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目的通过原核表达的方式制备人卵巢癌HO8910细胞株特异性结合短肽(ovarian cancer specific binding peptide 1,OSBP-1)和氨基酸顺序重排的短肽(scrambled peptide,OSBP-S),探讨其对卵巢癌HO8910细胞的靶向特异性。方法构建pGEX-6P-3/OSBP-1与pGEX-6P-3/OSBP-S原核表达载体,转化大肠埃希菌BL21(DE3),进行GST融合蛋白的诱导表达和纯化,纯化后的融合蛋白经SDS-PAGE和蛋白质印迹法分析无误后,进行融合蛋白的酶切及小分子肽Tricine-SDS-PAGE电泳鉴定,N端进行FITC标记。通过细胞免疫荧光和竞争性结合实验分析OSBP-1对人卵巢癌HO8910细胞株的靶向性,以及这种靶向性是否与特定的氨基酸顺序有关;通过亲和性试验研究OSBP-1与其他卵巢癌细胞、不同组织来源的肿瘤细胞及正常细胞的亲和性。结果成功构建了pGEX-6P-3/OSBP-1与pGEX-6P-3/OSBP-S原核表达载体;纯化后的融合蛋白经SDS-PAGE分析显示,出现单一的GST-OSBP-1和GST-OSBP-S蛋白条带;蛋白质印迹法分析证明,融合蛋白能被GST单克隆抗体识别;小分子肽Tricine-SDS-PAGE表明,成功获得OSBP-1和OSBP-S;标记后的FITC-OSBP-1与FITC-OSBP-S经高效液相色谱技术和质谱分析,纯度均>95%。细胞免疫荧光实验显示,FITC-OSBP-1对HO8910细胞有很强的结合能力,能进入细胞内显示很强的绿色荧光,但其与正常卵巢上皮细胞仅个别细胞显示绿光荧光,而FITC-OSBP-S与HO8910细胞和正常卵巢上皮细胞均显示很弱的绿色荧光。另外,随着FITC-OSBP-1浓度的增加,HO8910细胞的荧光强度增强;竞争性结合实验显示,FITC-OSBP-1与HO8910细胞的结合能力随着OSBP-1浓度的增加而下降,平均荧光强度(mean fluorescent intensity,MFI)递减,分别为0.140±0.002 1、0.062±0.001 5、0.027±0.002 0和0.009±0.001 5,差异有统计学意义(F=3 111,P<0.001),而加入不同浓度OSBP-S的MFI无明显变化,分别为0.142±0.004 2、0.142±0.005 7、0.139±0.002 5和0.138±0.001 5,差异无统计学意义,F=0.874,P=0.494;亲和性试验结果显示,FITC-OSBP-1与人卵巢癌HO8910细胞的MFI为0.157 4±0.001 1,明显大于FITC-OSBP-S阴性对照组0.006 2±0.000 5(t=219,P<0.001),与正常卵巢上皮细胞的MFI0.002±0.000 3,明显小于FITC-OSBP-S阴性对照组0.006 3±0.000 5(t=13.462,P<0.001),而与其他卵巢癌细胞OVCAR3、SKOV3、不同组织来源的肿瘤细胞LOVO、HepG2及正常细胞293T的MFI与FITC-OSBP-S阴性对照组分别比较,差异均无统计学意义,P>0.05。结论通过原核表达的方式成功制备OSBP-1和OSBP-S,证实OSBP-1对卵巢癌HO8910细胞具有浓度依赖性和氨基酸序列特异性靶向结合能力,且OSBP-1对正常卵巢上皮细胞几乎没有亲和力,对其他肿瘤细胞及正常细胞293T仅有很弱的亲和力,为卵巢癌的靶向治疗提供了理想的载体。
Objective To prepare ovarian cancer specific binding peptide 1 (OSBP-1) and scrambled peptide (OSBP-S) of human ovarian cancer cell HO8910 by prokaryotic expression, Targeting Specificity of Ovarian Cancer HO8910 Cells. Methods The prokaryotic expression vectors pGEX-6P-3 / OSBP-1 and pGEX-6P-3 / OSBP-S were constructed and transformed into Escherichia coli BL21 (DE3) for expression and purification of the GST fusion protein. The purified fusion protein After analysis by SDS-PAGE and Western blotting, the fusion protein was digested with Tricine-SDS-PAGE and identified by FITC on the N-terminus. The targeting of OSBP-1 to human ovarian cancer HO8910 cell line was analyzed by immunofluorescence and competitive binding assay, and whether this targeting was related to specific amino acid sequence. The affinity of OSBP-1 and other Ovarian cancer cells, tumor cells from different tissues and normal cells. Results The prokaryotic expression vectors pGEX-6P-3 / OSBP-1 and pGEX-6P-3 / OSBP-S were constructed successfully. SDS-PAGE analysis of the purified fusion protein showed that single GST-OSBP- OSBP-S protein bands. Western blot analysis showed that the fusion protein was recognized by GST monoclonal antibody. The small molecule peptide Tricine-SDS-PAGE showed that OSBP-1 and OSBP-S were successfully obtained. The labeled FITC-OSBP- 1 and FITC-OSBP-S were> 95% pure by HPLC and mass spectrometry. Immunofluorescence experiments showed that FITC-OSBP-1 had a strong binding ability to HO8910 cells and could enter the cells to show strong green fluorescence, but it showed green fluorescence only with individual cells of normal ovarian epithelial cells, whereas FITC- OSBP-S and HO8910 cells and normal ovarian epithelial cells showed weak green fluorescence. In addition, as the concentration of FITC-OSBP-1 increased, the fluorescence intensity of HO8910 cells increased; competitive binding assay showed that the binding capacity of FITC-OSBP-1 and HO8910 cells decreased with the increase of OSBP-1 concentration, the average fluorescence (MFI) decreased by 0.140 ± 0.002, 1.062 ± 0.001 5, 0.027 ± 0.002 0 and 0.009 ± 0.001 5, respectively, the difference was statistically significant (F = 3 111, P <0.001) There was no significant difference in the MFI of OSBP-S at different concentrations (0.142 ± 0.004, 2.0142 ± 0.005 7, 0.139 ± 0.0025 and 0.138 ± 0.001 5 respectively), F = 0.874, P = 0.494; The results showed that the MFI of FITC-OSBP-1 and human ovarian cancer HO8910 cells was 0.157 4 ± 0.001 1, which was significantly higher than that of FITC-OSBP-S negative control group 0.006 2 ± 0.000 5 (t = 219, P <0.001) The MFI of 0.002 ± 0.000 3 in normal ovarian epithelial cells was significantly lower than that in FITC-OSBP-S negative control group (0.006 3 ± 0.000 5, t = 13.462, P <0.001), but not with other ovarian cancer cell lines such as OVCAR3, SKOV3 Of the tumor cells LOVO, HepG2 and normal 293T MFI and FITC-OSBP-S negative control group respectively, the difference was not statistically significant, P> 0.05. CONCLUSIONS: OSBP-1 and OSBP-S were successfully prepared by prokaryotic expression. OSBP-1 was proved to have a concentration-dependent and amino acid sequence-specific binding ability to ovarian cancer cell line HO8910 and OSBP-1 had almost no effect on normal ovarian epithelial cells Affinity for other tumor cells and normal cells 293T only weak affinity for the targeted treatment of ovarian cancer provides an ideal carrier.