分泌型人白细胞介素18重组质粒的构建及其真核表达

目的：构建带IL—12 P40信号肽序列的分泌型IL—18质粒，使其在真核细胞中稳定表达。方法：根据IL—12 P40的信号肽cDNA序列及人成熟IL—18序列设计4条特异性引物，用RT—PCR法自人树突状细胞中分别扩增IL—12 P40信号肽序列(片段A)及成熟IL—18基因(片段B)，用重叠延伸PCR法拼接和扩增融合基因(片段C)，连接制备pGEM—T克隆载体，亚克隆至pcDNA3．1^ 。转染NCI—H460人肺癌细胞株，经RT—PCR和Western blot检测IL—18的表达。结果：表达质粒的酶切图谱符合预期的结果，测序结果与预期的cDNA序列完全一致，转染细胞株表达了IL—18。结论：成功地构建了含IL—12 P40信号肽序列的IL—18表达质粒，并在人肺癌细胞株中得到了表达。

Construction of secretory human interleukin 18 plasmid and its expression in eukaryotic cells

Objective To construct human IL 18 expression vector containing IL 12 P40 signal sequence,and to obtain a stable expressing cell line. Methods IL 12 P40 signal sequence and IL 18 gene were cloned by RT PCR. The hybrid cDNA of these two fragments was acquired by overlap extention PCR. Subclone techniques were used to construct expression plasmid. RT PCR and Western blot were used to detect the expression of IL 18 in gene transfected cells. Results Sequencing result was correct. The transfected cells expressed mature IL 18. Conclusion Secretory IL 18 vector has been constructed and the stable expressing cell line successfully obtained.