作　　者： ; ; ; ;

机构地区： 汕头大学医学院炎症与免疫性疾病研究所

出　　处： 《现代生物医学进展》 2010年第8期1440-1442,共3页

摘　　要： 目的:构建小鼠IL-35单链融合基因及其真核表达载体。方法:将10ugLPS和等体积的完全福氏佐剂乳化后皮下注射C57BL/6小鼠,7天后,取小鼠脾脏,提取脾细胞总RNA,通过RT-PCR克隆小鼠EBI3和IL-12p35cDNA;采用重叠延伸PCR,通过编码疏水性多肽接头(Gly4Ser)3的DNA序列连接小鼠EBI3全长基因和IL-12p35成熟肽基因,构建小鼠IL-35单链融合基因(mouse sigle chain IL-35,mscIL-35),并将其克隆至pcDNA3.1/V5-His訫TOPO誖TA载体。通过酶切和测序鉴定阳性重组载体。结果:DNA序列分析结果表明:小鼠IL-35单链融合基因中EBI3、IL-12p35和linker的连接顺序、方向及序列完全正确。结论:成功构建了小鼠IL-35单链融合基因及其真核表达载体,为进一步探讨IL-35的生物学功能奠定了基础。 Objective：To construct mouse sigle chain IL-35 interleukin-35（mscIL-35） fusion gene and its eukaryotic expression Vector.Methods：The cDNA encoding mouse EBI3 and IL-12p35 were amplified by RT-PCR from the total RNA extracted from spleen cells of C57BL/6 mice immunized with LPS.EBI3 gene and IL-12p35 mature peptide gene were fused via a hydrophobic polypeptide linker（Gly4Ser）3 by Overlap Extension PCR to obtain mscIL-35 fusion gene.The mscIL-35 fusion gene was cloned into eukaryotic expression vector pcDNA3.1/V5-His  TOPO  TA and the positive recombinant clone was analyzed by digestion of restriction endonuclease and DNA sequencing.Results：Sequence analysis showed that the splicing order,orientation and sequence of mscIL-35 fusion gene were completely correct.Conclusion：The mscIL-35 fusion gene was constructed successfully,which will be helpful for the further research on its biological function.

关 键 词： 小鼠白介素 融合基因 重叠延伸

领　　域： [生物学] [生物学]