血管性血友病因子裂解酶活性域的表达及其单克隆抗体的制备

血管性血友病因子裂解酶(vWF-cp)是一种新发现的金属蛋白酶,与血栓性微血管病的发生密切相关。本研究应用IPTG从pET28a( )-vWF-cp/MD质粒中诱导表达了vWF-cp的金属蛋白酶域,重组蛋白经Ni-NTA柱纯化,复性后免疫BALB/c小鼠,利用杂交瘤技术获得了稳定分泌抗vWF-cp金属蛋白酶域抗体的细胞株,进一步对其单克隆后,收集细胞接种于小鼠腹腔产生腹水,鉴定所获得的单克隆抗体,同时,利用单克隆抗体确定vWF-cp在组织细胞中的位置及其在各种正常组织中的表达情况。结果表明,重组蛋白主要以包涵体形式存在,占菌体总蛋白的28%。利用杂交瘤技术制备出3株抗vWF-cp单克隆抗体,对其中2株单克隆抗体作了进一步的鉴定。免疫双扩散和竞争ELISA结果显示,它们均属IgG1亚类,腹水中的含量约为4mg/ml,效价达1×10-5,而且识别vWF-cp金属蛋白酶域不同的表位。Westernblot和免疫沉淀结果表明,2株单克隆抗体能够识别重组蛋白和正常血小板中200kD的蛋白。在组织细胞中,2株单克隆抗体识别的蛋白位于细胞胞质内,而且在多种正常组织如肝、前列腺和卵巢等组织中表达,但脑组织中无该蛋白的表达。结论:本研究为了解vWF-cp在组织中的分布以及进一步研究该蛋白酶的结构和功能奠定了基础,并且可用所制备的单克隆抗体建立vWF-cp抗原的检测方法。

Expression of the Metalloproteinase Domain of von Willebrand Factor-Cleaving Protease and Preparation of Its McAb

The von Willebrand factor-cleaving protease (vWF-cp) is a newly identified plasma metalloproteinase and plays an important role in the pathogenesis of thrombotic microangiopathy. In the present study, the metalloproteinase domain of vWF-cp was expressed by IPTG-induced the recombinant engineered E.coli strain harbouring pET28a (+)-vWF-cp/MD and purified using chromatography on Ni-NTA column. Then the BALB/c mice were immunized with the recombinant protein to prepare the monoclonal antibodies (McAb) against vWF-cp and the obtained McAbs were characterized. Furthermore, the expression panels of vWF-cp in human normal tissues were investigated using immunohistochemistry. The results showed that high-level expression of the recombinant protein was achieved, which existed as inclusion body and amounted to 28% of total bacteria protein. Three monoclonal antibodies against the metalloproteinase domain of vWF-cp were obtained and two of them, SZ-112 and SZ-113, were further evaluated. Both of them belong to IgG(1). The concentration of them in ascites was 4 mg/ml, and their titers were as high as 1 x 10(-5). The data of ELISA showed that SZ-112 and SZ-113 recognized different epitopes of recombinant protein. The Western blot and immunoprecipitation data showed that the two monoclonal antibodies reacted not only with the recombinant protein, but also with a 200 kD protein in platelet lysate. Moreover, the vWF-cp was found to be present in the cytoplasm of many human tissues such as liver, prostate, ovary, etc. However, the protease could not be detected in brain tissue. In conclusion, the above-mentioned research work contributed not only to the further study of the structure and function of this protease, but also to the establishment of the method for quantifying the vWF-cp antigen in plasma.