Construction Of Fusion Protein Of Complement Receptor Type1Derivate And Its Protective Effects On Septic Inflammatory Injury Of Mice

As one of major reasons of death in intensive care units, sepsis is the systemicinflammatory response syndrome (SIRS) caused from infection. The mechanism of sepsis iscomplicated, which is associated to every system in body. One of the initial stage isexcessive activation of complement system that recognizes pathological microbes. Thesubstances of bacteria, endotoxin, exotoxin et al. make large amount of inflammatoryfragments from activated complement, and subsequent amplified inflammation plays a keyrole in development of sepsis. At present controlling complement excessive activation is animportant treatment method for the septic inflammatory injury.In complement classical, alternative and lectin pathways, all the initiate componentsare different, but all the downstream components are converged to C3to form C3/C5convertase and entry into terminal pathway. If the C3/C5convertase formation is inhibitedat C3, the releasing of C3a, C5a and C5b-9can be decreased, and SIRS occurrence andprocess can be shut down or reduced in a certain degree.Our group previously developed two functional segments of complement receptor type1(CR1), short consensus repeats (SCR)1-3and SCR15-17, being produced by PichiaPastoris and testified for anti-complement activity in vitro and in vivo. Each segment,however, separately combines C4b and C3b, only to inhibit one or two complementactivation pathways in limited controlling capality. Therefore, this study combined theseparate segments together to construct CR1derivate, called CR1-short consensus repeatsof two domains (CR1-SCR2D), followed by detecting anti-complement activity in vitro.Then further research was proceeded for testifying anti-inflammation effect in sepsis ofmice in vivo. Thus, our research achieved following results:1. Successful construction of pPICZαB-SCR2D expression plasmid. Renew the multiple cloning sites in pPIC9k into pPIC9k(+). PCR amplify for gene SCR1-3andSCR15-17using pPIC9k-SCR1-3and pPIC9k-SCR15-17plasmid template built previously.Clone each gene with an artificial linker into pPIC9k(+) to form target gene SCR2D. Clonethe gene SCR2D into pPICZαB to get pPICZαB-SCR2D expression plasmid. Identificationof double restriction enzymes digestion and sequencing to pPICZαB-SCR2D plasmid wereperformed.2. pPICZαB-SCR2D expression plasmid was electroporated to Pichia Pastorisfollowed by protein expression identification. Being linerazed by Sac I, pPICZαB-SCR2Dwas electroporated to Pichia Pastoris and the colonies on YPD plate containing Zeocin waspicked followed by PCR identification. Target protein CR1-SCR2D was expressed byrecombinant X-33/CR1-SCR2D in the inducing condition of1%methanol and156h.Western blot was performed to testify the target protein band located at90kDa using mouseanti-human CR1monoclonal antibody.3. Through Ni-NTA agarose metal-chelate-affinity chromatography, target proteinwas purified, followed by glycosylation identification. After supernatant of expression wasloaded through Ni-NTA column, other proteins were eluted with low concentration ofimidazole, target protein was eluted with high concentration of imidazole. SDS-PAGEelectrophoresis showed target protein at90kDa was pure. Treated with Endoglycosidase H(EndoH), CR1-SCR2D was shifted to45kDa, which proved glycosylation existing onCR1-SCR2D.4. Identify the target protein CR1-SCR2D anti-complement activity in vitro. CH50and AH50assays showed CR1-SCR2D had stronger inhibiting activity of complementclassical and alternative pathways than both SCR1-3and SCR15-17. DeglycosylatedCR1-SCR2D was the same activity as itself with glycosylation. The CR1-SCR2D couldinhibit complement components C4b and C3b deposition on zymosan in alternativepathway and lectin pathway.5. CR1-SCR2D had protective effect on septic inflammatory injury. Septic micemodel was successfully built using cecal ligation and puncture (CLP) method. Groupexperiments showed CR1-SCR2D could improve survival96h after CLP. CR1-SCR2Dreduced serum C5a, IL-6, TNF-α, lung MPO activity and blood bacterial load of mice24hafter CLP. Immunohistological assay showed CR1-SCR2D decreased C4b and C3b deposition on lung24h after CLP. The pathological section of lung showed CR1-SCR2Dattenuated septic injury in lung, such as attenuating congestion, edema, inflammatory cellsinfiltration and consolidation in alveolar septum and alveolus.In conclusion, this study successfully constructed pPICZαB-SCR2D expressionplasmid and target protein CR1-SCR2D was expressed through secretion by Pichia Pastoris.Activity study in vitro showed CR1-SCR2D inhibited three pathways of complementactivation. CR1-SCR2D possessed protective effect on septic inflammatory injury of mice.These results indicated that complement played an important role in septic inflammatoryinjury, and CR1-SCR2D had protective effect for septic mice through controllingcomplement excessive activation.Thus it provided a novel idea and approach for sepsistreatment research.