Expression Of Human CR1-SCR15-18 In Pichia Pastoris And Its Protective Effect On Intestinal Ischemia-Reperfusion Injury

Intestinal ischemia-reperfusion (I/R) injury occurs frequently in clinical and trauma conditions and is associated with high morbidity and mortality. The pathogenesis of intestinal of I/R is very complex, in recent years, more and more evidences indicate that the activation of complement plays a key role in intestinal ischemia-reperfusion injury. Inhibition of complement activation becomes a very useful strategy to prevent intestinal ischemia- reperfusion injury.Recent experimental studies have shown that I/R injury in intestinal results in activation of complement components through the antibody-dependent classical pathway, the alternative pathway, and the MBL pathway.Meanwhile, the complement activation leads to the release of biologically active substances including the anaphylatoxins, complement factor 3a (C3a) and 5a (C5a), and the cytolytic terminal membrane attack complement complex C5b-9 (MAC).These products are all able to directly or indirectly cause cellular activation,tissue inflammation and tissue injury.So,it is not enough to prevent injury mediated by complement by inhibiting just a single pathway or a single product during the complement activation. Although the three pathways are different in the initial activatin,they all converge at C3 and form the C3/C5 convertase and then activate the downstream of the complement pathway. Therefore,the C3/C5 convertase is a ideal inhibition target. Human complement receptor type 1(CR1) is a important inhibitor,which inhibits the C3/C5 convertase in vivo. CR1 comprises 30 repeating short consensus repeat (SCR) domains. In CR1, there are three active sites ,one of which is SCR15-18.CR1-SCR15-18 can not only bind the activated products C3b/C4b, inactivates C3/C5 convertase but also help the serum protease factor I to degrade C3b and C4b to complement fragments that play no further part in the convertase activity, resulting in the block of C5a and C5b-9 production. Therefore, CR1-SCR15-18 can be used as a therapeutic agent for inhibiting complement activation in many diseases such as ischemia- reperfusion injury,transplantation and autoimmune disease.In this study we aimed to construct pichia yeast recombinant expression vector of CR1-SCR15-18 based on our previous work and express CR1-SCR15-18 in Pichia pastoris as a secreted protein and investigate its activity of inhibiting of complement hemolysis in vitro and its protective effect on intestinal ischemia-reperfusion injury in a rat model. The major contents and results are as follows:1. Primer was designed based on DNA sequence of human CR1-SCR15-18 and the multiple clone site of pPIC9.Xhol I site was added into the upstream primer, which connected the target sequence behind the sequence of signal peptide; TAG termination codon,6x HIS tag and Not I site were added into the downstream primer. The DNA sequence of CR1-SCR15-18 was successfully obtained from pET32a-sCR1-SCR15-18 by PCR.The fragment was 756 bp.2. The obtained sequence and pPIC9 were digested with Xhol I and Not I and then linked with T4 ligase.The recombinant express vector named as pPIC9-CR1-SCR15-18 was successfully constructed and identified correctly by PCR, enzyme digestion and nucleotide sequencing.3. The recombinant plasmid pPIC9-CR1-SCR15-18 was digested with restriction enzyme Sal I .The linearized vector was transformed into Pichia Pastoris strain GS115 by electroporation followed by plating and screening of transformants on MD plates. Positive tranformants were conformed by colony PCR.Four positive tranformants were screened and named as pPIC9-CR1-SCR15-18/GS115.4. The four positive recombinants were fermented in shake flaskes and induced with methanol. The recombinant protein were identified with SDS-PAGE and Western blot analysis of the medium supernatants conformed that the target gene was successfully expressed in the yeast and the recombinant protein was successful secreted into the culture supernatants.5. The medium supernatants were purified by Ni2+-NTA agarose metal chelate affinity chromatography and the purity of CR1-SCR15-18 was very high. After purification and dry,the yield the recombinant protein was 31.8mg/L.The high yield was a foundation for high density fermentation.6. The biological activity of the recombinant protein was analysised in vitro after purification. CR1-SCR15-18 inhibited complement hemolysis in vitro.The lower concentration of protein CR1-SCR15-18, the weaker capacity of inhibiting complement hemolysis. The results indicate that CR1-SCR15-18 can inhibit complement activity in vitro.7. The rat intestinal ischemia-reperfusion injury model was successfully established. After I/R, the vascular permeability, the activity of MPO and the content of MDA in I/ R group were significantly increased; the activity of SOD was decreased; HE staining demonstrated severe intestinal histological damage; and a mount of complement 3 and its derivates were deposited in necrosis area. Compared with I/R group, in CR1 group, the vascular permeability, the activity of MPO and the content of MDA were decreased and the activity of SOD was increased significantly. CR1-SCR15-18 also significantly attenuated intestinal histological injury and reduced the deposition of C3 and its derivates in necrosis zone.In conclusion ,in this study, the recombinant express vector pPIC9-CR1-SCR15-18 was successfully constructed.CR1-SCR15-18 was successful expressed in Pichia Pastoris yeast and secreted into the culture supernatant. After purification ,the protein could inhibit complement hemolysis in vitro and exerted a protective effect against intestinal I/R injury in rat ,possibly by inhibiting activation of complement. The results of our study have paved a way for further studies of prevention and cure ischemia reperfusion injuries using CR1-SCR-15-18.