摘要
目的 :构建能在人舌癌细胞中高效表达Bax蛋白的bax嵌合基因。方法 :将RT PCR获得bax基因编码序列克隆至原核表达载体pGEX 5T ,再将baxcDNA插入人α1(Ⅰ )胶原基因顺式作用元件 (CIP)与报告基因CAT之间 ,构成 pSV CIP bax CAT嵌合基因 ;用阳离子脂质体DOSPER将此嵌合基因转染至人舌鳞癌细胞Tca8113;用免疫狭缝印迹和ELISA方法分析报告基因CAT和目的基因bax在细胞中的表达。结果 :bax基因编码序列及嵌合基因限制性酶谱正确 ;CAT表达的ELISA检测证实 ,转染是成功的 ,且CIP在Tca8113细胞中有促进其下游基因表达的作用 (为对照组的 15倍 ) ;免疫狭缝印迹和ELISA证实 ,转染细胞能高效表达Bax蛋白 (为对照组的 8倍 )。结论 :bax嵌合基因 pSV CIP bax CAT能在人舌癌细胞中高效表达Bax蛋白 ,为进一步研究bax基因对舌癌细胞生长与凋亡的影响奠定了基础。
Purpose:To construct a bax chimeric gene that can be expressed effectively in human squamous lingual carcinoma cell line.Methods:The coding sequence of bax gene was amplified from total RNA of human brain tissue by RT-PCR and cloned into the prokaryotic expression vector pGEX-5T. The cloned bax cDNA then was inserted between the regulatory sequences of human α1(I) collagen (CIP) and reporter gene (CAT) to construct a chimeric gene pSV-CIP-bax-CAT. Human squamous lingual carcinoma cell Tca8113 was transfected with this chimeric gene by using polyionic liposome DOSPER. The expression of bax and CAT genes was detected by immuno-slot blot and ELISA.Results:The coding sequence of bax and pSV-CIP-bax-CAT gene was characterized by restriction analysis. The results of immuno-slot blot showed that expression of CAT in the transfected carcinoma cells was remarkably stimulated by the CIP (pSV-CAT: 0.011 μg/g protein, pSV-CIP-CAT: 0.166 μg/g protein). It was confirmed by immuno-slot blot and ELISA that effective expression of Bax protein in the transfected carcinoma cells was mediated by pSV-CIP-bax-CAT.Conclusions:By transfecting bax chimeric gene, pSV-CIP-bax-CAT, Bax protein can be effectively expressed in the transfected carcinoma cells. It suggests that pSV-CIP-bax-CAT is a useful vector for studying bax gene inducing apoptosis of carcinoma cell.
出处
《中国癌症杂志》
CAS
CSCD
2002年第1期1-4,共4页
China Oncology
基金
国家自然科学基金 (No .39370 742 )
