EMMPRIN糖基化突变质粒的构建及功能检测

摘要

Objective To construct in the extracellular matrix metalloproteinase inducer (EMMPRIN)glycosylation single point mutation plasmid,and to explore its relationship with tumor cell proliferation.Methods PCR point mutantion technology was used to construct the mutantion plasimid of EMMPRIN glycosylation single point.After successful mutation, the function of mutantion plasmids were detected. Western blotting was used to detect the expression of EMMPRIN protein,immunofluorescence method was used to detemine the morphological changes of the cells,and MTT assay was performed to detect the relationship between mutantion pasmid and tumor cell proliferation.Results Confirmed by restriction enzyme digestion and sequencing,the 44th,the 152th,and the 186th Asn were successfully mutated to Gln in the sequence of EMMPRIN;EMMPRIN/GFP (N44Q), EMMPRIN/GFP(N152Q),and EMMPRIN/ GFP (N186Q)glycosylation single point mutation plasmids were constructed.Compared with wild-type,thel morphology of the cells was significantly changed,the core division of mutant-type cells was significantly reduced, the number of filopodia was reduced. The results of MTT assay showed that the survival rate of the cells in wild-type group were significantly increased compared with control group (P<0.05 );the survival rates of the cells in EMMPRIN (N44Q) group, EMMPRIN (N152Q) group and EMMPRIN(N186Q)were significantly decreased compared with wild-type group(P<0.05).Conclusion Mutant-type EMMPRIN can inhibit the proliferation of tumor cells;with the duration increasing, the inhibitory effect is weakened.There is a correlation between EMMPRIN glycosylation and proliferation of tumor cells.%目的：构建细胞外基质金属蛋白酶诱导因子（EMMPRIN）糖基化位点突变质粒，探讨其与肿瘤细胞增殖的关系。方法：利用PCR定点诱变技术构建 EMMPRIN糖基化位点突变质粒。突变成功后，对突变质粒进行功能检测，Western blotting 法检测 EMMPRIN蛋白表达；免疫荧光法检测细胞形态学变化；MTT法检测突变质粒与肿瘤细胞增殖的关系。结果：酶切鉴定和测序证实，将 EMMPRIN序列中第44、152和186位的天冬酰胺突变成谷氨酰胺，成功构建 EMMPRIN／GFP（N44Q）、EMMPRIN／GFP（N152Q）和 EMMPRIN／GFP（N186Q）糖基化单点突变质粒；糖基化位点突变后，突变型细胞核分裂相显著减少，伪足数减少；MTT法检测，与对照组比较，野生型组细胞存活率明显升高（P＜0.05）；而 EMMPRIN 糖基化位点突变后，与野生型比较， EMMPRIN（N44Q）、EMMPRIN （N152Q）和 EMMPRIN （N186Q）细胞存活率均明显降低（P ＜0.05）。结论：EMMPRIN突变型能抑制肿瘤细胞的增殖，且随作用时间增加，抑制作用减弱；EMMPRIN糖基化修饰与肿瘤细胞的增殖有关联。