摘要
目的在体外原核表达基质金属蛋白酶-2(MMP-2)纤维连接蛋白样片段,制备抗MMP-2特异性的单克隆抗体。方法体外掊养EAhy926内皮细胞,收集细胞后抽提总RNA,利用所合成引物经RT-PCR获得纤维连接蛋白样片段cDNA,与PET20b(+)表达载体重组后转化大肠杆菌BL21(DE3)plus,挑选阳性克隆后,经IPTG诱导表达蛋白。蛋白经次氮基三乙酸镍琼脂糖(Ni—NTAagarose)柱纯化浓缩后免疫Balb/c小鼠,尾静脉加强后取脾细胞融合,ELISA法筛选阳性克隆,制备腹水。结果原核表达MMP-2纤维连接蛋白样片段大小为21kd,表达量占菌体总蛋白25%,经Ni-NTAagarose柱纯化后,获得了部分纯化的蛋白。小鼠脾细胞与杂交瘤细胞融合后筛选获得1株单克隆抗体。结论抗MMP-2特异性抗体的获得为MMP-2测定提供了一个有效工具,为进一步研究MMP-2的功能及探讨MMP-2在实体瘤及血液肿瘤发病过程中的作用奠定了基础。
Objective To express the fibronectin-like domain of matrix metalloproteinase-2(MMP-2)(MFD)in vitro, and prepare the monoclonal antibody to MMP-2. Methods The RNA of EAhy926 cells was obtained after being dealt with Trizol and the cDNA of fibronectin-like domain was amplified by RT-PCR. After sequence analysis, the amplified DNA fragment was inserted into expression vector with 6xhis Tag-PET20b(+) .The recombinant expression vector was then transformed into E. coli BL21(strain DE3) plus and induced by IPTG. Before used to immunize Balb/c mouse, the recombinant protein was purified by chromatography on Ni-NTA agarose column and renatured by Tris-HCl buffer containing GSH and GSSG. Results The protein peptide of MMP-2 is 2.1 kd. The expression protein accounted for 25% of total bacterial protein. Purefied protein was obtained after chromatographyon Ni-NTA agarose. After the spleen cells of the mouse and hybridoma cells SP2/0 were hybridized, we characterized a piece of McAb. Conclusion It will afford a useful tool for testing MMP-2 and help to study the function of MMP-2 in tumor.
出处
《苏州大学学报（医学版）》
CAS
北大核心
2005年第1期27-30,62,共5页
Suzhou University Journal of Medical Science
基金
苏州大学青年教师研究基金资助项目(项目编号Q3134405)
