Cloning And Expression Of Gene Fragment Of The Functional Binding Domain Of Human Soluble Complement Receptor Type 1

Previous studies have proved that superactivated complement fragments, together with the effects of C5a, C3a, and C5b-9, can result in opportunistic infection by pathogens after severe traumatic (burn) injuries or injuries due to ischemia-reperfusion, so it is limited to aim directly at only one fragment in prophylaxis and therapy. C3 is the most important factor in three pathways of the complement activation. Soluble complement receptor type 1 (sCRl) can not only inactivate C3/C5 invertase, but also bind to C3b, and can help Factor I to split C3b, resulting in the block of production of C5a and C5b-9, etc. In this study, to construct the prokaryotic expression vector pET32-sCRl-SCR15-18 and obtain the activated gene fragment of the functional domain of human sCRl, we extracted total RNA from monocytes of human peripheral blood for the purpose of cloning of the gene fragment of the functional domain of SCR15-18 by RT-PCR, and then the target gene was inserted into pMD18-T vector for sequencing. After confirmation of the correctness of the cDNA, we inserted it into pET32a and transferred pET32- sCRl-SCR15-18 into BL21. After screening and identification by BL21/ pET32- sCR1-SCR15-18, the protein expression was induced by IPTG. At last, the protein was purified by Ni-NTA chromatography and the reactive capability of the expressed product in inhibition of hemolysis by the complement was observed. The results of the study are as follows:1. Total RNA, extracted from monocytes of human peripheral blood, was employed as the template to design primers. We succeeded in cloning the cDNA of SCR1-SCR15-18 (length: 753 bp), and inserted it into pMD18-T vector and sequenced. The sequence of the cDNA was identical to the sequence in the GeneBank.2. The expression plasmid (pET32- sCRl-SCR15-18) was successfully constructed by inserting the correct cDNA into pET32. The positive recombinant plasmid screened byaminobenzylpenicillin was identified by enzyme digestion.3. The recombinant vectors were transduced into Escherichia coli (E. co/f) BL21. The expression was induced by IPTG and the optimal conditions (EPTG of 1 mmol/L at 37 癈 for 4 h) of the induction were achieved. SDS-PAGE analysis showed that the recombinant fusion protein of about 43kDa was highly expressed as inclusion bodies. Western blot analysis showed a single positive band with molecular weight of 43 kDa.4. The expressed protein was purified and renatured by Ni-NTA chromatography and multistep dialysis. SDS-PAGE analysis showed a single band in the target protein.5. Analysis of the biological activity of the purified protein showed that the purifiedprotein could inhibit the hemolysis reaction of the complement.In conclusion, we have successfully constructed the prokaryotic expression plasmid (pET32- sCRl-SCR15-18) and obtained the gene fragment of the sCRl functional binding domain that could inhibit the activity of the complement. The results of. our study have provided the reliable experimental methods for the highly expressed and purified sCRl-SCR15-l8 protein in large scale as well as the foundation for the prevention and treatment of complement-mediated opportunistic infection by pathogens after severe traumatic (burn) injuries or ischemia-reperfusion injuries.