【摘要】 目的:克隆并在293T细胞中表达卡波济肉瘤相关疱疹病毒(KSHV)包膜糖蛋白编码基因K8.1、K8.1A与gL。方法:以BCBL-1细胞总DNA为模板,用PCR方法扩增出K8.1和gL基因;以BCBL-1细胞的总RNA为模板,通过RT-PCR的方法扩增出K8.1A基因。将PCR产物克隆进真核载体pcDNA3.1(+)内,分别构建含K8.1、K8.1A和gL基因的重组真核表达质粒。重组质粒转染293T细胞,用Westernblot的方法检测K8.1和K8.1A在293T细胞中的表达;RT-PCR方法检测gL基因在293T细胞中的转录。结果:核酸序列分析的结果表明,克隆的K8.1、K8.1A和gL基因与GenBank中已登记的KSHVK8.1、K8.1A和gL基因序列100%同源。Westernblot结果显示,在约35ku位置有目的条带,与预期的重组K8.1和K8.1A蛋白大小一致。RT-PCR结果显示约在500bp位置检测到特异性条带。结论:KSHVK8.1、K8.1A和gL编码基因在293T细胞中获得正确表达。更多还原

【Abstract】 Objective:To clone envelope glycoprotein K8.1,K8.1A and gL gene of Kaposi’s sarcoma-associated herpesvirus(KSHV)and express them in 293T cells.Methods:K8.1 and gL genes were amplified by PCR with the total DNA of BCBL-1 cells as template.K8.1A genes were amplified by RT-PCR with the total RNA of BCBL-1 cells as template.The PCR fragments were cloned into pcDNA3.1(+)vector to construct recombinant eukaryotic expression plasmids.293T cells were transfected with the recombinant plasmids.Western blot was performed to evaluate the expressions of K8.1 and K8.1A in 293T cells.RT-PCR was performed to evaluate the transcription of gL in 293T cells.Results:The sequence of K8.1,K8.1A and gL was 100% homology with K8.1,K8.1A and gL gene of KSHV previously registered in GenBank.An interesting band about 35 ku was visible in the result of Western blot,which was consistent with expected size of recombinate protein of K8.1 and K8.1A expressed in 293T.An interesting band about 500 bp was visible in the result of RT-PCR.Conclusion:Glycoprotein K8.1,K8.1A and gL of KSHV were correctly expressed in 293T cells.更多还原