maculifrons, Vespa velutina nigrithorax, Vespa magnified and of worker honey bees, Apis cerana cerana and A. mellifera, respectively. The PCR products were ligated into pGEM -T easy vector. The sequencing results showed that the amplified cDNAs containing the open reading frames of prepromelittin, and their lengths were all 213 bp. The ORFs were potential to encode polypeptides of 70 amino acid residues with predicted molecular weight of 7.7 kDa, including a signal peptide of 21 residues and a promelittin of 49 residues. Comparative analysis showed that the prepromelittins from 4 Vespoidea species and 2 honeybee species shared more than 92% identities in amino acid sequences with each other. The sequences of prepromelittins of Polistes hebraeus, Vespula maculifrons, Vespa velutina nigrithorax, Vespa magnifica,Apis cerana cerana and Apis mellifera shared 93%, 100%, 97.2%, 97.2%, 97.2% and 100% identities in amino acid sequences with that of Apis cerana indica, respectively. In conclusion, the prepromelittins are very conserved in the primary structure and the insects of Vespoidea also contain the melittins in their venoms, which are very similar to that ofhoneybee, although they belong to different superfamilies. It is the first report in the world that there are me/ittin genes of Vespoidea species. The /vepromelittin mRNA sequences of Polistes hebraeus, Vespula maculifrons, Vespa velutina nigrithorax, Vespa magnified and Apis cerana cerana reported here were all submitted to GenBank with accession No. AF487909, AF487911, AF487908, AF487910 and AF487907, respectively.The me/ittin coding regions of A. cenara cenara (AccM) and P. hebraeus (PhM)were inserted into the GST fusion expression vector pGEX-4T-2 to form therecombinant vectors, pGEX-AccM and pGEX-PhM, respectively. The 2 recombinantvectors were transformed into the E. coli BL21 and the target fusion proteins were thenhighly expressed with the induction of IPTG The expressed protein bands of about29kD, which were consistent to the expected molecular weight of the fusion proteins,GST-AccM and GST-PhM(28.5kD), appeared on the SDS-PAGE profiles. Theexpression products were confirmed by Western blotting and triple antibody sandwichELISA. At the same time, the expression conditions of GST-AccM and GST-PhMfusion proteins for E. coli BL21 transformants were optimized, respectively, indifferent bacterial concentrations (ODeoo), IPTG concentrations, temperatureconditions and induction durations. Thin layer scanning on the SDS-PAGE profiles ofGST-AccM and GST-PhM showed that the expressed proteins accumulated up to about12.7-14.1% and 11.7% of total protein of bacterial cells under the optimizedexpression conditions, respectively. Purified and recovered recombinant me/ittins of A.cerana cerana and P. hebraeus showed bioactivity in activating rabbit platelets toaggregate, accompanying by the formation of thromboxane B2.The restriction fragments of GSTAccM and GSTPhM were inserted into the multiple cloning site of the pBacFastHTb to construct recombinant donor plasmaids, pBacHT-GSTAccM and pBacHT-GSTPhM, which were used to transposed to the target Bacmid in E. coli(DH10) by Tn7 transposition funct...
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