淋巴细胞特有重组激活基因蛋白载体构建及功能鉴定

Construction and functional identification of lymphocytes specific recombination activating gene protein vector

目的:淋巴细胞特异性重组激活基因(RAG)蛋白1和2共同组成的RAG重组酶是造成淋巴细胞发育及产生抗体多样性的关键性蛋白.本研究构建并优化同时表达RAG1和RAG2的慢病毒载体,为研究RAG1和RAG2的结构和功能奠定基础.方法:构建同时表达RAG1、RAG2和绿色荧光蛋白(GFP)的载体;Western Blotting验证该载体RAG1和RAG2蛋白表达;体外重组实验证实该载体表达的RAG蛋白具有催化断裂DNA的功能.结果:构建了表达RAG重组酶的载体p WPI-RAG2-P2A-RAG1-IRES-GFP;Western Blotting验证了该载体表达RAG1和RAG2蛋白,并显示该载体RAG1和RAG2蛋白的表达量具有较高的一致性.体外重组实验证实该载体表达的RAG1和RAG2组成的重组酶具有更好的催化断裂DNA的活性.结论:优化后的p WPI-RAG2-P2A-RAG1-IRESGFP载体可以保证RAG1和RAG2同时有效表达,GFP可作为荧光标记,为后续细胞以及动物实验奠定基础.

AIM:To construct and optimize the vector expressing recombination activating gene(RAG)protein1and RAG protein2simultaneously and lay a solid foundation for studying the structure and function of RAG recombinase.METHODS:Vector expressing RAG1,RAG2and green fluorescent protein(GFP)simultaneously were constructed;The expression of RAG1and RAG2protein were identified by Western Blotting,then the function of RAG protein was detected by in vitro recombinant experiment.RESULTS:A recombinant vector expressing RAG recombinase.was successfully constructed,and Western Blotting indicated that the expression level of RAG1had a good consistency with the expression of RAG2protein in this vector.Vitro recombinant experiment suggested that pWPI-RAG2-P2A-RAGl-IRES-GFP vector has a better DNA cleavage activity,and GFP reporter gene of the optimized pWPI-RAG2-P2A-RAGl-IRES-GFP vector could be used as cell labeling.CONCLUSION:After optimization,the RAG vector can ensure the expression of RAG1and RAG2at the same level effectively,and the reporter gene GFP can be used as a fluorescent marker and lays the foundation for further animal and cell experiments.