人卵巢癌细胞特异性结合短肽的原核表达及靶向性分析  

Prokaryotic expression and targeting analysis of ovarian cancer specific binding peptide

作　　者：钟嘉莉[1] 康佳丽[1] 廖花[1] 刘启才[2] 周聪[1] 王小霞[1] 

机构地区：[1]广州医科大学附属广州市第一人民医院妇产科,广东广州510180 [2]广州医科大学基础学院分子生物学教研室,广东广州510180

出　　处：《中华肿瘤防治杂志》2014年第22期1800-1806,共7页Chinese Journal of Cancer Prevention and Treatment

基　　金：广东省自然科学基金(S2012010009341)

摘　　要：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=0.874OBJECTIVE To synthesize human ovarian cancer specific binding peptide 1 （OSBP-1）and scrambled peptide（OSBP-S） by prokaryotic expression and analyze the binding specificity of the peptides with ovarian cancer cell HO8910. METHODS The prokaryotic expression vectors of pGEX-6P-3/OSBP-1 and pGEX-6P-3/OSBP-S were con- structed and transfected into E. coli BL21（DE3）. The expression of the GST fusion gene was induced with IPTG, and the GST fusion protein was purified by GST purification column. The GST fusion protein after purification was analyzed by SDS-PAGE and Western-blot and then cleaved by Prescission Protease enzyme. The cleaved small molecular weight peptides（OSBP-1 and OSBP-S）were identified by Tricine-SDS-PAGE and then labeled with FITC. Next, the specific bind- ing ability of OSBP-I to ovarian cancer HO8910 cell lines and whether the binding specificity was associated with a specific amino-acid sequence was analyzed by cell immunofluorescence and competition binding assay, further the affinity of OSBP-1 for other ovarian cancer cell lines,tumor cell lines from different tissue and normal ceils were assessed by affinity assay. RESULTS The prokaryotic expression vectors, pGEX-6P-3/OSBP-1 and pGEX-6P-3/OSBP-S, was constructed successfully. SDS-PAGE analysis of the GST fusion protein after purification showed single GST-OSBP-1 and GST-OSBP-S protein. The result of Western blot indicated that the fusion protein can react with anti-GST monoclonal an- tibody. After enzymatic cleavage of the GST tail,the Tricine-SDS-PAGE of small molecular weight peptides showed that we successfully obtained OSBP-1 and OSBP-S. Through high performance liquid chromatography （HPLC） and mass spec- trometry,we knew that the purity of FITC labeled OSBP-1 and OSBP-S were greater than 95 ~. Cell immunofluorescence experiments demonstrated that FITC-OSBP-1 had high binding to HO8910 cells, which can enter the cells showed strong green fluorescence, but only the individual normal ovarian epithelial cells showed green flu

关 键 词：特异性结合短肽 卵巢肿瘤 原核表达 靶向性 

分 类 号：R737.31[医药卫生—肿瘤]