Research On The Construction Of Retrovirus Vector PBabeNeo-HBD-2 And The Gene Transfer Of HBD-2 Into Mammary Cells

Research on the construction of retrovirus vector pBabeNeo-HBD-2 and the gene transfer of HBD-2 into mammary cellsBackground: Infectious disease is still the most prevalent disease in the world, which consists variety of new challenges, including chronic infection, arthrosclerosis induced by infection, infection after transplantation, infection due to the resistance of bacteria, et al. Moreover, the overdose of antibiotics brings another problem that the resistant bacteria appears quickly after the administration of new kind of antibiotics. Defensin is a natural peptide, which were found among many kinds of creatures on the earth, and has a wide functional spectrum, such as bacteria, fungus, virus, PPLO, et al. The most interesting feature is that it is hard for the bacteria to generate a resistant type to the denfensins although they have met with each other for billions of years on the earth. Considering these properties the defensin really is a new kind of effective antibiotic.Objective: Human β defensin 2(HBD-2) is the first inducible human defensin which was discovered in 1997.This research is going to design a retrovirus vector carrying the gene of HBD-2 and infect different kinds of mammary cells. Then the functional properties of the cells and HBD-2 extracted were examined. And also the antibacterial biological function of transduced cells was examined in in vitro and in vivo model.Methods: The retrovirus vector pBabeNeo-HBD-2 was constructed and used to infect some kinds of mammary cells. The the genetic, protein, immune and biological functions were examined with Northern Blot, Western Blot, ELISA, CM Extraction, ZipTip and Geloverlay methods. And the in vitro tissue-engineering and SCID mice model were used to check the anti-bacteria function.Results: The retrovirus vector pBabeNeo-HBD-2 was constructed successfully and used to infect the cells. The HBD-2 extracted from the culture supernatant was proven to have the similar biological function as the original one from the human skin. And the anti-bacteria function were shown in both in vitro and in vivo model.Conclusions: The research is the first time to try to transfer the HBD-2 gene into mammary cell and examine its biological properties. It showed that many kinds of transduced mammary cell can express the functional HBD-2, which was similar as the original one from the human skin. This successful gene transfer of HBD-2 is the first step towards the clinical therapy use of defensins.