摘要
目的构建睾丸特异表达基因2(TSEG-2)的真核表达载体,探讨TSEG-2存细胞中的表达及其生物学功能。方法以小鼠睾丸组织cDNA为模版,设计携带HindⅢ和BamHI酶切位点的引物序列,聚合酶链反应(PCR)扩增基因片段,克隆至携带增强型绿色荧光蛋白(EGFP)的真核表达载体pEGFP—N1;在阳离子聚合物聚乙烯亚胺(PEI)的介导下,重组质粒转染体外培养的精母细胞株GC-2spd48h后,荧光显微镜下观察TSEG-2基因在细胞内的表达定位,噻唑蓝(MTT)比色法测定细胞生长活性,AO/EB荧光染色法、Hoechst33258荧光染色法、AnnexinV—FITC/PI双染流式细胞术检测细胞凋亡,实时定量PCR测定Fas、bcl-2、bax表达水平。结果成功构建了TSEG-2与EGFP的融合表达载体pEGFP—TSEG2,转染GC-2spd细胞后可见胞质内绿色荧光蛋白表达。转染pEGFP—TSEG248h后,GC-2spd细胞生长抑制39.2%(P〈0.05),出现细胞凋亡形态学改变,凋亡率为28.3%(P〈0.05);GC-2spd细胞中Fas和bcl-2的表达分别下调17.9%、64.8%(P〈0.05),而bax的表达上调25.1%(P〈0.05)。结论成功构建TSEG-2基因的真核表达载体,能在精母细胞株中表达并促进细胞凋亡。
Objective To construct the eukaryotic expression vector of Testis specific expressed gene 2 (TSEG-2) and explore its expression and function in cultured cells. Methods TSEG-2 gene fragment with restrictive sites Hind Ⅲ and BamH Ⅰwas cloned from mouse testis cDNA by reverse transcription-polymerase chain reaction (RT-PCR) , and inserted into eukaryotie expression vector pEGFP-N1. Under the induction of polyethylenimine, the recombinant vector was transfected into cultured spermatocyte GC-2spd cells. The expression of TSEG-2 was observed under a fluroseeent microscopy. The viabilities of GC-2spd cells were observed by MTT assay. Cell apoptosis was assayed by AO/EB fluorescent staining, Hoechst 33258 fluorescent staining, and Annexin V-FITC/propidium iodide staining flow cytometry. The mRNA expression levels of Fas, bcl-2 and bax were detected by real-time PCR. Results The fusion expression vector pEGFP-TSEG2 was constructed. Forty-eight h post-transfetion, fusion protein expression was observed in the cytoplasm of cultured cells. Transfeetion of TSEG-2 into GC-2spd cells resulted in the decrease of cell viabilities by 39.2% ( P 〈 0.05 ) , and obvious morphological changes of apoptosis. The apoptosis rate was 28.3% ( P 〈 0.05 ). The expression of Fas and bel-2 was downregulated by 17.9% and 64.8% (P 〈 0.05 ), respectively, while that of bax was increased by 25.1% (P 〈0.05 ). Conclusion The eukaryotic expression vector of TSEG-2 was successfully constructed, and can be expressed in cultured spermatocytes and promote apoptosis.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2010年第3期380-382,共3页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金资助项目(30200284,30600278,30772359)
教育部新世纪优秀人才支持计划资助项目(NCET-06-0641)
教育部留学回国人员科研启动基金资助项目(2008889)
关键词
睾丸特异表达基因2
真核表达载体
基因表达
脱噬作用
Testis specific expressed gene 2
Eukryotic expression vector
Gene expression
Apoptosis