摘要
炎症是机体的一种重要的基本病理过程。各种炎症虽然有其特殊性,但从局部的病理变化看来,它们有着不少共同点,即都是机体对致炎因子的损伤作用所产生的一种反应,最新的医学研究发现,当炎症的程度严重或发展成慢性炎症时,它可能导致肿瘤、心血管疾病、糖尿病、自身免疫性疾病以及与年龄相关的疾病(如关节炎、早老性痴呆病、骨质疏松症等)的发生。细胞间粘附分子-1(ICAM-1)作为重要的致炎因子,在炎症反应、免疫监督等过程中起着重要的作用。以上许多疾病过程中可见ICAM-1的异常表达,因此阻遏ICAM-1的表达为以上疾病的诊断和治疗提供了新的策略。
本研究在已制备的抗ICAM-1单克隆抗体的基础上,从分泌该单抗的杂交瘤细胞株中克隆出VH和VL基因,组装成ScFv基因,将ScFv基因插入原核表达载体pET-22b(+)中,转化宿主菌中进行表达,之后对表达的包涵体进行了复性和纯化。最后通过体外细胞实验和体内动物实验检测ScFv抗体的生物学活性。细胞实验表明,制备的ScFv能有效地抑制内皮细胞与单核细胞之间的粘附;动物实验证明此ScFv对小鼠无菌性炎症有明显的抑制作用。本课题为进一步治疗炎症性疾病奠定了基础。
Preparation and identification of anti-human ICAM-1 ScFv Adhesion molecules mediate the adhesion between cells and groundplasm, which produce a marked effect in the following a series of physiologic and pathologic processes, such as cellular signal transduction and activation, cellular spread and immigration, cellular growth and differentiation, inflammation, thrombopoiesis, tumor metastasis and wound healing. The functions are executed by the form of interaction between ligand and receptor.
ICAM-1 is a member of the immunoglobulin supergene family, which is a cell surface ligand for the lymphocyte function-associated antigen-1 (LFA-1). ICAM-1 expressed on the surface of activated lymphocytes in inflamed region,mainly on endothelial cells. The expressed amount in peripheral blood lymphocyte are very low in normal. The expression of ICAM-1 is up-regulated in the presence of various stimuli (e.g. inflammatory mediators, oxidative stress and viral infection).
ICAM-1 serves as an adhesion molecule, which participates in many pathologic and physiologic reactions. The interaction of ICAM-1 with the LFA-1 plays an important role in leukocyte adhesion and the execution of immunological and inflammatory functions. The elevated level of ICAM-1 result in the transmigration of neutrophils during the initial phase of inflammation. Inhibition of this process could decrease inflammatory response and thus reduce the tissue damage. Many diseases are associated with the over-expression of ICAM-1. Inhibiting its expression will reduce certain pathological injuries. In experimental model, the rolling of leucocytes were blocked by down-regulating of ICAM-1, which influence the aggregation of leucocytes, consequently, prevent the process of inflammation. These reports suggested that anti-ICAM-1 have a potential application in the treatment of ICAM-1 mediated immunological and inflammatory diseases. The strategy by down-regulation of ICAM-1 will provide a new method for the treatment of such diseases. In clinical,the monoclonal antibodies against CAMs were used usually. Anti-ICAM-1 monoclonal antibodies was effective in preventing the progression of ICAM-1 mediated diseases. But the mouse monoclonal antibodies will produce Human Anti-Mouse Antibody (HAMA) and can’t get into the target part of the patient body because of its big size. Nowadays, ScFv overcomes the limitations of monoclonal antibodies and was paid more attention. ScFv can lessen HAMA response in human body, but it still keeps the binding specificity and affinity comparable to that of its parent antibody. The ScFv strategy has become one of the most popular methods in antibody engineering because of it’s less immunogenicity. Furthermore, it’s small molecular size endows ScFv with better tissue penetration and shorter half life in plasma, therefore, at present ScFv has a wide range of applications in research, diagnosis and therapy. So there is a need to prepare anti-ICAM-1 ScFv whether in theory or practice.
VH and VL genes of anti-human ICAM-1 were cloned by RT-PCR from hybridoma cells. The ScFv gene was spliced with linker by sequence over lap extending (SOE) PCR. Sequences analysis showed that the VH and VL genes were spliced by 45 bases coding a (G4S)3 flexible linker. The length of ScFv fragment was 744bp, VH fragment was 357bp encoding 119 amino acids and VL fragment was 342bp encoding 114 amino acids. The VH and VL had integrity structure of FR and CDR, whidch was consistent with the characteristics of variable region of mouse immunoglobulin gene by comparing with the sequences in Genbank.
The ScFv gene was cloned into pET-22b (+) vector and constructed prokaryotic expressing plasmid pET22b-ScFv. The plasmid was transformed into E.coli BL-21(λDE3) and induced by IPTG, but the ScFv antibody was expressed in form of inclusion body. The yield of the ScFv accounted for about 40% in total cellular proteins. Meantime, we tried different expressed condition and determined the optimization condition for fermentation, which established foundation for a large scale production.
After fermentation, the inclusion body was extracted by centrifugalization and purified initially by washing buffer with 2M urea and 0.5% TritonX-100. During the process, about 150-200mg/L of inclusion bodies was obtained and the purity of ScFv in the pellet was up to 70%, which is determined by SDS–PAGE. Finally, the inclusion bodies were solubilized in 8M urea and the denatured solution were refolded by three methods, namely, dilution, dialysis and chromatography. As for the column chromatography, we chose the anion-exchange chromatography (Q HP) as the chromatographic media. Meanwhile, we groped the optimum condition for refolding by dilution. The results indicated that the column chromatography refolding method is suitable for large-scale production and the renatured product was more purer than refolding by other two methods.
The immunologic property and biologic activity of the renatured ScFv were assessed by ELISA, cytoactive and animal experiment, which showed that the refolded ScFv have same characteies of recognition and binding to human ICAM-1 as the parental mAb. In addition, the ScFv could also bind with mouse ICAM-1, which paved the way for further experiment. The ScFv against ICAM-1 can markly suppressed the MCs adhesion to LPS-stimulated EC-304 monolayers, moreover, the degree of inhibition was strengthened with the increasing of dosage. But the effectiveness of ScFv was a little weaker than McAb. In order to assess the effect of ScFv for inflammation, we established mouse model of aseprtic inflammation, from data, we saw the ScFv inhibited notablely inflamentory swelling of auricle in mice induced by dimethyl benzene.
We have successfully prepared anti-human ICAM-1 ScFv,which retained excellent biologic activity. The study establish the foundation for ICAM-1 ScFv in clinical application.
引文
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