Font Size: a A A

Cloning And Expression Of Genes Encoding Phospholipase A2 And Hyaluronidase Of Honeybee Bee Venom From Apls Cerana Cerana And A.Mellifera

Posted on:2003-05-14Degree:DoctorType:Dissertation
Country:ChinaCandidate:L R ShenFull Text:PDF
GTID:1100360062485187Subject:Agricultural Entomology and Pest Control
Abstract/Summary:PDF Full Text Request
Bee venom is a kind of toxic substances that man has had to live with since prehistoric time. As a natural medicine, it has been used for numerous health-related purposes at all times and in all over the world and has extensive therapeutic effects for many diseases, such as rheumatoid arthritis, cardiovascular disease .Meanwhile, as a type of rich apicultural products, it has important economic value. Researchers have identified more than 40 active components in bee venom, of which phosphlipase A2 (BvPLA2)and hyaluronidase (BvHya) are two of the important components. Recent 20 years, BvPLA2 and BvHya from Apis mellifera have been studied and used extensively as tool enzymes , active peptide medicines and natural pesticide components in cellular and molecular biological research, clinic medicinal and agricultural research. The molecular biology and functions of the both components have been paid more and more attention.In this study, two aspects are implemented.( I ) Cloning and expression of genes encoding BvPLA2 from Apis cerana cerana and A. melliferaTwo matured peptide coding regions of the bee venomous phospholipase A2 (BvPLA2 ) genes, AcPLA2 and AmPLA2 were amplified by RT桺CR from the total RNA of honey bees, Apis cerana cerana and A- mellifera worker bee venom glands , respectively, Their PCR products were ligated into pGEM畻T easy vector and their nucleotide sequences were analyzed. The results of sequencing showed that the cDNA length of AcPLA2 and AmPLA2 were all 405 bp encoding a 134 amino acid polypeptide with predicted molecular weight of 14.7kD, respectively. The alignment of 5 PLA2 genes showed that the amino acid sequence of AcPLA2 has 95%, 90%, 54%and 39% amino acid identity with AmPLA2, A.dorsala PLA2 , Bumbus pennsylvanicus PLA2, Drosophilia melanogasl PLA2 , respectively, 5 PLA2 polypeptides have common calcium binding sites, catalytic domain and disulfide bridges and showed high homology and conserved characters. A phylogenetic tree of 7 PLA2 drawn by using GENETYX program showed AcPLA2 and AmPLA2 have the highest homology. AcPLA2 cDNA sequence has been submitted to GenBank as a new sequence ( Accession number:AF321087).It is also the first reported functional gene from A. cerana cerana.The restriction fragments of AcPLA2 and AmPLA2 were inserted into the multiple cloning site of the pBacFastHTa under the control of Ph promoter and flanked by the left and right ends of Tn7 and with a His tag in their N terminal to constructed recombinant donor plasmids, pBacHT-AcPLA2 and pBacHT-Am PLA2 ,which were used to transposed to the target Bacmid in E.coli (OHIO) by Tn7 transposition function , respectively. Through screening of appropriate antibiotic resistance and white-blue colonies , PCR identification, the recombinant Bacmid-AcPLA2 and Bacmid-AmPLA2 were obtained. Then the both recombinant Bacmids were transfected into Tn-5Bl-4 cells mediated by Lipofectin. Their expressed protein bands of 18 kD were determined by SDS-PAGE, respectively. Through immunizing rabbit with the purified native AmPLA2 as antigen, the anti-AmPLA2 polyclonal antibodies were prepared. Western blot with the anti-AmPLA2 polyclonal antibodies as the first antibodies showed that the expected protein bands same as the native AmPLA2 and bee venom appeared on the profiles of AcPLA2 and AmPLA2 respectively. Thin layer scanning on the SDS-PAGE profile showed that the expressed protein bands of AcPLA2 and AmPLA2 were about 5.32 % and 5.35 % of total protein of Tn cells, respectively. Biologic assay showed the extracts of Tn cell protein infected with Bacmid-Ac PLA2 and Bacmid-AmfifcA^had PLA2 activity which was similar to the native AmPLA2 and bee venom. This was the first report on the successful expression of BvPLA2 genes in baculovirus-insect (cell) expression vector system.Then the both target genes amplified with PCR from the sequenced pGEM-AcPLA2 and pGEM-AmPLA2 were inserted into plasmid pETBlue-1 to constructrecombinant expression vector pET-AcPLA2 and pET-AmPLA2,respectively. After that both...
Keywords/Search Tags:Apis cerana cerana, A·mellifera, bee venom, AcPLA2, AmPLA2, AcHya, AmHya, nucleotide sequence, gene expression, baculovirus-insect (cell) expression vector system, E. coli expression system
PDF Full Text Request
Related items