Expression Of Bombyx Mori ABP Fusion Protein Indusing By Lactose

Anti-bacterial peptides(ABP)are a kind of small molecule peptides, which are produced by specific gene encoding when organisms are invaded by outside pathogen, which is also an intergral part of the host's defense barrier against invading bacteria. ABP can kill bacteria effectively, especially to those drug resistance pathogen, which has already attracted people's attention.Besides, ABP have many other characteristics including low molecular weight, soluble in water, thermal stability, a broad antimicrobial spectrum, difficult to develop drug resistance, and so on. They show a good practical perspective in the field of medicine, food and agriculture. However, isolation of ABP from natural sources is indfficient and time-consuming, while chemical synthesis of peptides is costly, genetic engineering techniques have been considered a better method. Now, researchers have already expressed ABP in different gene expression systems, as Escherichia coli, yeast, plant and mammal. However Escherichia coli is the best choice to mass produce ABP, because Escherichia coli has a clear genetic background, grows fast, which is good for the mass production of recombinant protein. In the E.coli gene expression system, IPTG is used to be the inducer, but it is expensive and has latent toxicity. In this thesis, from the points of mass producting cost and safety, we use lactose as the inducer instead of IPTG. We also study the optimized synthetizing of cecropin B, construction of expression vector, induced expression by lactose, purification of inclusion body and in vitro antibacterial activity. The research is devided into 3 parts:1 Based on the gene sequence of natural Bombyx mori ABP (cecropin B) and E.coli preferred codons, we artificially synthesized the optimized ABP gene. It turned out that the homology of the gene sequence was 78%, and the amino acid sequence did not change. We analyzed the gene codons by software DodonW, and found that CAI (Codon Adaptation Index) increased from 0.276 to 0.539, CBI (Codon Bias Index) increased from 0.335 to 0.537, FOP(Frequency of Optimumcodons) increased from 0.610 to 0.729. The result indicated the optimizing of gene is good for expression of cecropin B.By the restriction enzyme cutting sites EcoR I和Hind III, the synthesized gene was inserted into the prokaryotic expression vector pET-41a, and then transform the recombinant plasmid into XL10-Gold to clone the gene. The double digestion showed a 200bp stripe as expected, and sequencing result was exactly the same as we designed, which proved the expression vector pET41a-cecB had been successfully constructed.Meanwhile, we carried out the bioinformatics analysis of the expressed gene. The bioinformatics analysis shows: the gene of cecropin B codes a protein which consists of a 63 amino acid base, its relative molecular weight is 6831.2Da, theoretical isoelectric point is 10.45, (-) amino acid residues (Asp + Glu) are 4, and (+) amino acid residues (Arg + Lys) are 11. The molecular formula is C315H521N83O79S3 and the number of atoms is 1001. the half-life period is 30 h (mammal in vitro)﹥20 h(yeast in vivo)﹥10 h(E.coli in vivo), unstable index is 47.01, fat-soluble index is 106.98, GRAVY is 0.367. By the analysis of Tmpred and SMART, the former 23 amino acids of N-end are a transmembrane domain sequence, which helps the protein to move to the cell membrane, combine to the membrane and send the protein out of the cell. PSORTII also predicts that the protein is located outside of the cell, as secretory protein. The 57th amino acid Ser may be the locus of protein kinases phosphorylation.2 We enlarged the clone bacteria and extracted the recombinant plasmid to transform into the expression bacteria BL21(DE3), and then we got the protein of 42 kD by SDS-PAGE and Western Blot. We optimized the inducing conditions and got the result that the best condition to induce by lactose was 0.25 mM lactose induces the OD600 1.0 engineering bacteria 5 h at 37℃, the production of recombinant protein can reach 17.1%, better than induced by IPTG; there is no obvious difference between two kinds of adding lactose; almost all the product expresses as inclusion body.3 We purified the protein through GST affinity chromatography and got more than 95% purity target protein. In the test of antibacterial activity, inhibition zone appeared in cervus elaphus E.coli, domestic sheep pasteurella and swine staphylococcus; and the OD600 showed decreasing in cervus elaphus E.coli, domestic sheep pasteurella, domestic sheep salmonella and swine staphylococcus.This research optimized the conditions of lactose as a inducer in recombinant protein expression, achieved a high level expression, and purified the target protein and detected the antibacterial activity. The results showed that using lactose to induce ABP expression instead of IPTG is feasible, and also lowered the cost of use of ABP, which had the significance to recombinant ABP commercial production.