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目的 构建人基质金属蛋白酶 1(MMP 1)基因重组质粒的克隆 ,获得具有抗原性的人MMP 1融合蛋白。方法 从人肝组织提取总RNA ,以此为模板 ,逆转录巢式PCR扩增MMP 1全编码区基因片段 ,构建含目的片段的T载体克隆及原核表达载体 pMAL c2x重组质粒亚克隆 ,经异丙基 β D半乳糖苷酶 (IPTG)诱导表达MMP 1重组质粒菌 ,用十二烷基硫酸钠聚丙烯酰胺凝胶电泳 (SDS PAGE)和Western blot对重组蛋白进行分析鉴定。结果 经核苷酸序列测定和免疫印迹鉴定 ,成功地构建了人MMP 1重组质粒基因工程菌 ,并表达具有MMP 1抗原性的融合蛋白。结论 构建了人MMP 1重组质粒克隆 ,获得了具有抗原性的MMP 1融合蛋白 ,这将有助于今后制备MMP 1多克隆抗体。
Objective To construct a clone of recombinant plasmid of human matrix metalloproteinase 1 (MMP 1) gene and obtain human MMP 1 fusion protein with antigenicity. Methods Total RNA was extracted from human liver tissue and used as a template to amplify the full-length MMP-1 gene fragment by reverse transcription-nested PCR. The T vector clone containing the target fragment and the subclone of the recombinant plasmid pMAL c2x were constructed. The recombinant plasmids were induced by propyl β D galactosidase (IPTG). The recombinant proteins were identified by SDS PAGE and Western blot. Results The human MMP-1 recombinant plasmid genetically engineered bacteria were successfully constructed and identified by nucleotide sequencing and immunoblotting. The fusion protein with MMP1 antigen was expressed. Conclusion The recombinant human MMP 1 plasmid was constructed and the antigenic MMP 1 fusion protein was obtained. This will help to prepare the polyclonal antibody against MMP 1 in the future.