摘要
目的 构建大连蛇岛蝮蛇毒类凝血酶(Gloshedobin,GSB)基因真核表达载体,试图借助IL-2的分泌通路,使GSB分泌至胞外而达到溶栓和防栓的目的。方法 应用PCR重组技术,将GSB的5’端和3’端分别与IL-2基因的信号肽和poly A尾相连接,将此重组基因克隆入逆转录病毒载体 pLNCX,以脂质体介导转染人外用血淋巴细胞,并以绿色荧光蛋白(GFP)基因作为报告基因检测转染效率、确定DNA和脂质体的比例。转染24h后从mR-NA、DNA和蛋白质水平检测GSB的表达情况。结果 mRNA和DNA水平均有外源基因GSB的表达;SDS-PAGE显示在相对分子质量 30 000左右出现一条带,且转染上清具有精氨酸酯酶活性。结论 构建的真核表达载体可表达具有活性的GSB,为GSB基因治疗血栓的体内研究打下基础。
Objective To construct an eukaryotic expression vector for Gloshedobin (GSB) and dissolve or prevent the formation of thrombus by promoting the secretion of GSB to the extracellular domain through the secrecting path of IL-2. Methods Link the 5' and 3' terminals of GSB gene to the signal peptide and ployA tail of IL-2 gene by PCR respectively. Clone the recombinant GBS gene into a reverse transcription virus vector pLNCX, then transfer to human peripheral blood lymphocytes in the mediation of liposome. Detect transfection efficiency and determine the ratio of DNA to liposome using green fluorescin gene as report gene. Detect the expression of GSB at mRNA, DNA and protein levels 24h after transfection. Results The expression of GSB were detected at mRNA and DNA levels. SDS-PAGE profile showed a single band with a relative molecular weight of about 30000. The supernatant of infected cells showed the activity of arginase. Conclusion The constructed eukaryotic expression vector expressed the GSB with activity. The study laid a foundation of treating thrombus with GSB gene in vivo.
出处
《中国生物制品学杂志》
CAS
CSCD
2003年第3期161-164,共4页
Chinese Journal of Biologicals
