真核表达载体pVAX-LAT的构建及其在Vero细胞的瞬时表达.docVIP

真核表达载体pVAX-LAT的构建及其在SH-SY5Y神经细胞中的瞬时表达 仕瑶慧，杨慧兰*，樊建勇，赵举锋，黄小宴，周翠 广州军区广州总医院皮肤科,广州 510010 目的 构建表达疱疹病毒Ⅱ型潜伏相关转录体（Latency-associated transcripts,LATs）基因的真核表达载体，并验证其在SH-SY5Y细胞里的表达情况。 方法 从疱疹病毒Ⅱ型基因组里，PCR扩增出长为3200 bp的LAT基因。利用LAT基因与pVAX载体共有的BamHⅠ酶切位点，将LAT基因分为1200bp和2000bp两段，分段克隆，构建载体pVAX - HindⅢ-BamHⅠ和pVAX -BamHⅠ-PstⅠ。测序验证插入片段正确后，利用HindⅢ、BamHⅠ双酶切载体pVAX - HindⅢ-BamHⅠ，将长约1200bp的HindⅢ-BamHⅠ片段插入到经HindⅢ、BamHⅠ双酶切的pVAX -BamHⅠ-PstⅠ载体中，双酶切鉴定，阳性重组子即为pVAX-LAT载体。将pVAX-LAT载体瞬时转染SH-SY5Y细胞，RT-PCR验证LAT基因在真核细胞里的表达情况。结果 双酶切及测序结果表明pVAX-LAT载体构建成功；RT-PCR结果显示LAT基因在Vero细胞里表达。 结论 成功构建了国内首个可在真核细胞内表达疱疹病毒Ⅱ型LAT基因的真核表达载体。 关键词 单纯疱疹病毒；潜伏相关转录体；瞬时转染 Construction of eukaryotic expression vector pVAX-LAT and its expression in SH-SY5Y cells [Abstract] Objective To construct eukaryotic expression vector for express the latency-associated transcripts gene of herpes simplex virus type 2, and investigate its expression in SH-SY5Y cells. Methods Plasmid pVAX-LAT, which include a 3,200bp genomic DNA fragment of HSV-2 strain 333, this DNA encompasses of latency-associated promoter 1(LAP1) and entire sequences of LAP2 and major latency-associated transcript gene. The plasmid may express the major LAT strongly under the control of cytomegalovirus (CMV) IE promoter. To generate pVAX-LAT, we constructed plasmid pVAX-HindⅢ-BamHⅠand pVAX-BamHⅠ-PstⅠfirst, these plasmid have 1,200bp and 2,000bp portions of HSV-2 LAT gene respective. Then the sequences between HindⅢ and BamHⅠ cleavase site of pVAX-BamHⅠ-PstⅠ were replaced with the HindⅢ-BamHⅠ fragment of pVAX-HindⅢ-BamHⅠ. If the replace success, the pVAX-LAT were constructed. Results pVAX-LAT was successfully constructed. SH-SY5Y cells were successfully transfected with pVAX-LAT, and the LAT gene had been expressed, could observed by RT-PCR. Conclusions The successful construction of pVAX-LAT and transfection of pVAX-LAT of eukaryon expression vector would lay foundation for futher study on the role of LAT. [Key words] herpes simplex virus; latency-associated transcripts; transi