纤维连接蛋白N端肝素结合域多肽的制备及其对DIC大鼠的治疗作用  

Preparation of Recombinant Polypeptide of N-terminal Heparin-binding Domain of Fibronectin and Its effect on Disseminated Intravascular Coagulation in Rats

作　　者：邹起练[1,2] 郭江睿[1] 陈小芳[1] 黄美娟[1] 吴勇[1] 陈元仲[2] 

机构地区：[1]福建省血液病研究所,福建医科大学附属协和医院血液科,福建福州350001 [2]福建医科大学基础医学院细胞生物学与遗传学系,福建福州350004

出　　处：《中国实验血液学杂志》2010年第3期698-703,共6页Journal of Experimental Hematology

基　　金：福建省科技创新平台建设项目(编号2009J1004);福建医科大学科研基金资助项目(编号FJGXT04033)

摘　　要：本研究利用酵母表达系统制备纤维连接蛋白(fibronectin,FN)N端肝素结合域多肽,检测多肽的生物学活性,进行多肽治疗DIC大鼠的实验研究。利用PCR技术从FNcDNA中扩增FN N端肝素结合域序列,将获得的目的基因片段克隆至T载体,筛选后转移至酵母表达pAo815SM载体进行选择,再构建重组酵母表达pPIC9K载体,线性化重组载体并转染GS115酵母细胞,在GS115酵母细胞表达目的多肽。发酵液经80%硫酸胺沉淀,沉淀物过S-100柱分离纯化,纯化物再经过SP柱纯化,测定多肽结合肝素等生物学活性。股静脉注射内毒素脂多糖(LPS)50mg/kg建立大鼠DIC模型。建立的DICSD大鼠40只,随机分2组,每组20只,一组为rhFNHN-29多肽治疗组,另一组为生理盐水对照组。治疗组分别于注射LPS前半小时、注射后2小时、4小时尾静脉注射rhFNHN-29多肽(10mg/kg);生理盐水对照组注射等体积生理盐水。另设正常对照组SD大鼠20只,注射等体积生理盐水作为正常对照组。各组大鼠于股静脉注射LPS后6小时,对侧股静脉抽血测血常规,分离血浆检测血浆TNFa和凝血象。于注射LPS后72小时处死大鼠,取肝、肾、脾、肺、心、脑组织进行病理组织学检查。结果表明:成功地在酵母表达系中表达了目的多肽,其表达量达到30mg/L,所表达的多肽具有结合肝素活性。在多肽抗DIC作用的动物实验中发现多肽治疗组的血浆TNFa水平、血常规指标、凝血象和病理组织学的改变都较生理盐水对照组更接近正常对照组。结论:成功地制备了rhFNHN-29多肽,这种多肽能在一定程度上预防和治疗DIC。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 transfomed 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 μl 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 μl blood. The rest 450 μl 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

关 键 词：纤维连接蛋白 肝素结合域多肽 酵母表达系统 弥漫性血管内凝血 

分 类 号：R554[医药卫生—血液循环系统疾病] R554.8[医药卫生—内科学]