TSEG-2基因真核表达载体的构建及其在细胞内的表达

Construction of the eukaryotic expression vector of TSEG-2 and its expression in cultured cells

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.