摘要
构建了透明质酸合成酶2真核表达载体,并观察其在真核细胞中的表达。利用RT-PCR法从MG63细胞总RNA中扩增,获得人透明质酸合成酶2cDNA,并克隆至真核表达载体pEGFP-N3,将经双酶切鉴定和DNA测序证实的阳性重组子命名为pEGFP-N3-HAS2。脂质体法转染HEK293细胞,用倒置荧光显微镜检测、分析其在HEK293细胞表达及定位。双酶切鉴定和DNA测序证实成功构建pEGFP-N3-HAS2真核表达载体。转染HEK293细胞后可见HAS2-EGFP融合蛋白高表达并定位于浆膜。该载体的成功构建为进一步探讨透明质酸合成酶2在治疗骨性关节炎、修复关节软骨缺损中的应用价值奠定了基础。
Eukaryotic expression vector of pEGFP-N3-HAS2 is constructed and its expression in HEK293 cells is investigated. Hyaluronan synthase-2 cDNA was obtained by RT-PCR using total RNA from MG63 cells as template and further cloned into pEGFP-N3. After confirmed by restriction endonuclease digest analysis and DNA sequencing, the positive recombinant was named as pEGFP-N3-HAS2. Then the recombinant vector was transfected into HEK293 cells by liposome and the expression of the fusion protein HAS2-EGFP was identified by fluorescent microscope. Restriction enzyme digest analysis and DNA sequencing confirmed that recombinant plasmid was successfully construeted. The fusion protein was shown to be mainly located in plasma membrane by fluorescent microscope. The fusion protein is highly expressed in HEK293 cells and mainly located in plasma membrane.
出处
《科学技术与工程》
2007年第8期1573-1576,共4页
Science Technology and Engineering
