CAJ | 학술논문

目的制备针对人基质金属蛋白酶抑制剂Ⅰ (TIMP-Ⅰ)融合蛋白的单克隆抗体.方法用逆转录聚合酶链反应法从纤维化的肝组织中扩增TIMP-Ⅰ编码序列,产物克隆入pQE31载体,转化大肠埃希菌BL21,诱导表达后通过6×His标签进行纯化,用纯化后的蛋白免疫BALB/c小鼠,采用常规的细胞融合技术制备单克隆抗体,并进行鉴定.结果成功构建了TIMP-Ⅰ原核表达体系,并获得了重组蛋白.筛选出4株能稳定分泌抗TIMP-Ⅰ的单克隆抗体杂交瘤细胞株,免疫球蛋白类型有3株为IgGl类.Western Blot印迹显示这些单抗能特异结合TIMP-1蛋白.结论成功构建了人基质金属蛋白酶抑制剂Ⅰ原核表达体系,并获得了单克隆抗体.
목적 제비침대인기질금속단백매억제제Ⅰ (TIMP-Ⅰ)융합단백적단극륭항체.방법 용역전록취합매련반응법종섬유화적간조직중확증TIMP-Ⅰ편마서렬,산물극륭입pQE31재체,전화대장애희균BL21,유도표체후통과6×His표첨진행순화,용순화후적단백면역BALB/c소서,채용상규적세포융합기술제비단극륭항체,병진행감정.결과 성공구건료TIMP-Ⅰ원핵표체체계,병획득료중조단백.사선출4주능은정분비항TIMP-Ⅰ적단극륭항체잡교류세포주,면역구단백류형유3주위IgGl류.Western Blot인적현시저사단항능특이결합TIMP-1단백.결론 성공구건료인기질금속단백매억제제Ⅰ원핵표체체계,병획득료단극륭항체.
Objective To prepare the monoclonal antibody (mAb) against tissue inhibitor of metalloproteinases Ⅰ (TIMP-Ⅰ)fusion protein.Methods TⅠMP-Ⅰ gene was amplified from fibrotic human liver tissue by RT-PCR,then ligated with pQE31 to form recombinant plasmid pQE-TIMP-Ⅰ and transformed into E.coli BL21.The protein induced by IPTG was purified by 6 × His-tag and used to immunize the BALB/c mice.The specific monoclonal antibodies (mAbs) were prepared by the cell fusion technique.Western Blot were used to detect specificity of mAbs.Results The prokaryotic plasmid expressing the recombinant protein was constructed,and the TIMP-Ⅰ recombinant protein was expressed and purified.Four hybridoma cell lines that secreted anti-TIMP-Ⅰ mAbs were obtained.3 of 4 mAbs were the IgG1 subtype.Western Blot indicated the mAbs showed specific combination with TIMP-Ⅰ protein.Conclusion The TIMP-Ⅰ recombinant protein is highly purified and has strong antigenicity.The anti-TIMP-Ⅰ mAbs were prepared successfully.