Preparation of recombinant polypeptide of N-terminal heparin-binding domain of fibronectin and its effect on disseminated intravascular coagulation in rats / 中国实验血液学杂志

ABSTRACT

This study was aimed to prepare the polypeptide of N-terminal heparin-binding domain of fibronectin(rhFNHN-29 polypeptide) with pichia expression system, to detect biological activity of recombinant polypeptide and investigate its effect on disseminated intravascular coagulation (DIC) in rats. The sequence of N-terminal heparin-binding domain of fibronectin was amplified from FNcDNA by PCR. The aim gene was cloned into T vector for selection. Then it was cloned into pAo815SM and pPIC9K vectors.Lined pPIC9K vectors were transformed into GS115 Pichia cells so as to express the aim polypeptide in Pichia expression system. The fermentation liquid were precipitated by 80% ammonium sulfate, and the further dissolved sediment were purified using S-100 column and SP column. Its activity of binding with heparin were detected by Western-blot. The established DIC rats (40 rats) were randomly divided into two groups. One group was treated with rhFNHN-29 polypeptide, and the other was treated with normal saline. The rats in the former group were injected with rhFNHN-29 polypeptide (10 mg/kg) through tail vein at 0.5 hour before, 2 hours and 4 hours after injection of LPS respectively. The rats in latter group were injected with equal volume saline. In addition, 20 normal rats injected with normal saline were as normal controls. 500 microl blood was taken from the rat vein, at 6 hours after the injection of LPS. White blood cell (WBC), hemoglobin (Hb) and platelets were tested from 50 microl blood. The rest 450 microl blood was used to isolate plasma for detecting TNFa level and coagulogram. The rats were killed at 24 hours after injection with LPS. Their livers, lungs, hearts, kidneys, and brain tissues were taken for histopathologic examination. The results showed that the aim polypeptide was successfully expressed in Pichia expression system. The expression level reached approximately 30 mg/L. The polypeptide had activity of binding with heparin antibody. In the experiment study of polypeptide effect on DIC in rats, the plasma TNFa level in polypeptide-treated group was lower than that in saline control group, the hemogram, coagulogram and histopathology were more obviously improved in polypeptide-treated group as compared with saline control group. It is concluded that the rhFNHN-29 polypeptide is successfully prepared, this polypeptide can antagonize DIC induced by endotoxin in rats.