Screening And Expression Of Anti-Bacterial Peptid From Venom Gland CDNA Library Of Pteromalid, Pteromalus Puparum

Insect antibacterial peptide is produced as immuno-reporter when insect has been invaded by microoganisms.It is a kind of polypeptide that found in hameolymph,which is produced by fat body or haemocyte.It is board-spectrum antibiotic,highly effective against bacteria,fungi,viruses and protozoan parasites and also act as tumcides.To date, over 200 insect antibacterial peptides have been found in Lepidoptera,Diptera, Coleoptera,Hemiptera,Isoptera and Hymenoptera,but none of the research concerning peromalid wasps.In this dissertation,we construct a cDNA expression library of venom gland from Ptermalus puparum,gain a new antibacterial peptide gene through screening, and express the gene in different E.coli cell expression system.We also test the anti-microbial activity of the fusion protein.Main results of this dissertation included as following:1.A cDNA expression library of venom gland was constructed by using venom gland from Ptermalus puparum.The titer of the original library was 89.5x10~4 pfu/ml with a recombinant rate of 97.6%,the amplified titer was 3.1×10~7 pfu/ml.PCR amplification suggested that the inserted cDNA fragments ranged from 0.3 to 1.4 kb.All of the data showed that the venom gland cDNA library had a high titer,high recombinant percentage and large inserted fragments.2.Use high throughput screening method for identifying insect antibacterial peptide, based on cDNA expression library.Through screening and testing repeatedly,three positive cloning with certain anti-bacterial activity were obtained.After sequencing their nucleotide sequence,comparing the results in the data base of Genbank and AMP,we found there is no sequence with high identity,so we inferred that these positive cloning were new anti-bacterial peptide gene.3.Both genes PP2a with signal sequence and PP2b without signal sequence were successfully sub-cloned into different prokaryotic expression vector pET28a and pGEX-4T-2,forming the recombinant expression vectors,pET28a-PP2a,pET28a-PP2b, pGEX-PP2a and pGEX-PP2b,respectively,then introduced into E.coli BL21(DE3)for expression.In the pET28a/BL21 system,His-PP2a fusion protein was high expressed. And in the pGEX-4T-2/BL21 system,GST-PP2b,GST-PP2a also high expressed,but the latter presented almost as inclusion body.All the fusion proteins were confirmed by Western blotting analysis with anti-His-antibody and anti-GST-body,respectively. Antimicrobial activity assays demonstrated that the crude fusion proteins had certain activity against bacteria,especially the GST-PP2b fusion protein.