摘要
研制抗胃泌素释放肽(Gastrin-releasing peptide,GRP)单克隆抗体,并且初步评估其在体外对乳腺癌细胞生长的抑制作用。纯化重组的GRP融合蛋白,免疫BALB/c小鼠,取经免疫的小鼠脾细胞与小鼠骨髓细胞SP2/0进行细胞融合,对杂交瘤细胞进行筛选,阳性孔经3次有限稀释法亚克隆,获得稳定分泌GRP抗体的杂交瘤细胞株,进一步从腹水中用硫酸铵沉淀法纯化单抗,并以间接ELISA、Western免疫印迹以及亚类分型等方法进行抗体鉴定,之后在体外用MTT比色法测定该单抗抑制乳腺癌细胞EMT-6生长的作用。结果表明通过筛选获得了一株阳性杂交瘤细胞,命名为W9,这株杂交瘤细胞分泌的单抗具有效价高,特异性好的特点,其亚型为IgG1,体外的细胞实验也表明这种单克隆抗体能够显著地抑制EMT-6细胞的增殖。
In order to obtain mouse anti-human GRP monoclonal antibodies and to estimate their inhibition effect on Breast Cancer Cell EMT-6 in vitro,BALB/c mice were immunized with purified recombinant GRP protein as the first step.Then spleen cells from the immunized mice were fused with SP2/0 myeloma cells.Those fused cells were selected by HAT medium and screened by ELISA for obtaining GRP monoclonal antibodies which were identified by ELISA,Western blotting and subtype verification in the next step.Later,this inhibition effect on cancer cells growth was measured by the method of MTT chromatometry.One clone of hybirdoma,named W9,stable in secreting GRP mAbs was obtained through the experiment.This kind of antibodies showed the high specificity to GRP with strong affinity and efficient suppression on proliferation of EMT-6 cells in vitro,so the GRP mAb provided a useful reagent for the clinical diagnosis and the further research.
出处
《药物生物技术》
CAS
CSCD
2007年第3期173-177,共5页
Pharmaceutical Biotechnology
基金
国家自然科学基金(No.30500458No.30270298No.30672464)
关键词
胃泌素释放肽
单克隆抗体
乳腺癌
Gastrin-releasing peptide,Monoclonal antibody,Breast cancer
