人溶酶体酸性β-葡萄糖脑苷脂酶基因的克隆及其在COS7细胞中的表达  

Cloning of human lysosomal acid β-glucosidase gene and its expression in COS7 cells

作　　者：张艳丽[1] 许丹[1] 王子玉[1] 孟立[1] 王锋[1] 

机构地区：[1]南京农业大学动物胚胎工程技术中心,南京210095

出　　处：《生物工程学报》2009年第2期263-267,共5页Chinese Journal of Biotechnology

基　　金：国家转基因生物新品种培育重大专项(No.2008ZX08008-004);江苏省国际合作项目(No.BZ2007065)资助~~

摘　　要：从人胎盘组织提取总RNA,采用RT-PCR扩增人溶酶体酸性β-葡萄糖脑苷脂酶(Lysosomal acid β-glucosidase,GlcCerase)基因编码区的全部序列,并克隆到pMD-19T载体上,构建克隆载体pMD-GlcCerase。经测序验证后,将GlcCerase亚克隆至表达载体pEGFP-C1上,构建了人GlcCerase绿色荧光蛋白真核表达载体pEGFP-GlcCerase。采用脂质体法将其瞬时转染至COS7细胞系后,在细胞中检测到了GlcCerase基因,并在细胞裂解产物中检测到了GlcCerase生物活性的表达。GlcCerase基因的克隆及其表达,为进一步了解GlcCerase基因的功能以及利用转基因动物乳腺生物反应器高效生产GlcCerase奠定了基础。In this study, we amplified human lysosomal acid β-glucosidase （GlcCerase） gene by RT-PCR from human placenta, and analyzed the sequence of the PCR product cloned in pMD-19T. The gene identity was 99% comparable to that of the reported human GlcCerase cDNA sequence in GenBank. The GlcCerase gene digested with Xho I was subcloned into eukaryotic express vector pEGFP-C1 to generate recombinant expression vector pEGFP-GlcCerase. After identified the recombinant plasmid by restriction enzyme digestion, we transfected pEGFP-GlcCerase into COS7 cells by liposome. GlcCerase mRNA was expressed and the activity of GtcCerase was also detected in COS7 cells. This study would lay a foundation for the function of GlcCerase and its production by transgenic bioreactor.

关 键 词：人溶酶体酸性β-葡萄糖脑苷脂酶基因 克隆 真核表达载体 COS7细胞 转染 

分 类 号：Q78[生物学—分子生物学]