Construction Of Mammary-specific Expression Vector Of Human α-defensin-1 (HNP-1) Gene

Objective: Defensins, also called human neutrophil peptides(HNP), are small cationic peptides with broad antimicrobial activity. They are highly abundant in the cytoplasmic granules of polymorphonuclear neutrophils. a-defensin-1 is an important mediator in anti-HIV. It can be developed to be an ideal new method to cure acquired immune default syndrome (AIDS). It is difficult to achieve amount of antimicrobial peptides from nature sources. Transgenic mammary gland bioreactors offer a safe and cost effective source to produce important proteins. The purpose of this study was to construct a mammary-specific expression plasmid containing beta-lactoglobulin (BLG) gene promoter and human a-defensin-1 (HNP-1) gene.Methods: The BLG gene amplified from genome of goat peripheral blood by PCR was digested with Kpnl and EcoRI, the HNP-1 gene amplified from genome of human peripheral blood by PCR was digested with EcoRI and Xhol. The two fragments were connected through a ligation reaction and cloned into pcDNA3.1 to construct a mammary-specific expression vector pcDNA3.1 -BLG-HNP-1.Results: l.A 880bp DNA fragment and a 865bp DNA fragment were successfully amplified by PCR from genome of goat peripheral blood and genome of human peripheral blood separately. 2. Restriction enzyme digesting confirmed the enzyme digesting map was according to the expected figure and the three fragments were successfully connected. 3. PCR from the positive plasmid confirmed the existence of the three fragments and the correction of their position. 4. DNA sequencing confirmed that the recombinant eukaryotic expression plasmid (pcDNA3.1-BLG-HNP-1) containing BLG and HNP-1 had been constructed correctly.Conclusions: A mammary-specific expression vector pcDNA3.1-BLG-HNP-1 was constructed successfully.