卡波济肉瘤相关疱疹病毒包膜糖蛋白K8.1B全长cDNA的克隆及在真核细胞中的表达  

Cloning of glycoprotein K8.1B cDNA of Kaposi’s sarcoma-associated herpesvirus and its expression in the eukaryotic cells

作　　者：秦娣[1] 卢春[1] 钱超[1] 曾怡[1] 唐桂霞[1] 张玲[1] 

机构地区：[1]南京医科大学微生物学与免疫学系,江苏南京210029

出　　处：《南京医科大学学报（自然科学版）》2005年第9期616-619,共4页Journal of Nanjing Medical University(Natural Sciences)

基　　金：国家自然科学基金资助项目(30100160;30271179)

摘　　要：目的:分离、克隆卡波济肉瘤相关疱疹病毒(KSHV)包膜糖蛋白K8.1B全长cDNA,并将其置于真核细胞中进行表达。方法:用K8.1B基因设计一对PCR引物,其5’端分别引入BamHI和XhoI酶切位点。以佛波酯(TPA)刺激的原发性渗出性淋巴瘤(PEL)细胞系BCBL-1细胞总RNA为模板,通过RT-PCR扩增KSHVK8.1B编码序列,经双酶切后克隆进pcDNA3.1(+)载体中,构建含K8.1BcDNA的重组真核表达质粒。重组质粒经酶切鉴定、核酸序列测定和分析后转染人胚肾293细胞,经G418筛选获细胞克隆。用间接免疫荧光染色(IFA)检测K8.1BcDNA编码蛋白在293细胞中的表达状况。结果:RT-PCR分离、克隆的K8.1BcDNA全长501bp,核酸序列分析显示,该基因与Genbank中已登记的K8.1B编码基因呈现100%同源性。经IFA证实该重组蛋白为KSHVK8.1B特异性蛋白。结论:KSHV包膜糖蛋白K8.1B编码cDNA在293细胞中获得了正确表达。Objective： To isolate and clone glycoprotein K8.1B eDNA of Kaposi＇s sarcoma-associated herpesvirus （KSHV） and express it in the eukaryotic cells. Methods- A pair of PCR primers for K8.1B ,in which BamH Ⅰ and Xho Ⅰ sites were introduced at the 5＇ end of the primer,respectively,was designed based on the sequence registered in GenBank. K8.1B encoded gene was amplified with reverse transcriptase-polymerase chain reaction （RT-PCR）,taking total RNA from BCBL-1 cells treated by phorbal esters （TPA） of primary effusion lymphoma （PEL） as template. Amplified PCR fragments ,sere digested with the above mentioned two enzymes and then subcloned into pcDNA3.1 （＋） vector to construct recombinant eukaryotic expression plasmid. Human embryonic kidney cells 293 were transfected with the recombinant plasmid,which had been identified with enzyme digestion and nucleotide sequences analysis. The stably transfected cell clones were obtained through G418 filtration and immunofluorescence assay （IFA） were further performed to evaluate the expression of K8.1B in 293 cells. Results： A 501-base-pair （bp） sequence isolated and cloned in this study was 100% homology with K8.1B cDNA of KSHV,which was previously registered in GenBank,and recombinant protein was substantiated to be the specific K8.1B of KSHV by IFA. Conclusion： Glycoprotein K8.1B cDNA of KSHV was correctly expressed in 293 cells.

关 键 词：卡波济肉瘤相关疱疹病毒 K8.1B RT-PCR 免疫荧光染色 

分 类 号：R739.5[医药卫生—肿瘤] R373.11[医药卫生—临床医学]