谷氨酸转运体GLT-1绿色荧光蛋白表达载体的构建及功能分析

北京理工大学珠海学院2020届本科生毕业论文Construction and function analysis of glutamate transporter GLT-1gene expression vector with green fluorescent proteinAbstractObjective:GLT 1(Glutamate transporter-1,with a gene name called solute carrier familymember 2,SLC/A2),is a vital membrane transporter,mediates about 90%glutamate transport.However,as an important excitatory neurotransmitter in the brain,if glutamate is nottransported by GLT-1 in time and has enriched in the synaptic gap,it will cause excitotoxiceffects.Therefore,normal expression and function of GLT-I are significant for maintainingregular physiological functions of the cerebrum.Centering on the relationship between theexpression of GLT-1 and its transport function,this study is aimed to analyze whether theexpression of GLT-1 affects its function.Methods:To study the relevance between theexpression level of GLT-1 and its transport function,the recombinant expression vectorpcDNA3.1-EGFP2-SLC1A2 was constructed by molecular cloning.After amplification ofrecombinant vector and transfection into HeLa,its expression level and transport function weredetected by confocal microscope and activity experiments under the time and the quality ofplasmid gradient respectively.Results:HeLa cells were transfected with pcDNA3.1-EGFP2-SLC1A2 recombinant vector,the expression level was the highest at 18h,and the expressionlevel dropped by degrees with the time extension.Also,the expressed protein had the transportactivity,which was the highest at 18h,and the transport activity gradually decreased with thetime extension.The expression and transport activity gradually decreased with the reduction ofthe quality of the transfection recombinant vector.Conclusion:the results suggest thatpcDNA3.1-EGFP2-SLC1A2 recombinant vector can be expressed in HeLa cells,and theexpressed protein has transport function,and the transport activity is correlated with theexpression amount.Key Words:pcDNA 3.1-EGFP2-SLC1A2;molecular cloning;glutamic acid;GLT-1