The Preliminary Research Of Human Neutrophil Peptide 1 Expression In E.coli BL21(DE3) Strains

Objective:HNP1 is the ever found most abundant antimicrobial peptides with highest activity,its ability to resist infection and the cell killing effect of malignant proliferation is not only its application value,but also the root cause why genetic engineering can't express HNP 1 efficiently in the host.Our subject aims to construct a pET-28a(+)-preproHNP1?a pET-28a(+)-proHNP1 and a pGEX-4T-3-preproHNP1 prokaryotic expression vector to explore HNP1 expression in BL21(DE3)strains,to find the activation mechanism and relevant influence factors and to lay the foundation of gene engineering strain to obtain precursor HNP1 efficiently.Methods:Construct pET-28a(+)-preproHNP1?pET-a(+)-28 proHNP1 and pGEX-4T-3-preproHNP1 prokaryotic expression vector by fast site-directed mutagenesis and gene engineering technology;Coomassie brilliant blue staining and Western Blot compare prepro1-HNP1 with proHNP1 of the expression efficiency in BL21(DE3);preproHNP1 protein purify by Nickel metal ion affinity chromatography;Detect the bacteriostatic activity of preproHNP1 proteins;Detect bacterial growth state after IPTG induction;Detect precursor and mature HNP1 expressed in prokaryotic system by mass spectrometry;Detect HNP 1 glycosylation modification by glycoprotein staining;Build a knockout system by Red homologous recombination.Results:The results manifested the recombinant prokaryotic expression plasmid pET-28-a(?)-preproHNP1?pET-28(+)-proHNP1 and pGEX-4T-3-preproHNPl have been constructed correctly;In BL21(DE3)prokaryotic system,preproHNP1 expresses more efficiency than proHNP1;Purified preproHNP1 protein has no obvious bacteriostatic activity;Bacterial growth is suppressed after IPTG induction,but no difference between preproHNP1 and proHNP1;Precursor and mature HNP1 protein are detected by mass spectrometry in the prokaryotic expression system;HNP1 glycosylation modification is found after IPTG induction;Red knockout system is built successfully.Conclusion:May be there exist a activation mechanism in E.coli BL21(DE3)similar to the human body of HNP1 protein,that is to say,precursor HNP1 has certain sterilization ability is just a illusion,it works by activating mature HNP1;it has no absolute effect of 19 amino acid signal sequence on the formation of inclusion body;glycosylation has nothing to do with the signal sequence,but may be associated with HNP1 activation.