| Ischemic cerebrovascular disease and cerebral reperfusion Inflammatory injury hasbeen one of the major diseases which have put a serious threat to human life and health inthe worldwide. Its Pathogenesis is complex, and researches have shown that theinflammatory effective fragments produced by complement activation are the main initialfactors which cause cerebral ischemia and reperfusion inflammatory injury. In the threecomplement activation pathways which are known as classic, mannose-binding lectin(MBL)and alternative pathway respectively, although they are activated at different starting points,they coverage at C3and form C3/C5convertase. So a molecule that can combine with C4b,C3b and inhibit the activity of C3/C5converting enzyme, would block the over-generationof pro-inflammatory complement fragments, such as C3a, C4a, C5a and C5b-9, eventuallypreventing the occurrence and development of the CI/R damage at the initial stage. TheSCR1-3functional domain of human complement receptor type1which has a capacity ofbinding C4b and inhibiting the activity of C3/C5converting enzyme, would reduce theexcess-generation of C3a, C5a and C5b-9in control of excessive activation of complementsystem at the early stage, and prevent the occurrence and development of CI/R injury.In this study, the total RNA was prepared from human peripheral blood, and the geneencoding sequence of CR1-SCR1-3was obtained by RT-PCR amplification. Afterrecombinant expression plasmid of pPIC9k-CR1-SCR1-3was constructed and conformedto be correct. Then it was electrotransported into Pichia pastoris for protein expression.Finally, the target protein was analyzed by SDS-PAGE and Western-Blot. NI-NTA agarose metal-chelate-affinity chromatography was used for protein purification. For further study,Complement control activity of target protein was investigated by SRBC haemolytic assayin vitro and its protective effects on acute cerebral ischemia reperfusion injury wereobserved in rats. Results were as follows:1. With the total RNA extracted from human peripheral blood as template, a pair ofprimers were designed by Primer Premier5.0on the basis of human CR1-SCR1-3genesequence, SnaBⅠrestriction site was added to the upstream primer, which could make surethat the target gene should be cloned in frame with the α-factor signal sequence of pPIC9kvector, and the downstream primer contained6×His tag, stop codon and NotⅠrestrictionsite. the617bp length of gene encoding sequence of CR1-SCR1-3was obtained by RT-PCR.2. After CR1-SCR1-3and pPIC9k were digested with SnaBⅠand NotⅠ, then theywere linked together by T4DNA ligase to construct recombinant expression plasmid.Chemical method was adopted for transforming Escherichia coli DH5α. Finally, therecombinant plasmid was identified by direct colony PCR, restriction enzyme digestion andDNA sequence, which showed a homology of100%with the CR1sequence released byGenebank, and it was named as pPIC9k-CR1-SCR1-3.3. The linearized plasmid of pPIC9k-CR1-SCR1-3digested with sal Ⅰ waselectrotransported into competent cells of Pichia pastoris, and the MD plates were took forscreening of the Mutstransformants, multiple inserts were selected by plates of YPD atdifferent concentrations of G418. After direct colony PCR identificatio n, nine multipleinserts of the positive mutstransformants were attained and named as pPIC9K-CR1-SCR1-3/KM71.4. Positive recombinants were selected for fermentation in shake flask and inducedwith1%methanol. Then the expressed protein was analyzed by SDS-PAGE andWestern-Blot, results demonstrated that the gene of CR1-SCR1-3was successfullyexpressed in Pichia pastoris.5. After the expressed protein was purified by NI-NTA agarose metal-chelate-affinitychromatography, SDS-PAGE indicated that the target protein had a high purity. Then BCAkit was used for determination of protein concentration before and after purification, resultsshowed that CR1-SCR1-3reached about35%of the supernatant total proteins.6. The biological activity of CR1-SCR1-3was analyzed by CH50method in vitro, results showed: with the increment of protein concentration, the complement controlactivity of CR1-SCR1-3was strengthening. When it reached about50μg/ml, CR1-SCR1-3could inhibit50%of total hemolytic complement activity. It indicated that CR1-SCR1-3was proved to possess a well complement control activity.7. The middle cerebral artery occlusion model of rat was successfully established. Andresults showed that: the neurological motor deficit score and infarct volume in protectiongroup were significantly lower than those in CI/R operation group. In biochemistry index,the activity of MPO and content of MDA in protection group were also significantly lowerthan those in CI/R operation group, while the activity of SOD markedly increased.Western-Blot indicated a down-regulation of C4b expression in protection group, andimmunohistochemistry also suggested that C4b deposition in cerebral cortex wassignificantly reduced in protection group. Furthermore, pathologic detection had found thatthere were no obvious pathological changes in sham operation group, and a series ofpathological damage were observed in CI/R operation group, while pathological damage inprotection group was significantly reduced. All indicated that CR1-SCR1-3exerted aprotective effect on complement-mediated CI/R injury.To sum up, we successfully construct a recombinant expression plasmid ofpPIC9k-CR1-SCR1-3, and a molecular weight of about27KD of CR1-SCR1-3proteincould be highly expressed in the Pichia pastoris. After purification by NI-NTA agarosemetal-chelate-affinity chromatography, a high purity of target protein is achieved. In vitro,Biological activity assay of CR1-SCR1-3suggests that the purified protein possesses a wellcomplement control activity. In a rat model of middle cerebral artery occlusion, we havefound that there is a protective effect on acute cerebral ischemia and reperfusion injury,which provides a promising insight into the prevention and treatment of CI/R injury forfurther study. |
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