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目的运用单克隆抗体技术制备抗人Toll样受体4(TLR4)单克隆抗体(mhTLR4抗体),并鉴定其生物学活性。方法以重组人TLR4(rhTLR4)作为抗原,腹腔注射免疫Balb/c小鼠。分离小鼠脾脏B淋巴细胞与骨髓瘤细胞融合并培养,挑选阳性杂交瘤细胞,扩大培养,制备和鉴定mhTLR4其生物学效应。结果经3次免疫后的小鼠血清中抗rhTLR4抗体的效价具有较高水平的表达。1∶500的小鼠免疫血清可明显抑制人PBMC的TNF-α表达。分离小鼠脾细胞与骨髓瘤细胞进行融合,获得3株阳性单克隆杂交瘤细胞。其中2株经过增殖培养、纯化浓缩制备出较高产量的鼠抗人TLR4单克隆抗体。生物学活性测定表明,产生的抗rhTLR4单克隆抗体,能够明显地阻断脂多糖(LPS)诱导的人PBMC细胞对TNF-α的表达。结论本文制备的鼠抗人TLR4单克隆抗体,经鉴定具有较高的阻断内毒素信号传导的效应。为进一步研制人源化抗TLR4抗体、研制新一代抗内毒素靶向药物奠定了基础。
Objective To prepare anti-human Toll-like receptor 4 (TLR4) monoclonal antibody (mhTLR4 antibody) using monoclonal antibody technology and identify its biological activity. Methods Balb / c mice were immunized intraperitoneally with recombinant human TLR4 (rhTLR4) as antigen. The splenic B lymphocytes of mice were fused with myeloma cells and cultured. The positive hybridoma cells were selected and expanded for culture. The biological effects of mTLR4 were identified. Results The titer of the anti-rhTLR4 antibody in mouse sera after 3 immunizations showed a higher level of expression. Mouse immune serum at 1: 500 significantly inhibited TNF-α expression in human PBMCs. Isolated mouse spleen cells were fused with myeloma cells to obtain 3 positive monoclonal hybridoma cells. Two of them were proliferated and purified to obtain a higher yield of mouse anti-human TLR4 monoclonal antibody. The biological activity assay showed that the produced anti-rhTLR4 monoclonal antibody could obviously block the lipopolysaccharide (LPS) induced human PBMC cells to TNF-α expression. Conclusions The mouse anti-human TLR4 monoclonal antibody prepared in this paper has been identified to have a higher effect of blocking endotoxin signaling. To further develop humanized anti-TLR4 antibody, the development of a new generation of anti-endotoxin targeted drugs laid the foundation.