Large scale production, purification, and functional characterization of recombinant murine properdin; its utility as a therapeutic agent against N. meningitidis

Abstract

The alternative pathway of complement activation is known to play a very important role in innate immune response to Neisseria meningitidis infection. Properdin is a protein which has been identified as the essential positive regulatory component of this activation pathway. It has been found that inherited and acquired properdin deficiencies are associated with a high predisposition for infections with N. meningitidis. This final year research project aims to show the therapeutic potential of functionally active recombinant properdin in N. meningitidis diseases. In this final year research project, recombinant murine properdin will be expressed using CHOk1, a mammalian cell line. We will initially design a pair of primers in order to amplify the cDNA ORF (open reading frame) encoding murine properdin. Then, to increase the amount of DNA copies we will clone the PCR product into a pGEM-T Easy vector and afterwards clone it into a pSecTag/hygromicinB expression vector. Finally, we will transfect the cells with this expression vector in order to obtain and purify recombinant active murine properdin. The functional activity of recombinant murine properdin will be evaluated by haemolytic assay and flow cytometry. The aim of acquiring this protein is to assess the therapeutic utility of functionally active recombinant properdin in an in vivo mouse model of N. meningitides infection This project will evaluate the reconstitution of properdin knock-out mice using recombinant murine properdin to restore the alternative pathway functional activity. C57BL/6 mice will be submitted to an intra-peritoneal injection. This injection will contain 100μg of recombinant murine properdin or placebo. The given dose of murine properdin is expected to increase the concentration of properdin in plasma. 6 hours later, mice will be challenged with N. meningitidis. Both experimental groups of mice will be compared to see if there is a visible difference between them after infection. With this final year project we aim to show the importance of properdin’s key role in promoting the alternative pathway functional activity by assessing the effect of the administration of recombinant properdin to enhance bacterial clearance from mice infected with N. meningitidis. Moreover, we want to show the possible therapeutic interest of recombinant murine properdin as a treatment against N. meningitidis infections.