大鼠睾丸GABA_A受体b3t拼接变异体在CHO细胞中的异源表达  

Heterologous Expression of Rat Testis GABAA Receptorb3t Variant in CHO Cells

作　　者：李世峰[1] 陈宇光[2] 严缘昌[1] 李逸平[1] 

机构地区：[1]中国科学院上海生命科学研究院生物化学与细胞生物学研究所分子细胞生物学重点实验室,上海200031 [2]上海大学生命科学学院,上海200436

出　　处：《生殖与避孕》2004年第4期200-203,231,i009,共6页Reproduction and Contraception

基　　金：国家重点基础研究发展规划项目973计划(No. G199905592);上海高校模式动物E-研究院基金资助。

摘　　要：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。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.

关 键 词：大鼠 睾丸 GABAA受体 β3t 变异体 CHO细胞 异源表达 内质网 

分 类 号：Q785[生物学—分子生物学]