前炎症细胞因子MDC和IL-18基因的快速克隆

A FAST WAY FOR THE CLONING OF PREINFLAMMATORY FACTOR MDC AND IL-18

目的改进巨噬细胞源趋化因子(macrophage-derived chemokine,MDC)基因和白细胞介素-18(interleukin-18,IL-18)基因的克隆方法。方法BALB/c小鼠随机分为烫伤组和非烫伤组。烫伤组小鼠脾细胞经脂多糖刺激培养48h提取总RNA,其肺组织直接提取总RNA;非烫伤组脾细胞用前列腺素E2刺激培养72h后提取总RNA,肺细胞用脂多糖刺激培养12h后提取总RNA。两组小鼠脾细胞的总RNA分别经反转录-聚合酶链反应(reverse trascription-polymerase chain reaction,RT-PCR)扩增MDC基因片段,肺组织的总RNA分别用RT-PCR扩增IL-18基因片段。将扩增片段分别与pGEM-T载体连接,通过酶切和测序鉴定。结果烫伤组成功扩增出预期大小的DNA片段,序列测序证实为MDC cDNA和IL-18cDNA。非烫伤组均未扩增成功。结论烫伤组比非烫伤组易获得前炎症细胞因子,其有可能成为快速有效获取前炎症细胞因子的手段。

Objective To improve the method for the cloning of macrophage-derived chemokine（MDC） and interleukin-18（IL-18）. Methods BALB/c mice were randomly divided into scald and non-scald groups. In scald group, total RNA were extracted from splenic cells which were stimulated with lipopolysaccharide for 48 h and directly extracted from lung tissue. In nonscald group, total RNA were extracted from splenic cells which were cultured with lipopolysaccharde for 72 h and from the lung cells which were stimulated with lipopolysaccharde for 12 h. In two groups, MDC gene was obtained from splenic RNA by reverse trascriptionpolymerase chain reaction（RT-PCR）, IL-18 gene was amplified from lung tissue RNA by RT- PCR. The DNA fragments of MDC and IL-18 were inserted into pGEM-T vector respectively,and identified by cutting with enzyme and DNA sequencing. Results In scald group, the DNA fragments of MDC and IL-18 were amplified successfully. DNA sequencing showed that the sequences are identical to the sequences recorded in Genbank. In the other group, it＇s failed. Conclusion Scalding will be a fast,effective means to obtain the preinflammatory cytokine genes.