| Ischemic cerebrovascular disease has been damaging the health of mankind seriously for its high morbidity, disability and mortality. Cerebral ischemia-Reperfusion (CI/R) injury was regarded as the key issue for the treatment of cerebral ischemia disease. A growing body of evidence indicates that: complement system plays an important role in CI/R injury, and over-activation of complement system is the initial factor for the CI/R inflammatory injury. Activated complement fragments are the key factors in mediating neutrophil collection, activation and endothelial cell injury. Therefore, inhibiting the activation of complement will provide a new strategy for the prevention of CI/R Injury. The complement system can be activated by three pathways, known as classical, alternative, and mannose-binding lectin pathways. Although the three pathways have different starting points, they converge at the point of cleavage of C3 and form C3 convertase,leading to initiation of the production of a series of pro-inflammatory complement fragments. sCR1 functional domain SCR15-18 can specifically block C3b and inhibit C3/C5 convertase formation, which will prevent the over-activation of inflammation-inducing complement fragments, such as C5a and C5b-9, eventually preventing the CI/R damage at the initial portion.In our study, on the basis of constructed recombinant engineering bacteria pET32a-sCR1-SCR15-18/BL21, a mass production of the sCR1-SCR-15-18 protein was achieved by high-density fermentation. The parameters of expression, purification and refolding of sCR1-SCR-15-18 protein were optimized. Ni-NTA affinity chromatography method was exploited for protein purification. The purified protein was renaturated by gradually decreasing the urea concentration of the dialysis solution in the glutathione redox system. The suppressive effect of the yield protein was determined by haemolysis assay. The role of SCR15-18 during the course of cerebral ischemic reperfusion injury(CI/R)was evaluated in a rat model of middle cerebral artery occlusion (MCAO). The main experimental contents and results are as follows:1. The recombinant PET32a-sCR1-SCR15-18/BL21 of engineering bacteria was resuscitated. We optimized the culture media, temperature, pH and concentrations of IPTG for fermentation in culture bottle. Based on this, the inducing occasion and inducing time were also optimized in a 3.7L fermenter. Finally, the best induced conditions were screened: the optimum culture media, temperature and concentration of IPTG were modified M9-CA, 37℃and 0.2mmol/L, respectively. The inducing occasion was 4 hours after growing, and the inducing time by IPTG lasted for 4 hours. Glycerol was chosen as carbon source. The flow rates of feeding at the growth and induction phases were kept at the speeds of 40 ml/h and 70ml/h, respectively. The culture's pH was maintained at 7.5 by adding ammonia water, while dissolved oxygen was controlled about 40% by regulating rotating speed. Under the optimal conditions mentioned above, highest yield of over 40g/L SCR15-18 protein was achieved, accounting 28% of the total bacteria protein. SDS-PAGE and Western blot analysis showed a single positive band with molecular weight of 43 kD.2. After engineering bacteria pET32a-sCR1-SCR15-18/BL21 was induced by IPTG, the cell pellet was split by ultrasonication. SDS-PAGE analysis showed that recombinant protein primarily expressed in inclusion bodies. The recombinant SCR15-18 protein was purified by Ni-NTA column. After SDS-PAGE analysis, it showed that using affinity chromatography could purify recombinant protein by one step. We obtained better activity by generally decreasing urea concentration in the glutathione redox system. (The concentrations of the oxidized form and the reduced form of glutathione were 0.3 mmol/L and 0.7 mmol/L, respectively).3. The suppressive effect of the yield protein was investigated by haemolytic assay, and it displayed that the interest protein (sCR1 -SCR15-18) we prepared possessed complement inhibition activity.4. Seventy five male rats were randomized divided into three groups: Sham group,I/R group and sCR1 -SCR15-18 protective group. One hour prior to the operation, the caudal vein injection of phosphate buffer saline (PBS) was given to both Sham group and I/R group; and sCR1 -SCR15-18 protein was injected to the protective group. A rat model of MCAO was obtained according to Suture Method, ischemia and reperfusion were treated for 2h and 24h respectively. Neurological score, cerebral infarction size and the activity of MPO were measured. The deposition of C3b in cerebral cortex and pathological changes were observed. Results showed that, neurological scores of SCR15-18 protective group was lower than I/R group (p<0.05), cerebral infarction size and the activity of MPO of SCR15-18 protective group were also lower than I/R group (p<0.05). C3b deposition and pathological injury were significantly reduced. It indicated that SCR15-18 exerted a protective effect on the CI/R injury.In conclusion, on the basis of a constructed recombinant engineering bacteria pET32a-sCR1- SCR15-18/BL21, we have optimized the culture conditions by fermentation, and a mass production of SCR-15-18 proteins is obtained. The SCR-15-18 protein is confirmed to possess the inhibitive activity. And it also exerts protective effects on brain in a rat model of I/R experiment, which provides a promising insight into the further study of I / R injury.
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