In this study, norcanthridin （NCTD）-encapsulated liposomes were modified with a novel murine anti-human CD19 monoclonal antibody 2E8 （2E8-NCTD-liposomes） and the targeting efficiency and specific cytotoxicity of 2E8-NCTD-liposomes to CD19^＋ leukemia cells were evaluated. BALB/c mice were injected with 2E8 hybridoma cells to obtain 2E8 monoclonal antibody （mAb）. NCTD-liposomes were prepared by using film dispersion method. 2E8 mAbs were linked to NCTD-liposomes using post-incorporation technology. Flow cytometry showed that the targeting efficiency of purified 2E8 mAbs on CD19＋ Nalm-6 cells was 99.93%. The purified 2E8 mAbs were conjugated with NCTD-liposomes to prepare 2E8-NCTD-liposomes whose targeting efficiency on CD19^＋ Nalm-6 was also 95.82%. The average size of 2E8-NCTD-liposomes was 118.32 nm in diameter. HPLC showed that the encapsulation efficiency of NCTD was 46.51%. When the molar ratio of 2E8/Mal-PEG2000-DSPE reached 1：50, we obtained the liposomes with 9 2E8 molecules per liposome. The targeting efficiency of 2E8-NCTD-liposomes on CD19^＋ leukemia cells was significantly higher than that on CD19-1eukemia cells. Similarly, the targeting efficiency of the immtmoliposomes was also higher than that of the NCTDAiposomes on CD 19^＋ leukemia cells. Those results were consistent with those observed by laser scanning confocal microscopy. 3-（4, 5-Dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay demonstrated that 2E8-NCTD-liposomes specifically killed Nalm-6 cells in a dose- and time-dependent manner. The viability of Nalm-6 cells treated by 2E8-NCTD-liposomes was significantly lower than that of Molt-3 cells and it was also significantly lower than that of Nahn-6 cells treated with the same concentration of NCTD-liposomes or free NCTD. We are led to concluded that 2E8 antigen can serve as a specific targeting molecule of B lineage hematopoietic malignancies for liposome targeting, and 2E8-NCTD-liposomes can be used as a new and effective means for the treatment o
目的:构建ZCH-7-2F9(简称2F9)单链抗体(ScFv2F9)原核表达载体,获得活性目的蛋白,为2F9免疫毒素及其它类型的基因工程抗体的研究奠定基础。方法:根据VH2F9、VL2F9、(G4S)3基因序列以及pIVEX2.3-MCS空载体多克隆位点内切酶位点NdeI和SmaI的核酸序列设计引物,采用高保真的Taq酶,通过重叠延伸拚接法(splicing by overlap extension,SOE)扩增克隆到ScFv2F9基因,T-A克隆、测序核实后克隆到pIVEX2.3-MCS空载体中,阳性重组质粒转化大肠杆菌菌株E.coli BL21star(DE3)plysS,IPTG诱导目的蛋白表达,镍树脂纯化后体外复性浓缩,流式细胞术观察其识别和结合CD14的能力。结果:成功构建ScFv2F9的原核表达载体pIVEX2.3-MCS/ScFv2F9;对大肠杆菌的包涵体进行纯化复性,其复性率为32.6%;通过流式细胞术观察到复性ScFv2F9对亲本单抗2F9-FITC有部分的阻滞效果,复性ScFv2F9阻滞1次后阳性细胞百分数、平均荧光强度(MFI)和高峰频道(peakCh)分别下降了11.73%、11.96%和31.57%,阻滞2次后分别下降了26.44%、21.75%和42.11%。结论:ScFv2F9在原核细胞中获得成功表达,复性后目的蛋白对人CD14有一定的识别和结合能力,为2F9单抗的进一步改造打下了坚实的基础。
