中文摘要：

目的：分离克隆卡波济肉瘤相关疱疹病毒（KSHV）编码IL-6基因（vIL-6），并导入NIH3T3细胞中进行真核表达。方法：根据KSHV编码IL-6基因核苷酸序列设计一对引物，在引物的5’端分别引入BamHI、HindⅢ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vIL-6基因．PCR产物经双酶切克隆进真核载体pcDNA3．1（＋）。进一步在原有的下游引物5’端加上一段flag序列，以经过序列测定正确的重组质粒为模板，PCR扩增vIL-6-flag融合基因，构建含vIL-6-flag的重组质粒。将该质粒转染NIH3T3细胞，经G418筛选获细胞抗性克隆。最后用抗flagM2单克隆抗体进行Westernblot检测vIL-6在NIH3T3细胞中的表达。结果：获得vIL-6-flag融合基因，全长671bp，其中vIL-6基因核苷酸序列与GenBank中所登记的KSHVvIL-6基因呈现100％同源性。Westernblot结果显示，在约25ku位置有目的条带，与预期的重组vIL-6-flag融合蛋白大小一致。结论：KSHVvIL-6编码基因在NIH3T3细胞中初步获得表达。

英文摘要：

Objective： To isolate and clone vIL-6 gene of Kaposi＇s sarcoma-associated herpesvirus （KSHV） and transfect it in NIH3T3 cells. Methods： A pair of PCR primers for vIL-6 was designed based on the sequence registered in GenBank BamH I and Hind Ⅲ sites were introduced into the 5＇ ends of the primers, respectively, vIL-6 gene was amplified with PCR, using the total DNA of BCBL-1 cells from primary effusion lymphoma（PEL） as template. The PCR fragments were digested with the above two enzymes and then cloned into pcDNA3.1 （＋） vector to construct recombinant plasmid pvIL-6. Furthermore, a flag sequence was added to the 5＇ ends of original downstream primer, vIL-6-flag gene was amplified with PCR, using pvIL-6 as template. Recombinant plasmid containing vIL-6-flag gene was constructed. NIH3T3 were transfected with vIL-6-flag recombinant plasmid. Subsequently,the stably transfected cell clones were obtained through G418 filtration. Finally, Western blot was performed to evaluate the expression of vIL-6-flag in NIH3T3 cells by anti-flag M2 monoclonal antibody. Results： The sequence of vIL-6 gene was 100% homology with vIL-6 gene of KSHV previously registered in GenBank. An interested band about 25 ku was visible, which was consistent with the expected size of vIL-6-flag fused protein expressed in NIH3T3. Conclusion： vIL-6 gene of KSHV could be isolated and cloned successfully and expressed in NIH3T3 cell correctly.