Cloning And Expression Of The Venom Genes Encoding Secapin, Apamin, MCDP And Ag5 From Honeybee And Vespoidea Species

The venoms from the gland of the honeybee and vespid have been widely studied and used in many fields worldwide. The venoms are significantly valuable to be exploited and utilized as a natural medicine. The honeybee venom contains many enzymes and peptides, of which apamin, MCDP and secapin are three of the important peptides and have been studied in medicine, immunology and molecular biology fields. The allergen antigen 5 is one of three major allergens found in vespid venom, which is valuable for venom allergic diagnose and immunotherapy. Up to date, limited reports on molecular biology of the honeybee venom are available and none of the research concerneing the molecular biology of vespid venom has been conducted in China. In this dissertation, we report the cloning of the venom genes encoding apamin, secapin, MCDP and Ag5 from honeybee and Vespoidea species, and their expressions in E.coli and Bac-to-Bac baculovirus-insect cell expression system. We have confirmed the venom glands of wasps contained the peptides of apamin, secapin and MCDP firstly. At the same time, we have prepared the anti-MCDP and anti-Ag5 polyclonal antibodies in this study.Main results of this dissertation included the following four parts: 1. Cloning and expression of the venom gene encoding apamin from honeybee andVespoidea Species(1) The preproapamin genes were amplified by RT-PCR from the total RNA of venom gland of 2 honeybee species, Apis mellifera ligustica, A. cerana cerana, and 4 wasp species, Vespa magnified, V. velutina nigrothorax, Vespula maculifrons and Polistes hebraeus, respectively. Their PCR products were ligated into pGEM?T easy vector and the nucleotide sequences were analyzed. The six fragments were all 141bp in length, containing an ORF coding the precursor of apamin. The deduced amino acid sequences of preproapamins from six species were all 46 amino acid residues in length with predicted molecular weight of 5.1 kD. The preproapamin could be clustered into two types, and the apamin (mature peptideregion) was completely conserved among the six species. The 3'-noncoding region of the gene was also amplified by 3'RACE from the total RNA of A. cerana cerana. The sequence of the 3'-noncoding region shared 91.4% homology with that of A. mellifera ligustica, withlbp deletion in the position of 226 and 2bp inserts in the position of 237, respectively.(2) Both the coding regions for the matured peptide (apamin) and the preproapamin together with the 3'-noncoding region of A.cerana cerana were successfully sub-cloned into the prokaryotic expression vector pGEX-4T-2, forming the recombinant expression vectors, pGEX/AccPPapamin and pGEX/AccApamin, respectively. The recombinant vectors were then introduced into E.coli BL21 (DE3) for expression. Their expressed protein bands of 28kD and 31kD, the same as the expected sizes, were examed by SDS-PAGE, respectively. Western blotting analysis with anti-GST-antibody confirmed that the bands of 28kD and 31kD were the GST-Apamin and GST-Preproapamin, respectively. GST-Apamin fusion protein accumulated up to about 30% of total bacterial protein. Most of recombinant protein was soluble after the expression conditions were optimized. The apamin was recovered by cleaving the fusion protein with thrombin protease.(3) The EGFP-AccApamin was expressed in baculovirus-insect cell expression system. The expressed protein band of 35 kD was determined by SDS-PAGE. It was showed that the expressed protein band of EGFP-AccApamin was about 10.9% of total protein of Tn cells. Probing with the mouse anti-His monoclonal antibodies as the first antibodies showed that the molecular weight of expressed protein band was the same as the estimated.2. Cloning and expression of the venom gene encoding secapin from honeybee and Vespoidea species(1) The preprosecapin genes were amplified by RT-PCR from the total RNA of venom gland of 2 honeybee species, A. mellifera ligustica, A. cerana cerana, and 4 wasp species, V. magnifica, V. velutina nigrothorax, V. maculifrons and P. hebraeus, re...