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目的探讨编码含有关键模序HGK的高分子激肽原(high molecular weight kininogen,HK)第5结构区域(D5H)的野生型与突变型的蛋白质对抑制肿瘤细胞转移能力的影响。方法利用PCR介导的定点突变技术分别将D5H的模序His485-Gly-Lys487中的每个氨基酸依次被Ala替代获得3个突变体。将这些突变体构建到p GEX-4T-1的表达载体上,并在大肠杆菌中进行融合蛋白的表达,利用亲和色谱纯化这些蛋白。通过细胞黏附、侵袭实验及免疫细胞化学SP法观察野生型与突变型GST-D5H融合蛋白对肿瘤细胞黏附及侵袭能力的影响。结果对抑制肿瘤细胞黏附能力的强弱顺序为:GST-D5H>GST-D5H(G486-A)>GST-D5H(H485-A)>GST-D5H(K487-A)。结论 Lys487是在D5H模序(His485-Gly-Lys487)中发挥生物学活性的关键氨基酸。
Objective To investigate the effect of wild-type and mutant-type proteins encoding the fifth domain (D5H) of HKK, a key motif of HGK, on the inhibition of tumor cell metastasis. Methods Three mutants were obtained by PCR-mediated site-directed mutagenesis. Each amino acid in D5H motif His485-Gly-Lys487 was replaced by Ala. These mutants were constructed on the expression vector pGEX-4T-1 and the expression of the fusion protein was carried out in E. coli and the proteins were purified by affinity chromatography. The effects of wild-type and mutant GST-D5H fusion proteins on the adhesion and invasion ability of tumor cells were observed by cell adhesion, invasion assay and immunocytochemical SP method. Results The order of the inhibition of tumor cell adhesion was GST-D5H> GST-D5H (G486-A)> GST-D5H (H485-A)> GST-D5H (K487-A). Conclusion Lys487 is a key amino acid that exerts biological activity in the D5H motif (His485-Gly-Lys487).