人类疱疹病毒8型病毒干扰素调节因子编码基因的克隆及在真核细胞中的表达

Clone and eukaryotic expression of interferon regulatory factor encoding gene of human herpesvirus 8

目的:分离、克隆人类疱疹病毒8型(HHV-8)编码的病毒干扰素调节因子(virusinterferonregulatoryfactor,vIRF)基因K9,并将其导入真核细胞中进行表达。方法:根据GenBank登记的K9基因核苷酸序列设计一对PCR引物,其5′端分别引入HindⅢ和BamHⅠ酶切位点。以原发性渗出性淋巴瘤(PEL)细胞系BCBL-1细胞总DNA为模板,PCR扩增K9基因,经双酶切后克隆进真核表达载体pcDNA3.1(+)中,构建重组真核表达质粒pK9。为了便于蛋白检测,再设计第二对引物,上游引物不变,在下游引物5′端引入了Flag序列。以质粒pK9为模板再次PCR,扩增K9-Flag基因,并把K9-Flag序列克隆入pcDNA3.1(+)中,得到重组质粒pK9F。重组质粒经酶切鉴定,核酸序列测定和分析后瞬时转染NIH3T3细胞,用RT-PCR、Westernblot分别从核酸和蛋白水平检测K9基因的表达情况。结果:PCR分离、克隆的K9-Flag序列全长1380bp,核酸序列分析显示克隆的K9基因与GenBank中己经登记的K9基因序列100%同源,引入的Flag序列完全正确;RT-PCR和Westernblot都在K9预期位置检测到特异性条带。结论:HHV-8K9基因在NIH3T3细胞中获得了正确表达。

Objective： To isolate and clone the interferon regulatory factor （vlRF） encoding K9 gene of human herpesvirns 8 （HHV-8）, and to investigate the expression in the eukaryotic cells. Methods：A pair of PCR primers for K9 gene with HindⅢ and BamH Ⅰ restriction enzyme cut sites were designed according to the sequence registered in GenBank, Then the genome of primary effusion lymphomas（PEL, also termed body cavity-based lymphomas or BCBL） was taken as template and K9 gene was amplified using PCR. Subsequently, amplified gcne fragments were digested with the two enzymes mentioned above and then cloned into pcDNA3.1 （＋） vector to create recombinant eukaryotic expression plasmid designated as pK9. To facilitate the detection of protein, another pair of PCR primers was designed with the same forward primer and Flag sequence was introduced at the 5＇ end of the reverse primer, Then K9-Flag fusion gene was amplified by using PCR, taking plasmid pK9 as template and were further cloned into pcDNA3,1 （＋） vector to construct recumbinant eukaryotic expression plasmid designated as pK9F. After identification with enzyme digestion and nucleotide sequences analysis, recombinant pK9F was transfected into NIH3T3 cells. The expression of pK9F mRNA and protein in NIH3T3 cells was detected by RT-PCR and western blot, respectively, Results： Nucleotide sequences analysis indicated that the isolated and cloned K9-Flag sequem：e length was 1 380 bp. The isolated K9 sequence was 100% homology with K9 gene previously registercd in GenBank. The specific bands were both detected at the expected place by RT-PCR and Western blot. Conclusion： K9 gene could be correctly expressed in NIH3T3 cells.