The Expression And Purification Of C1INH(14-32)-HSA And Preliminary Study

As the complement system inhibitors, Cl inhibitor (C1INH) which the N-terminal amino acid can be combined with LPS, with endotoxemia and endotoxin shock has a positive therapeutic significance.Experimental preliminary work:the N-terminal amino acid could bind with LPS after ELISA. Seven peptides were designed according to the C1INH (1-59). Then the seven peptides were tested via ELISA. The peptide of the N-terminal18to30amino acid C1INH (18-30) showed well in binging with LPS in vitro.6amino acids were increased on the two sides of C1INH (18-30), three of them are hydrophobic amino acids, two low hydrophilic, one hydrophilic. Then, to prolong the half-life of C1INH (14-32) in vivo, developed a long-acting C1INH (14-32)[C1INH (14-32)-HSA] by albumin fusion technology.Therefore, in this study, the C1INH (14-32)-HSA fusion protein was successfully expressed in Pichia pastoris. On this basis, the thesis continue to research the culture conditions of recombinant strain P. pastoris GS115/pPIH,from the expression time and methanol concentration, we desiged the gradient expriment, then, the result has been seen that:the best induction conditions for72hours, the final concentration of1%methanol. Since then. the C1NH(14-32)-HSA fusion protein was salted out by ammonium sulfate precipitation, used Blue Sepharose affinity chromatography to purity the C1INH(14-32)-HSA. then, concentrateed this protein by ultrafiltration tube. Finally. we can get the C1INH(14-32)-HSA which has the dieal purity and concentration, the concentration of519μg/ml.Next, immunizing rabits with C1INH(14-32)-HSA fusion protein prepared by above according to the conventional methods to prepare the polyclonal antibody, anti-C1INH(14-32)-HAS. Since then, determined the titer by indirect ELISA, be the optimal working dilution:the antigen was1:200, the antibody was1:1000. Then, this anti-C1INH (14-32) was detected by western blot, the map of the binding capacity has two clear bands, the58KDa and the27Kda. indicating that the anti-C1INH (14-32)-of HSA’s specific comparation is good. After that used Protein A affinity chromatography to purity the blood serum, dientified the purity by SDS-PAGE. In the SDS-PAGE map of anti- C1INH(14-32)-HSA existed in two protein bands which indicating that the high purity anti-C1INH(14-32)-HSA was successfully obtained. The molecular weight of heavy chain was about70kDa, the light one was about25kDa. Finally, detected the specificity of anti-C1INH(14-32)-HSA by Western blot.