A New Highly Efficient Cloning Method For The Peptide Gene Encoding

Antimicrobial peptide produced by the host defense system, which against infection of pathogens, is an important part of host immune defense system. Antimicrobial peptide not only has a broad antibacterial spectrum, but also inhibits the growth of cancer cells. Importantly, it does not have the characteristics of drug resistance. These make the development and utilization of antimicrobial peptides as a research focus in recent years. But the research and use of antibacterial peptide is not easy matter, first, the natural antibacterial peptide resources are limited, the extraction is complex; Next, chemical synthesis is not only costs very high, but also causes the serious pollution to the environment, therefore it urgently needs one kind of project fungus to highly effective express the antibacterial peptide. In the construction antibacterial peptide project fungus's process, the key technologies question is how to fast highly effective clone and how to express accurately antibacterial peptide.The highly effective clone is the first bottleneck in the present studies on the antibacterial peptide, because the antibacterial peptide's molecular weight is quite small, and the code gene's cDNA fragment of antibacterial peptide is small, it is usually below several hundred bp, some even is only several dozens bp, this brings enormous difficulty for the antibacterial peptide genetic engineering operation.Using the conventional method of enzyme cut and link toconstruction the expression vector is time-consuming and uneconomical, moreover, the antibacterial peptide gene is small, the laboratory general material particle examination and the PCR examination's method for the expression is very difficult. Therefore, how to highly effective clone the antibacterial peptide becomes to be another barrier to restrict the antibacterial peptide development application.This research using the unique illumination nature of the green fluorescence protein(GFP). and using the Classical swine fever virus (CSFV) fixed-point sudden change coat protein (EDDIE) as the fusion protein, constructed one kind of vecter pET30a-HDDH-GFP.which is able to highly effective and accurate express antibacterial peptide. Firs we use PCR to increases the GFP gene, simultaneously take pET30a-EDDIE-CAD as the template to reverse PCR, reverse PCR product does not include the CAD gene, and the both sides include both sides homologous sequence of the GFP gene, then transfer the increasion product of the GFP gene and reversion PCR product together into the XL-GOLD to homologous reorganizate, at last construct the cloning vecter pET30a-EDDIE-GFP.Then we select six peptide of ifferent molecules size for case, and take the pET30a-EDDIE-GFP cloning vecter as the template to overlaps reverse PCR, and directly transfer the overlaps reverse PCR product into the XL-GOLD competence cell to homologous reorganizate, obtaining the antibacterial peptide expression vecter. Then transfor it into coli BL21(DE3) to express, through examination of the antibacterial peptide bacteriostasis activety, it finally discovered these six antibacterial peptides could accurately express through this method.