GIT1-WT及GIT1-Y293F慢病毒载体的构建及其对成骨细胞迁移能力的影响

Construction of GIT1-WT and GIT1-Y293F lentivirus vectors and investigation the role of GIT1-Y293F in osteoblasts migration

目的:设计构建G蛋白偶联受体激酶结合蛋白1(G protein coupled receptor kinase interacting protein 1,GIT1)野生型(WT)及点突变型(Y293F)慢病毒载体,探讨293位点对GIT1功能的影响。方法:用PCR从小鼠cDNA文库中扩增GIT1-WT,将其连接到慢病毒载体PLJM-GFP中,构建质粒PLJM-GFP-GIT1-WT,测序鉴定。利用TaKaRa MutanBEST Kit试剂盒,对PLJM-GFP-GIT1-WT进行定点突变,构建质粒PLJM-GFP-GIT1-Y293F,测序鉴定。重组慢病毒载体转染包装细胞293T,获取病毒上清感染培养至第4代的小鼠成骨细胞。划痕愈合试验检测成骨细胞迁移能力的变化。结果:通过PCR鉴定、双酶切鉴定及测序鉴定,成功构建了PLJM-GFP-GIT1-WT与PLJM-GFP-GIT1-Y293F。划痕愈合试验观察,与PLJM-GFP-GIT1-WT相比,PLJM-GFP-GIT1-Y293F明显抑制成骨细胞迁移。结论:GIT1功能的正常发挥有赖于293位点适时的磷酸化。

Objective：To construct GIT1-WT（full-length avian GIT1）and GIT1-Y293F lentivirus vectors and to investigate the role of 293 site in GIT1. Methods：The GIT1-WT gene was obtained by polymerase chain traction（PCR）from mouse gene bank. The PCR products were inserted into PLJM-GFP plasmid. The PLJM-GFP-GIT1-Y293F was obtained by TaKaRa MutanBEST Kit. Both PLJM- GFP-GIT1-WT and PLJM-GFP-GIT1-Y293F were evaluated by PCR and sequencing. The virus obtained from transfected 293T cells was infected into osteoblasts. The role of GIT1-Y293F in ostroblasts migration was determined by wound healing assay. Results：The PLJM-GFP-GIT1-WT and PLJM-GFP-GIT1-Y293F were constructed successfully. The results of wound healing assay showed that GIT1-Y293F lentivirus vectors significantly inhibited osteoblast migration compared with GIT1-WT. Conclusion：The functions of GIT1 dependent on the phosphorylation of 293 sites.