ABSTRACT
AIM: To explore the effect of complement on the cerebral ischemia/reperfusion injury in rat and the protection by sCR1-SCR15-18. METHODS: 75 male SD rats were randomly divided into three groups: sham operation group (SO, n=15), middle cerebral artery occlusion and reperfusion (MCAO) without treatment group (I/R, n=30); MCAO treated with sCR1-SCR15-18 group (sCR1-SCR15-18, n=30). After the MCAO for 2 h, then reperfusion for 24 h, the scores of neural behavioral functional deficits were determined. Infarction area was measured by TTC staining. Activity of MPO in cerebral cortex was detected. C3b deposition and pathological change were observed by immunohistochemial staining and HE staining, respectively. RESULTS: After reperfusion for 24 h, the neurological deficits score, infarction area and activity of MPO in sCR1-SCR15-18 group were decreased compared to I/R group. In sCR1-SCR15-18 group, C3b deposition in ischemic area was decreased and pathological injury was improved compared to I/R group. CONCLUSION: Complement plays a role in cerebral ischemia-reperfusion injury and sCR1-SCR15-18 exerts a protective effect by inhibiting the excessive activation of complement.
ABSTRACT
Objective To construct and express a small complement receptor type 1 (CR1) deriv-ative which contained two functional domains. Methods Total RNA was isolated from the peripheral blood mononuclear cell. The functional fragment Ⅰ of CR1 was amplified using the RT-PCR. The functional frag-ment Ⅱ was amplified with the plasmid of pET-32a-CR1-SCR15-18 as template which had been already con-structed in our laboratory. Then the chimeric gene that contained the two fragments was constructed with spli-cing overlap extention PCR. The chimeric gene was then inserted into the plasmid of pET-32a (+) and transformed into E. coli Rosetta(DE3). The inserted gene was verified by enzyme digestion and DNA se-quencing. After induced by IPTG, the expressed protein was analyzed by SDS-PAGE and Western blot. Then the fusion protein was purified by Ni-NTA affinity column and renatured through dialysis. The comple-ment inhibition activity was determined by CH50 method. Results The chimeric gene was successfully cloned into pET-32a(+). The result of SDS-PAGE and Western blot confirmed the expressed protein and showed that the molecule mass(Mr) of the expressed protein was 63×103. The purity of the recombinant protein was up to 92% after Ni-NTA column affinity chromatography. The bioactivity assay showed the fusion protein had a concentration-dependent complement inhibition activity within the concentration range of 0-200 μg/ml. Conclusion The two functional domains contained small CR1 derivative was successfully construc-ted and expressed in E. coli Rosetta. The fusion protein had a relative high bioactivity, providing a basis for further function experiment in vivo.
ABSTRACT
Objective To express the short consensus repeat 15-18 (SCR15-18) domain of human complement receptor 1 (CR1) in Pichia pastoris as a secreted protein in order to found a base for large-scale industry fermentation. Methods The gene fragment of CR1-SCR15-18 was amplified by PCR from plasmid pET32a-sCR1-SCR15-18. The obtained sequence was subcloned into Pichia pastoris secretory expression vector pPIC9. After identification the recombinant plasmid pPIC9-CR1-SCR15-18 was electrotransported into the yeast. Positive clones were identified using colony PCR. Positive recombinants were fermented in shake flaskes and induced with methanol. The recombinant proteins were identified with SDS-PAGE and Western blot analysis. After the recombinant protein was purified by Ni-NTA agarose metal chelate affinity chromatography, inhibiting complement hemolysis testing was used to detect the biological activity. Results Recombinant expression plasmid pPIC9-CR1-SCR15-18 was successfully constructed. SDS-PAGE and Western blot analysis revealed that the target gene was successfully expressed in the yeast and the recombinant protein was successful secreted in the culture supernatant. After purification, the protein inhibited complement hemolysis in vitro. Conclusion CR1-SCR15-18 is successful expressed in Pichia Pastoris with high activity of inhibiting complement hemolysis.