HAb18Gedomab1单抗的制备及功能研究

Preparation and functional characterization of the monoclonal antibody HAb18Gedomab1

目的:制备抗人HAb18G/CD147胞外区蛋白(HAb18Ged)的单抗HAb18Gedomab1,并对其理化性质和生物学功能进行分析鉴定. 方法:用HAb18Ged免疫Balb/c小鼠,通过杂交瘤技术, 融合并筛选分泌抗人HAb18Ged蛋白单抗的杂交瘤细胞株, 制备腹水,采用离子交换层析法纯化该抗体.采用流式细胞术、免疫组化等方法对其特异性进行鉴定,并通过明胶酶谱、I型胶原酶谱、重组基底膜降解实验研究该抗体的体外生物学功能. 结果:获得一株稳定分泌抗人HAb18G/CD147胞外区蛋白单抗的杂交瘤细胞株HAb18Gedomab1,滴度为:1:106, 纯化后HAb18Gedomab1单抗的纯度大于90%,抗体亚型属IgG1型.流式细胞术及免疫组化结果证明,该抗体与FHCC-98细胞膜抗原及肝癌组织均呈特异性结合.生物学功能研究表明,HAb18Gedomab1可刺激鼠成纤维细胞(3T3)分泌明胶酶MMP-2,MMP-9及胶原酶MMP-1, MMP-8,并具有促进基底膜降解的作用. 结论:HAb18Gedomabl单抗可特异识别HAb18Ged,刺激MMPs分泌,促进基底膜降解.

AIM: To obtain mouse anti-human monoclonal antibodies against recombinant extracellular domain of HAb18G (HAb18Ged), and to analyze and identify its character and biological function. METHODS: Balb/c mice were immunized with HAb18Ged. Hybridoma cell was screened by cell fusion and subcloning approach. The monoclonal antibody in the ascites was purified by ion exchange chromatography and was identified by fluorescence-activated cell sorting analysis (FACs) and immunohistochemistry. Gelatin zymography and collage-nase type I zymography were used to analyze the effects of HAb18Gedomab1 on activation and production of matrix metalloproteinase (MMPs); Matrigel-boyden degradation chamber method was used to evaluate the infiltrative cells ratio. RESULTS: A hybridoma cell HAb18Gedomab1 stably secreting anti-HAb18Ged monoclonal antibody was obtained. The titer of this McAb in ascites was 1:106. The purity of the McAb was higher than 90%. The McAb belonged to IgG1 subclass. HAb18Gedomab1 showed high specificity and affinity to the antigen of FHCC-98 cell membrane and the tissue of hepatocellular carcinoma. The McAb induced production and activation of MMP-2, MMP-9, MMP-1 and MMP-8 in mouse fibroblast cells (3T3), and also promoted the degradation of reconstituted basement membrane. CONCLUSION: HAb18Gedomab1 can bind specifically to HAb18Ged protein. The McAb can also induce production and activation of MMPs and promote the degradation of reconstituted basement membrane.