Preliminary Study On Mammal Kallistatin Protein Expression,Purification And Monoclonal Antibody Production

Human tissue kallikrein binding protein(Kallistatin, Kal), as a kind of serine protease inhibitors, plays an important role in inflammation, angiogenesis, and damage repair, and other physiological and pathological process.Our laboratory previously study showed that Kal was of good resistance to liver fibrosis by inhibiting the activation of rat hepatic stellate cells, cutting stellate cells alpha smooth muscle actin(alpha SMA) expression, and inhibiting the formation of reactive oxygen species stellate cells induced by H202. In addition, Kal should be an excellent biomarker for monitoring the development of liver fibrosis.The aim of this study was attempting to establish an ELISA kit for clinical diagnosing liver fibrosis. The design and makeup of the standard ELISA-Kal kit were carried on according to stardard regulations of the Ministry of Health, China. That is,in addition to in vitro detect antibody kit, the clinical used kit were generally produced by double antibody sandwich method, with a biotin-streptavidin affinity system.First of all, pichia yeast fermentation and mammalian cells were used for express Kal protein. Neverthless, the results showed that Kal protein expressed by Pichia pastoris was lack of glycosylation in comparison to that of those nature products.Therefore in this study, the Kal protein obtained for this study came from yeast fermentation and mammalian expression system.The evidences from laboratory experiments also revealed that the high purity recombinant Kal protein could obtained through the yeast fermentation for Kal antibody production. However, it was found that the Kal protein was less immunogenic, thus, the immune dosage of Kal increased to 40 u g / ml with Freund’s adjuvant mixed at 1:1 ratio, and the best immune effect was achieved. We found that it was better to use subcutaneous immunization rather then intrasplenic immunization for Kal monoclonal antibody production, although it might be take a much longer time, the high titer monoclonal antibody production could be obtaineded in comparison to subcutaneous immunization and intrasplenic immunization methods in this study.The Kal mammalian expression vector was also constructed. The data showed that Kal protein well expressed in CHO cells, and subsequently, the experimental conditions of Kal protein production were optimized in CHO cell lines.The establishment of standard ELISA Kal kit for clinical application is underway in our laboratory, the present study provided some useful reference for producing a high sensitivity liver fibrosis Kal diagnostic kit and information how to handle Kal biological antibodies for clinical application.