作　　者： ; ; ; ; ; ;

机构地区： 广州军区广州总医院

出　　处： 《医学研究生学报》 2008年第9期899-903,I0001,共6页

摘　　要： 目的:克隆单纯疱疹病毒2型(HSV-2)潜伏相关转录子蛋白读码框2(LAT ORF2)基因,构建真核表达载体并在真核细胞中表达,用于进一步研究LAT介导的HSV-2潜伏及激活感染机制。方法:以构建成功的pVAX-LAT重组质粒为模板,根据GenBank中HSV-2LATORF2基因的核苷酸序列设计引物,并在引物的5′端分别引入KpnⅠ和PstⅠ酶切位点。用PCR特异性扩增LAT基因ORF2片段,PCR产物纯化回收后经KpnⅠ、PstⅠ双酶切,再次回收后产物与同样经双酶切的pcDNA 6/myc-His B质粒进行连接,Amp抗性筛选阳性重组子,双酶切及测序鉴定连接产物。在脂质体介导下瞬时转染Vero细胞,用RT-PCR及SDS-PAGE检测其在Vero细胞中的表达。结果:重组质粒经KpnⅠ和PstⅠ双酶切获得预期大小的两条片段,测序表明ORF2正确,RT-PCR及SDS-PAGE显示转染了重组质粒的Vero细胞内有目的基因及其编码蛋白的表达。结论:成功构建了ORF2-pcDNA6/myc-His B重组质粒,并可在Vero细胞内有效表达。 Objective： To construct the eukaryotic expression plasmid containing the open reading frame （ORF）2 of the herpes simplex virus （HSV）-2 latency associated transcript （LAT） gene and to detect its expression in Vero cells in order to lay a foundation for further research on the mechanism of LAT medicating HSV-2＇s latent and recurrent infection. Methods ： A pair of primers were designed and synthesized according to the published sequence of I_AT ORF2 in GenBank. After amplification with PCR, the amplified products were cloned into the plasmid pcDNA 6/myc-His B to construct a recombinant plasmid and then sequenced and identified by restrictive endonuclease digestion. The constructed recombinant plasmid was finally transfected into Vero cells with the lipid transfection reagent and the expression of ORF2 was analyzed by RT-PCR and SDS-PAGE. Results： The successful construction of the recombinant plasmid ORF2-pcDNA 6/myc-His B was verified by restrictive endonuclease assay and sequence analysis with the same target fragment HSV-2 LAT ORF2 in GenBank, and the expression of the target fragment was detected in the lysate of the transfected Vero cells by RT-PCR and SDS-PAGE. Conclusion： The eukaryotic expression plasmid for LAT ORF2 was successfully constructed and its expression in Vero cells confirmed.

关 键 词： 单纯疱疹病毒 型 潜伏相关转录子 开放读码框 重组载体

领　　域： [生物学]