大鼠睾丸GABA_A受体b3t拼接变异体在CHO细胞中的异源表达

目的:研究大鼠睾丸GABAA受体b 3t拼接变异体特有5端的特性以及可能的细胞内定位作用。方法:将b 3t拼接变异体的全长cDNA克隆到真核表达载体pEGFP-N1和pEGFP-C1中,用磷酸钙共沉淀方法转染CHO细胞,表达后用荧光显微镜和激光共聚焦显微镜观察分析,并用ConA-Texas-Red标记转染后的细胞,进一步研究其在胞内的定位分布情况。结果:大鼠睾丸GABAA受体b 3t拼接变异体在CHO细胞中表达时呈现两种表达类型,但均不能定位到细胞膜上,而大鼠脑b 3亚基能定位分布到细胞膜上。ConA标记的结果显示b 3t拼接变异体滞留在内质网上。结论:大鼠睾丸GABAA受体b 3t拼接变异体N端的25 aa序列中可能存在内质网滞留信号。

Heterologous Expression of Rat Testis GABAA Receptorb3t Variant in CHO Cells

Objective: To study the character and possible retention function of specific sequence in the5'end of rat testis GABAA receptor b 3t variant. Methods: Rat testis GABAA receptor b 3t variant cDNA was clonedand inserted into two eukaryotic expression vectors of pEGFP-N1 and pEGFP-C1 respectively, which have EGFPreporter gene. The recombinant plasmids were transfected into CHO cells with calcium phosphate co-precipitationmethod. Fluorescence microscope and laser confocal microscope were used to analyze the transfected cells. ConA-Texas-Red was used to label cell ER and study the located area of rat testis b 3t variant in CHO cells. Results: Whenrat testis b 3t variant express in CHO cells, there were two expression pattern, the even and concentrated expressionpattern. Although rat brain b 3 can reach cell membrane, the rat testis b 3t variant cant target to the cell membrane.It may retain in the ER of CHO cells. Conclusion: There may exist an ER retention signal in the 25 specific aminoacid sequence in the N terminal of rat testis b 3t variant.