Silkworm Lysozvme Expression, Purification And Activity Detection By Fusion With Polyhedrin

Lysozyme(LZM，E.C.3.2.1.17) was found in snotter in the first time by theBritish Fleming in1922. Abraham and Robinson is the first people who isolatedlysozyme crystals from ovalbumin until1937. The full name of lysozyme is1,4-β-N-lysozyme, which can cut off β-1,4glycosidic bond between the N-NAG andN-NAM in peptidoglycan and damage peptidoglycan framework, under internalosmotic pressure the bacteria cracking and cells burst. Lysozyme as the naturalprotein which in natural world widely, have many functions of pharmacological,health care, antiseptic and so on. In addition, lysozyme can clear away cell debrisafter by other antimicrobial protein killed bacteria in immune defense system, it playsa very important role.When screening silkworm cDNA library, we found a gene which has38%homology with lysozyme in humanity, from the silkworm fat-body organizationcDNA amplification obtained the Open Reading Frame of silkworm lysozyme geneby PCR, named BmLZM(Bombyx mori lysozyme), GenBank accession number isL37416.1. BmLZM gene sequence length360bp, coding119amino acids, predictionmolecular weight is13.75kD after removed signal peptide. We used BaculoviralPolyhedrin(Polh) as a Novel Fusion Partner for facilitating efficient expression of theBmLZM, between the Polh and BmLZM insert nucleotide sequence ofThrombin(Thb), we constructed the prokaryotic expression vectorpET-28a-Polh-Thb-BmLZM. by PCR, double digestion and nucleic acid sequencingdemonstrated recombinant plasmid is right.The recombinant plasmid was transformed into the E.coli BL21(DE3), afterinduced by IPTG, SDS-PAGE showed have a specificity protein stripe nearby45kDis corresponded to expected value.The fusion protein form is inclusion body, it is used differential pH buffer anddifferential centrifugation to purified fusion protein based on the fusion proteinshowed character of polyhedrin. To polyhedrin protein as the antigen immunized male New Zealand rabbit. Use of Polyhedrin protein polyclonal antibody detection offusion protein, the result of Western Blot showed the fusion protein expression hasbeen successful. Purified the fusion protein Polh-Thb-BmLZM was digested bythrombin protease after that centrifugation and ultrafiltration obtained the protein ofBmLZM.E. coli heat shock method activity detection, macrography results show thatbacteria suspension liquid divided into two-layer, this phenomenon due to cellsinclusions released after cell wall dissociation, we have got the activity BmLZMprotein. Agar diffusion experiment demonstrated the BmLZM protein we obteinedhave biological activity that is appeared zones of inhibition. The result ofMicrococcus lysodeikticus turbidimetry mensurated average quality enzyme activityis334.75U/mg.The study based on polyhedrin expression technology, fusion expression,purificaction of bioactivity BmLZM, not only provides the prokaryotic expression theprotein that the method of scale manufacture antibacterial activity class technology,but also the silkworm lysozyme related basic research and application developmentestablished a theoretical basis.