摘要
目的:克隆和表达中国人金属基质蛋白酶组织抑制因子-1(tissue inhibitor of metalloproteinases-1;TIMP-1)基因,获得具有抗原性的人TIMP-1蛋白。方法:用RT-nest-PCR扩增TIMP-1编码区基因片段,用基因重组技术构建含该片段的重组质粒并进行序列分析。在大肠杆菌E.coli中表达融合蛋白MBP-TIMP-1,用SDS-PAGE和Western-blot对重组蛋白进行分析鉴定,并用亲和层析试剂盒纯化融合蛋白MBP-TIMP-1。结果:经核苷酸序列分析表明,本研究克隆的中国人TIMP-1为624bp,与报道的国外TIMP-1基因序列同源。经SDS-PAGE和Western blot表明,表达的融合蛋白NBP-TIMP-1分子质量为66Ku,具有TIMP-1的抗原性,并可进行亲和层析纯化。结论:克隆了中国人TIMP-1基因,表达和纯化了具有免疫原性的融合蛋白MBP-TIMP-1,他将对肝纤维化的诊断有一定作用。
AIM: To clone the Chinese gene of TIMP-1 and express the fusion protein MBP-TIMP-1. METHODS: The fragment of TIMP-1 gene from the liver tissue of indigenous Hunan residents was amplified by RT-nest-PCR and it was cloned, sequenced by gene recombinations techniques. Fusion protein MBP-TIMP-1 was expressed in Ecoli, analysed by SDS-PAGE and Western blot and purified by affinity chromatography. RESULTS: The sequence of cloned Chinese TIMP-1 gene was 624 bp. It is homologous with the reported other human TIMP-1 gene sequence. Fusion protein MBP-TIMP-1 was 66 kd and is of the antigenicity of TIMP-1. The MBP-TIMP-1 was purified by affinity chromatography. CONCLUSION: The Chinese gene of TIMP-1 was cloned and MBP-TIMP-1 was expressed aod purified successfully in vitro. It may be useful to the diagnosis of liver fibrosis.
出处
《世界华人消化杂志》
CAS
2003年第8期1164-1167,共4页
World Chinese Journal of Digestology
基金
卫生部科研基金课题
No.98-1-120
