人IL-2信号肽和大鼠心肌肌凝蛋白α重链片段融合基因的构建及分泌表达

Construction and secretive expression of fused gene containing partial rat cardiac myosin α heavy chain cDNA sequence and signal peptide of human IL-2

目的 :克隆编码大鼠心肌肌凝蛋白α重链aa736～ 96 0的cDNA片段 ,构建携带融合基因的逆转录病毒分泌表达载体。方法 :以RT PCR方法从幼龄大鼠心肌组织中扩增出 6 81bp的目的基因 ,与hIL 2cDNA序列拼接 ,构建融合基因hIL 2 myosin及其逆转录病毒载体。采用脂质体法转染NIH3T3和PA317细胞 ,经G4 18筛选后 ,用RT PCR、免疫组化、免疫电镜和Dot blot法分析检测融合基因的表达。结果 :核苷酸序列测定显示克隆基因的核苷酸及推导的氨基酸序列与报告序列一致 ,开放阅读框正确 ;RT PCR检测提示转染细胞有融合基因mRNA转录 ;免疫组化、免疫电镜和Dot blot检测表明转染细胞有该肌凝蛋白重链片段的分泌表达。结论 :大鼠心肌肌凝蛋白α重链基因逆转录病毒载体构建成功 ,并可分泌表达 ,为心肌肌凝蛋白转基因诱导机体特异性免疫耐受及对心肌免疫损伤的研究打下了基础。

Objective:To clone the rat cardiac myosin α heavy chain cDNA fragment encoding aa736 960 and construct its recombinant retrovirus vector Methods:The 681 bp target gene was amplified from heart tissue of young rats with RT PCR Fusion gene of hIL 2/myosin was constructed by splicing with preserved region of hIL 2 cDNA using ligation methods and subsequently the plasmid pLNC hIL 2 myosin was constructed NIH3T3 cells and PA317 cells were transfected with plasmid pLNC hIL 2 myosin using Lipofectamine After screening with medium containing G418, the positive clone was chosen and was detected using RT PCR, immunohistochemistry, immune electron microscope and dot blot Results:The determination of nucleotide sequence showed that the nucleotide and amino acid sequence of the gene cloned was the same as the reported sequence, and its open reading frame was correct RT PCR analysis indicated that mRNA of the fused gene was present in the positive clone Immunohistochemistry, immune electron microscope and dot blot showed that the fused gene IL 2 myosin was successfully expressed Conclusion:The fused gene of rat cardiac α heavy chain fragment and the preserved region of human IL 2 was constructed and expressed successfully