IMP-4基因克隆、原核表达及活性测定.doc

IMP-4基因克隆、原核表达及活性测定 　【摘要】 目的:采用RT-PCR法获取TIMP-4基因，并通过原核表达获取具有生物活性的TIMP-4融合蛋白。方法:从SD大鼠心肌组织中提取总RNA，利用RT-PCR法扩增出TIMP-4基因，经基因序列分析后构建表达载体PET-28a(+)-TIMP-4，转化大肠杆菌BL21，IPTG诱导TIMP-4的表达。利用N2+-NTA纯化和复性后，根据其骨肉瘤细胞迁移能力的影响检测其活性。结果:克隆到编码序列完全正确的大鼠TIMP-4基因，能在大肠杆菌中获得高效表达，表达产物占全菌总蛋白的23.5%，N2+-NTA纯化和复性后，纯度达90%以上。纯化后的融合蛋白使骨肉瘤细胞株的迁移能力得到显著抑制。结论:通过基因克隆和原核表达的方式可以大量获得具有生物活性的TIMP-4融合蛋白。 【关键词】 TIMP-4基因 克隆 基因表达 免疫活性 Abstract: Objective:To extract the gene of TIMP-4 with RT-PCR methods and obtain the bioactive rat TIMP-4 fusion protein expressed in E. Coli. Methods: Total RNA was extracted from cardiac muscle of SD rat. TIMP-4 gene was obtained and amplified with RT-PCR method. The isolated fragment was sequenced, recombined with plasmid pET-28a(+) at EcoRI and HindⅢ sites and transformed into E.coli cells. The expression of TIMP-4 was induced by IPTG. TIMP-4 protein was purified and renaturalized by N2+-NTA. The capability of inhibiting migration of osteosarcoma cell line was used as a measure to assay its viability. Results: The TIMP-4 gene of the rat, which was cloned and correctly coded, could be highly expressed in the E.coli strain. The expressed product of the TIMP-4 amounted for 23.5% of the total bacterial protein. The TIMP-4 amounted for at least 90% after N2+-NTA purification. The purified TIMP-4 protein could significantly inhibit the migration of osteosarcoma cell line. Conclusion: TIMP-4 fusion protein with bioactivity can be obtained profusely by gene cloning and expression in E. Coli. Key words: TIMP-4; gene clone; expression; viability assay 目前对恶性肿瘤的治疗尚缺乏非常有效的措施，其主要原因在于肿瘤具有侵袭、转移和基因变异等特性，对肿瘤侵袭转移机制方面的研究成为肿瘤治疗的关键。组织基质结构的破坏和肿瘤新生血管的形成是肿瘤生长、转移得以实现的先决条件。基质金属蛋白酶（matrix metalloproteinase， MMP）为一类蛋白水解酶家族，它能降解多种胶原蛋白、纤维连接蛋白和弹性蛋白等血管基膜和基质结构，为肿瘤的生长、转移创造合适的环境。金属蛋白酶组织抑制剂（tissue inhibitor of matrix metalloproteinase，TIMP）的发现是肿瘤研究上的一大突破，它是MMP的天然抑制剂，目前根据发现的时间顺序分别称为TIMP（1～4）。国内外对于TIMP-4的功能已经有了一些研究，但结果有所差异。Jiang等[1]的研究发现，TIMP-4可以抑制乳腺癌细胞的凋亡，而更多的实验表明TIMP-4为肿瘤生长的抑制因素[2，3]。为进一步开展对TIMP-4在骨肉瘤和软骨肉瘤方面的作用及