The Expression Of Artificial-modified Cecropin B-shiva-2in Tobacco

Antimicrobial peptides (AMPs) are small molecule proteins or short peptides which canresist foreign invading pathogens. Studies have indicated that AMP has high-potencyinhibitory activity for the bacteria and killed part of fungus, virus and cancer cells; but hasless toxicity for eukaryotic cells and difficulty to get drug resistance. Recently, breakthroughsare made in producing pharmaceutical proteins and recombinant vaccine by using transgenicplant plants. Plants are usually used as bio-reactor in producing pharmaceutical proteins, so asto provide a safe and low-cost production system for AMP. Therefore, using plants to produceAMP have wide prospects.AMP Shiva-1is the analogue of Cecropin B, which can be very destructive to thegram-positive bacterium and gram-negative bacterium but not toxic for eukaryotic cells andfungi. This study utilized genetic engineering, the amino acid sequence of antibacterialpeptide Shiva-1as a template, the purpose of the synthetic modified gene was named Shiv-2.This research used tobacco as bioreactor to produce AMP Shiva-2. This experiment wasexpected to express active AMP.The research got the following results:Based on the amino acid sequence of the mutant Shiva-1of cecropin B, our research tookthe tobacco preference codons and added a Kozak sequence in the upstream of the target geneto enhance the transcription and expression of the gene. We added Gly codon on thedownstream of the gene to ensure its biological activity, and added enzyme digestion sitesused to construct the vector. We obtained the target gene via whole-genome synthesistechnology.Shiva-2was cloned into efficient plant binary expression vector pPZP211to constructthe nuclear expression vector pPZP211-Shiva-2and we transffered the vector intoagrobacterium MP90through triparental mating. Then, we transffered the target gene intotobacco by agrobacterium-mediated transformation to transient expression, and the extractedprotein was deteced without biological activity.In order to find out whether the expressed protein of synthesis gene has biologicallyactive or not, we constructed prokaryotic expression vector pET-28a-Shiva-2, and transformedinto host bacteria BL21, expressed the active target protein. But the expression product has suppression to the host bacteria.We Constructed the intermediate expression vector pKS-PA and laid the foundation oftobacco chloroplast as bioreactor to express the purpose gene Shiva-2.