人类疱疹病毒8型肿瘤转化基因K12的分离及在昆虫细胞中的表达

Isolation of Oncogenic Transforming Gene(Open Reading Frame K12)in Human Her-pesvirus 8(HHV-8)and Its Expression in Insect Cells

目的:分离人类疱疹病毒8型(HHV-8)肿瘤转化基因 K12,克隆至杆状病毒载体并在昆虫细胞中作尝试性表达。方法:设计一对引物,在引物的5′端分别引入BamHI、EcoRI酶切位点,以佛波脂刺激的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HHV-8肿瘤转化基因K12。将经序列测定后的PCR产物克隆进杆状病毒转移载体pVL1393中,构建成重K12杆状病毒转移载体pVL-K12,并与线性BaculoGold病毒 DNA共转染昆虫细胞Sf9,获得重组病毒。RT-PCR检测K12 mRNA转录水平;间接免疫荧光染色(IFA)检测K12编码蛋白在Sf9细胞中的表达状况。结果:含 HHV-8 K12基因重组杆状病毒在昆虫细胞Sf9中获得表达,经IFA证实该重组蛋白为HHV-8特异性蛋白。结论:杆状病毒载体能够介导HHV-8肿瘤转化基因K12编码的蛋白在昆虫细胞中表达。

Objective: To isolate oncogenic transforming gene (open reading frame K12) of human herpesvinis 8 (HHV-8) and evaluate expression of K12 in insect cells using baculovirus express system after transfection. Methods: Open reading frame K12 of HHV-8 was amplified by PCR taking the total DNA of BCBL-1 cells, which was obtained from primary effusion lymphoma(PEL)after stimulation of TPA, as template. The specific primers were engineered with the cut sites of BamHI and EcoRI restriction enzyme on the 5' ends to facilitate directional cloning. The PCR product was confirmed by sequence determination and analysis and cloned into the baculovirus transfer vector pVL1393 to obtain the recombinant plasmid named pVL-K12. Recombinant pVL-K12 DNA and linearized baculovirus DNA (BaculoGold?viral DNA) was co-transfected into insect cells Sf9 to produce recombinant virus. Reverse transcriptase PCR (RT-PCR) and immunofluoscence assay (1FA) were performed to evaluate mRNA transcription level and protein expression of HHV-8 K12 respectively. Result: The level of mRNA transcription from K12 was significantly high in the method of RT-PCR and recombinant protein was substantiated to be HHV-8 specific protein by IFA. Conclusion: Baculovirus transfer vector may mediate the coding protein of HHV-8 K12 gene expressed in insect cells.