猪血液中三种凝血因子的提取及凝血因子X的蛋白分析研究
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摘要
研究目的：凝血因子X作为凝血过程中一个重要的因子,其蛋白质结构的分析对于解决器官移植中机体排异现象有着重要意义；纤维蛋白原和凝血酶作为止血药已经广泛应用于外科领域,随着医学研究的飞速发展,动物性止血药的应用越来越多,提高凝血酶等凝血因子的提取率和原料的利用率对于今后凝血因子在医疗方面的应用有着重要意义。
实验方法：本研究以猪血为原料,采用连续提取法依次提取纤维蛋白原等三种凝血因子。先用冻融法提取纤维蛋白原；然后用等电点沉淀法及离子交换层析法分离提取凝血酶；再用柠檬酸钡吸附法以及离子交换层析提取纯化凝血因子X；对纯化后的凝血因子X样品采用质谱法测定其氨基酸序列并进行蛋白同源性分析；最后建立动物外伤模型,测定凝血酶的止血效果。
实验结果：采用连续提取法分离得到的纤维蛋白原的含量2.81g／L,其提取率(%)为89.05%；离子交换层析等方法所得凝血酶的总活性为604.4U,比活为37.8U／mg；用飞行质谱法测得凝血因子X的纯化样品的小肽序列,在数据库中的编号为(NCBIABF82361.1);通过对该序列进行不同物种间的比较及同源性分析,发现猪FX的氨基酸序列与牛的同源性最高,与牛的亲缘性也最高；在家兔的三种外伤模型的止血实验中,凝血酶的止血效果明显,在耳外伤止血效果最好,其止血时间平均为48.6秒。
本研究的创新性在于：采用连续提取法从猪血中依次提取纤维蛋白原、凝血酶及凝血因子X,节约了原料,简化了提取过程,为将来工业化生产打下基础；采用质谱法测得凝血因子X的小肽序列,比较分析序列同源性等,为解决机体的排异现象及相关器官移植等研究奠定了基础。
Research objectives:Analyzing the protein structure of blood coagulation factor X which is a predominant molecule of coagulation cascade is vital for resolving body rejection in organ transplantation. Fibrinogen and thrombin used as hemostyptic are widely utilized in surgical department. As the rapid development of medical research, hemostyptics drugs used in animal medical field will be increasing. Enhancing extraction percentage of blood coagulation such as thrombin and utilization rate of raw materials is important for their medical application in future.
Research methods:three coagulation factors were extracted successively by continuous extraction method prepared from porcine blood. First of all, fibrinogen was extracted by freeze-thawed nerve, thrombin by isoelectric point precipitation and ion-exchange chromatography, factor X was extracted and purified by barium citrate adsorption method and ion-exchange chromatography. Then amino acid sequences of factor X were obtained by the mass spectrography and the protein homology was analyzed. At last established animal external injury model and determined hemostatic effects of thrombin.
Results:Fibrinogen content is 2.81g/L and extraction percentage is 89.05% using continuous extraction method. Gross and specific activity of thrombin is 604.4U and 37.8U/mg by ion-exchange chromatography. Applying MALDI-TOFMS method we had obtained polypeptide sequences.(the number is ABF82361.1 in NCBI database) Homology analysis and multiple alignments of amino acid sequences of Factor X were performed between pig and other species. The results indicated that porcine factor X is highly homologous to bovine factor X and related to bovine factor X. There were three kinds of rabbit external injury models in our hemostatic experiment. The hemostatic effects of thrombin were distinct and the effect of external injury in the ear was best, the hemostatic time was 48.6 second on the average.
Originality:This technology that fibrinogen, thrombin and coagulation factor X are extracted in turn by continuous extraction method could save the materials, reduce the process and lay the foundation of industrial production in the future. Applying MALDI-TOFMS method we had obtained polypeptide sequences, analysed homology of amino acid sequences, laid the foundation for resolving body rejection in organ transplantation.
实验方法：本研究以猪血为原料,采用连续提取法依次提取纤维蛋白原等三种凝血因子。先用冻融法提取纤维蛋白原；然后用等电点沉淀法及离子交换层析法分离提取凝血酶；再用柠檬酸钡吸附法以及离子交换层析提取纯化凝血因子X；对纯化后的凝血因子X样品采用质谱法测定其氨基酸序列并进行蛋白同源性分析；最后建立动物外伤模型,测定凝血酶的止血效果。
实验结果：采用连续提取法分离得到的纤维蛋白原的含量2.81g／L,其提取率(%)为89.05%；离子交换层析等方法所得凝血酶的总活性为604.4U,比活为37.8U／mg；用飞行质谱法测得凝血因子X的纯化样品的小肽序列,在数据库中的编号为(NCBIABF82361.1);通过对该序列进行不同物种间的比较及同源性分析,发现猪FX的氨基酸序列与牛的同源性最高,与牛的亲缘性也最高；在家兔的三种外伤模型的止血实验中,凝血酶的止血效果明显,在耳外伤止血效果最好,其止血时间平均为48.6秒。
本研究的创新性在于：采用连续提取法从猪血中依次提取纤维蛋白原、凝血酶及凝血因子X,节约了原料,简化了提取过程,为将来工业化生产打下基础；采用质谱法测得凝血因子X的小肽序列,比较分析序列同源性等,为解决机体的排异现象及相关器官移植等研究奠定了基础。
Research objectives:Analyzing the protein structure of blood coagulation factor X which is a predominant molecule of coagulation cascade is vital for resolving body rejection in organ transplantation. Fibrinogen and thrombin used as hemostyptic are widely utilized in surgical department. As the rapid development of medical research, hemostyptics drugs used in animal medical field will be increasing. Enhancing extraction percentage of blood coagulation such as thrombin and utilization rate of raw materials is important for their medical application in future.
Research methods:three coagulation factors were extracted successively by continuous extraction method prepared from porcine blood. First of all, fibrinogen was extracted by freeze-thawed nerve, thrombin by isoelectric point precipitation and ion-exchange chromatography, factor X was extracted and purified by barium citrate adsorption method and ion-exchange chromatography. Then amino acid sequences of factor X were obtained by the mass spectrography and the protein homology was analyzed. At last established animal external injury model and determined hemostatic effects of thrombin.
Results:Fibrinogen content is 2.81g/L and extraction percentage is 89.05% using continuous extraction method. Gross and specific activity of thrombin is 604.4U and 37.8U/mg by ion-exchange chromatography. Applying MALDI-TOFMS method we had obtained polypeptide sequences.(the number is ABF82361.1 in NCBI database) Homology analysis and multiple alignments of amino acid sequences of Factor X were performed between pig and other species. The results indicated that porcine factor X is highly homologous to bovine factor X and related to bovine factor X. There were three kinds of rabbit external injury models in our hemostatic experiment. The hemostatic effects of thrombin were distinct and the effect of external injury in the ear was best, the hemostatic time was 48.6 second on the average.
Originality:This technology that fibrinogen, thrombin and coagulation factor X are extracted in turn by continuous extraction method could save the materials, reduce the process and lay the foundation of industrial production in the future. Applying MALDI-TOFMS method we had obtained polypeptide sequences, analysed homology of amino acid sequences, laid the foundation for resolving body rejection in organ transplantation.
引文
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