人蛋白C的研制及其相关研究
|
摘要
人蛋白C(human Protein C，hPC)是一种维生素K依赖的丝氨酸蛋白酶原，
属血浆糖蛋白，相对分子量是62KDa。蛋白C系统是一种重要的抗凝机制之一，
该系统由蛋白C(PC)、凝血调节蛋白(TM)、蛋白S(PS)、蛋白C抑制物及新近
发现的内皮细胞蛋白C受体(EPCR)组成。活化蛋白C的主要功能是抗凝、抗炎和
促纤溶。近年来，活化蛋白C在感染性疾病，特别是在严重脓毒症、内毒素血症
和弥散性血管内凝血等严重感染性疾病中的治疗备受关注。
随着人血浆PC及重组人蛋白C(recombinant human Protein C，rhPC)在疾病
治疗应用范围的扩展，建立简便、快速、灵敏和特异的人PC检测方法显得尤为
重要。目前，国外的蛋白C制品(血浆浓缩剂及重组制剂)和诊断试剂价格较昂
贵，不适合临床的推广应用。国内蛋白C的研究处于起步阶段，因此对蛋白C制
品及诊断试剂的研究将有广阔的市场前景。
本研究包括：
1 物理化学方法纯化人血浆蛋白C：将3000ml新鲜冷冻血浆为起始材料，以
DEAE-Sephadex A 50为吸附介质，收集洗脱产物，再经DEAE-Sepharose FF及肝
素-Sepharose CL 6B层析，对收集产物进行APTT、SDS-PAGE及Western blotting
检测，获得纯度大于95％的人血浆蛋白C。
2 人蛋白C cDN_A在CHO-dhfr细胞中的表达：利用脂质体转染技术将含有人
蛋白C cDNA基因和二氢叶酸还原酶基因的载体转入CHO细胞中，经过MTX
中文摘要博士学位论文
(6.4umol/L)抗性筛选，APTT、SDS一PAGE及westem blotting检测获得了6株
高表达的细胞株。
3人蛋白C单克隆抗体的制备与鉴定:利用人血浆蛋白C纯化产物免疫
BALB/C小鼠制备了5株人蛋白C单克隆抗体细胞株，分别进行了效价、亚类、
特异性及亲和力等鉴定。
4人蛋白C单克隆抗体的初步应用
l)酶联免疫分析法检测人血浆蛋白C:利用两株识别不同人蛋白C位点的单
克隆抗体细胞株建立了夹心ELISA检测人血浆蛋白C。此方法可用于检测血浆中
3.12~ZO0ng/mL范围内蛋白C，实验可在4小时内完成，实验的批内和批间变异
不超过7%，测得102份正常血浆样品的参考范围是4.02士0.295林留mL
2)亲合层析法纯化人血浆蛋白C:先用DEAE一S叩hadexA一50吸附人血浆，
然后将吸附产物过人蛋白C单克隆抗体与澳化氢活化的SePharose 4B偶联制备亲
和层析柱，获得人蛋白C的纯化产物。此方法可使纯化产物浓缩16700倍，纯度
超过99%。
本研究建立了人血浆蛋白C的纯化(物理化学纯化与亲和层析)方法，有望用
于规模化生产;在国内首次实现人蛋白C CDNA在dhfr缺陷型CHO细胞中的高效
表达，为今后蛋白C的进一步研究打下了基础;建立了抗人蛋白C单克隆抗体杂
交瘤细胞系，利用两株识别不同抗原位点的单克隆抗体建立了夹心EL工SA检测人
血浆蛋白C的方法，为临床检测人血浆蛋白C奠定了基础。
Human protein C, a vitamin K-dependent serine protease, is a plasma glycoprotein with relative molecular mass(Mr) of 62x103. Activated protein C potently inhibits coagulation and inflammation by inactivating factors V and VIII and facilitates fibrinolysis in vivo. In recent years, activated protein C attracted more attention in the treatment of infectious diseases, especially in the treatment and prevention of pyaemia, endotoxin blood poisoning and disseminated intravascular coagulation in endotoxemia.
With the extensive application of human protein C products (protein C purified from human plasma and recombinant protein C) in the treatment of varied diseases, it is urgent to establish a simple, fast, sensitive and specific measure for protein C detection. At present, abroad protein C products (protein C purified from human plasma and recombinant protein C) and diagnostic kits are very expensive, which is impossible for extensive clinical application.
There are four parts in this experiment:
1 Purifying protein C by physical-chemical methods: The major steps are as follows: After collecting elution products from DEAE-Sephadex A-50 absorbed, DEAE-Sepharose Fast Flow and Heparin-Sepharose CL 6B chromatography, protein C was confirmed by APTT, SDS-PAGE and Western blotting. The purity of protein C was estimated to be more than 95% by SDS-PAGE.
2 Expression of human protein C cDNA in CHO cells: By using lipofectamine-mediated gene transfer technique, recombinant expression vector containing human protein C cDNA gene and dhfr gene was transfected into CHO cells.
Six higher expression cells were obtained by MIX (6.4umol/L) selecting. Protein C was confirmed by APTT, SDS-PAGE and Western blotting.
3 Preparation and identification of monoclonal antibodies to human protein C: Five monoclonal antibodies designated as PC1 to PC5 were obtained from hybridoma cells cloned after the fusion of mouse myeloma cells with spleen cells of mice immunized with purified human protein C and the titer, subclass, specificity and affinity were detected.
4 Application of monoclonal antibodies to human protein C.
1) Method of human protein C detection through ELISA (Enzyme-linked immunoadsordent assay): The ELISA method was established through two monoclonal antibodies (McAb) identification with different antigenic sites of human protein C which enabled the determination of protein C in concentrations range of 3.12~ 200ng/mL in less than 4 hours with a single antigen-antibody reaction. Within-run and between-run coefficients of variation were less than 7%. The reference range of protein C in plasma of 102 normal samples was 4.02?.295ug/mL.
2) Immunoaffinity purification of protein C by monoclonal antibody to human protein C: Protein C was isolated from DEAE-Sephadex A-50 adsorbed fresh plasma by immunoaffinity chromatography on a column of Sepharose 4B coupled with monoclonal antibody to protein C. This immunopurification resulted in a 16700-fold purification of fully functional zymogen from plasma. The purity of human protein C was estimated to be more than 99% by SDS-PAGE.
The research established purification methods (physical-chemical method and affinity chromatography) for human protein C, which is hopeful for large scale protein C production; High expression of human protein C cDNA in CHO-dhfr" cells laid a foundation for the research of protein C; In addition, we developed Five hybridoma cell lines of monoclonal antibodies to human protein C and established ELISA to detect human plasma protein C.
属血浆糖蛋白，相对分子量是62KDa。蛋白C系统是一种重要的抗凝机制之一，
该系统由蛋白C(PC)、凝血调节蛋白(TM)、蛋白S(PS)、蛋白C抑制物及新近
发现的内皮细胞蛋白C受体(EPCR)组成。活化蛋白C的主要功能是抗凝、抗炎和
促纤溶。近年来，活化蛋白C在感染性疾病，特别是在严重脓毒症、内毒素血症
和弥散性血管内凝血等严重感染性疾病中的治疗备受关注。
随着人血浆PC及重组人蛋白C(recombinant human Protein C，rhPC)在疾病
治疗应用范围的扩展，建立简便、快速、灵敏和特异的人PC检测方法显得尤为
重要。目前，国外的蛋白C制品(血浆浓缩剂及重组制剂)和诊断试剂价格较昂
贵，不适合临床的推广应用。国内蛋白C的研究处于起步阶段，因此对蛋白C制
品及诊断试剂的研究将有广阔的市场前景。
本研究包括：
1 物理化学方法纯化人血浆蛋白C：将3000ml新鲜冷冻血浆为起始材料，以
DEAE-Sephadex A 50为吸附介质，收集洗脱产物，再经DEAE-Sepharose FF及肝
素-Sepharose CL 6B层析，对收集产物进行APTT、SDS-PAGE及Western blotting
检测，获得纯度大于95％的人血浆蛋白C。
2 人蛋白C cDN_A在CHO-dhfr细胞中的表达：利用脂质体转染技术将含有人
蛋白C cDNA基因和二氢叶酸还原酶基因的载体转入CHO细胞中，经过MTX
中文摘要博士学位论文
(6.4umol/L)抗性筛选，APTT、SDS一PAGE及westem blotting检测获得了6株
高表达的细胞株。
3人蛋白C单克隆抗体的制备与鉴定:利用人血浆蛋白C纯化产物免疫
BALB/C小鼠制备了5株人蛋白C单克隆抗体细胞株，分别进行了效价、亚类、
特异性及亲和力等鉴定。
4人蛋白C单克隆抗体的初步应用
l)酶联免疫分析法检测人血浆蛋白C:利用两株识别不同人蛋白C位点的单
克隆抗体细胞株建立了夹心ELISA检测人血浆蛋白C。此方法可用于检测血浆中
3.12~ZO0ng/mL范围内蛋白C，实验可在4小时内完成，实验的批内和批间变异
不超过7%，测得102份正常血浆样品的参考范围是4.02士0.295林留mL
2)亲合层析法纯化人血浆蛋白C:先用DEAE一S叩hadexA一50吸附人血浆，
然后将吸附产物过人蛋白C单克隆抗体与澳化氢活化的SePharose 4B偶联制备亲
和层析柱，获得人蛋白C的纯化产物。此方法可使纯化产物浓缩16700倍，纯度
超过99%。
本研究建立了人血浆蛋白C的纯化(物理化学纯化与亲和层析)方法，有望用
于规模化生产;在国内首次实现人蛋白C CDNA在dhfr缺陷型CHO细胞中的高效
表达，为今后蛋白C的进一步研究打下了基础;建立了抗人蛋白C单克隆抗体杂
交瘤细胞系，利用两株识别不同抗原位点的单克隆抗体建立了夹心EL工SA检测人
血浆蛋白C的方法，为临床检测人血浆蛋白C奠定了基础。
Human protein C, a vitamin K-dependent serine protease, is a plasma glycoprotein with relative molecular mass(Mr) of 62x103. Activated protein C potently inhibits coagulation and inflammation by inactivating factors V and VIII and facilitates fibrinolysis in vivo. In recent years, activated protein C attracted more attention in the treatment of infectious diseases, especially in the treatment and prevention of pyaemia, endotoxin blood poisoning and disseminated intravascular coagulation in endotoxemia.
With the extensive application of human protein C products (protein C purified from human plasma and recombinant protein C) in the treatment of varied diseases, it is urgent to establish a simple, fast, sensitive and specific measure for protein C detection. At present, abroad protein C products (protein C purified from human plasma and recombinant protein C) and diagnostic kits are very expensive, which is impossible for extensive clinical application.
There are four parts in this experiment:
1 Purifying protein C by physical-chemical methods: The major steps are as follows: After collecting elution products from DEAE-Sephadex A-50 absorbed, DEAE-Sepharose Fast Flow and Heparin-Sepharose CL 6B chromatography, protein C was confirmed by APTT, SDS-PAGE and Western blotting. The purity of protein C was estimated to be more than 95% by SDS-PAGE.
2 Expression of human protein C cDNA in CHO cells: By using lipofectamine-mediated gene transfer technique, recombinant expression vector containing human protein C cDNA gene and dhfr gene was transfected into CHO cells.
Six higher expression cells were obtained by MIX (6.4umol/L) selecting. Protein C was confirmed by APTT, SDS-PAGE and Western blotting.
3 Preparation and identification of monoclonal antibodies to human protein C: Five monoclonal antibodies designated as PC1 to PC5 were obtained from hybridoma cells cloned after the fusion of mouse myeloma cells with spleen cells of mice immunized with purified human protein C and the titer, subclass, specificity and affinity were detected.
4 Application of monoclonal antibodies to human protein C.
1) Method of human protein C detection through ELISA (Enzyme-linked immunoadsordent assay): The ELISA method was established through two monoclonal antibodies (McAb) identification with different antigenic sites of human protein C which enabled the determination of protein C in concentrations range of 3.12~ 200ng/mL in less than 4 hours with a single antigen-antibody reaction. Within-run and between-run coefficients of variation were less than 7%. The reference range of protein C in plasma of 102 normal samples was 4.02?.295ug/mL.
2) Immunoaffinity purification of protein C by monoclonal antibody to human protein C: Protein C was isolated from DEAE-Sephadex A-50 adsorbed fresh plasma by immunoaffinity chromatography on a column of Sepharose 4B coupled with monoclonal antibody to protein C. This immunopurification resulted in a 16700-fold purification of fully functional zymogen from plasma. The purity of human protein C was estimated to be more than 99% by SDS-PAGE.
The research established purification methods (physical-chemical method and affinity chromatography) for human protein C, which is hopeful for large scale protein C production; High expression of human protein C cDNA in CHO-dhfr" cells laid a foundation for the research of protein C; In addition, we developed Five hybridoma cell lines of monoclonal antibodies to human protein C and established ELISA to detect human plasma protein C.
引文
1 Kisiel W. Human Plasma ProteinC-Isolation, Characterization, And Mechanism Of Activation By ct-Thrombin. J Clin Invest. 1979,761．
2 Sakata Y, David JL, Candece L, etc al. Mechanism of Protein C-Dependent Clot Lysis: Role of Plasminogen Activator Inhibitor. Blood 1986,68 (6) , 1218．
3 Hoogendoorn H., Nesheim ME, and Giles AR. A Qualitative and Quantitative Analysis of the Activation and Inactivation of Protein C In Vivo in a Promate Model.Blood, 1990,75 (11) , 2164．
4 王博诚，李金泉，金坚，等．人血浆蛋白C的分离纯化与鉴定．生物化学杂 志．1993，9(2) ，204．
5 CharlesT.Esmon, KenjiFukudome, TimMather, etcal. Inflammation, Sepsis, and coagulation. Hamatologica.1999, 84,254．
6 Satoko N and Sakata Y. Immunoaffinity purification of protein C by using conformation-specific monoclonal antibodies to protein C-calciumion complex. Biochimica et Biophysica Acta, 1987,925,85．
7 Holger H and Walkinshaw MD. Separation of human vitamin K-dependent coagulation proteins using hydrophobic interaction chromatography. Journal of Chromatography. 1999, 736, 77．
2 Sakata Y, David JL, Candece L, etc al. Mechanism of Protein C-Dependent Clot Lysis: Role of Plasminogen Activator Inhibitor. Blood 1986,68 (6) , 1218．
3 Hoogendoorn H., Nesheim ME, and Giles AR. A Qualitative and Quantitative Analysis of the Activation and Inactivation of Protein C In Vivo in a Promate Model.Blood, 1990,75 (11) , 2164．
4 王博诚，李金泉，金坚，等．人血浆蛋白C的分离纯化与鉴定．生物化学杂 志．1993，9(2) ，204．
5 CharlesT.Esmon, KenjiFukudome, TimMather, etcal. Inflammation, Sepsis, and coagulation. Hamatologica.1999, 84,254．
6 Satoko N and Sakata Y. Immunoaffinity purification of protein C by using conformation-specific monoclonal antibodies to protein C-calciumion complex. Biochimica et Biophysica Acta, 1987,925,85．
7 Holger H and Walkinshaw MD. Separation of human vitamin K-dependent coagulation proteins using hydrophobic interaction chromatography. Journal of Chromatography. 1999, 736, 77．