人IL-18结合蛋白cDNA的克隆及其逆转录病毒载体的构建

Cloning of human interleukin-18 binding protein and the construction of its retroviral vector

目的 克隆人白细胞介素 - 18结合蛋白 (hIL - 18BP)的cDNA及构建hIL - 18BP逆转录病毒载体 ,以研究IL - 18BP与IL - 18相互作用的机制及自身免疫性疾病的基因治疗。方法 从中国汉族人胎盘组织中提取总RNA ,用RT -PCR方法扩增hIL - 18BPcDNA ,与载体pcDNA3连接 ,转化大肠杆菌DH5α。得到的重组质粒经鉴定正确后 ,再作为模板进行PCR。PCR产物插入逆转录病毒载体pLNCX中。结果 RT -PCR扩增出全长 5 85bp的hIL - 18BPcDNA ,2次酶切鉴定证明hIL - 18BP已分别与pcDNA3、pLNCX重组。 2次测序结果均与国外文献报道的hIL - 18BP的序列一致。结论 成功地克隆了中国人IL - 18BPcDNA ,并构建了重组hIL -

Objective To clone the cDNA of human interleukin-18 binding protein (hIL-18BP) and construct the recombined hIL-18BP retrovirus as a theoretical and preparatory approach to the IL-18BP gene therapy for autoimmune diseases. Methods The total RNA was extracted from the placenta of a Chinese individual and the intact cDNA of hIL-18BP was amplified by RT-PCR. The cDNA obtained was first linked with pcDNA3 vector and then transferred into E.coli DH5(. After being identified, the recombined plasmid (pcDNA3/ hIL-18BP) was used as the template in PCR, and finally, the 585 bp PCR product was inserted into the retroviral vector pLNCX. Results Recombined pcDNA3/ hIL-18BP, pLNCX/ hIL-18BP were identified by double-enzyme digestion and the results of sequencing were consistent with that of the published hIL-18BP cDNA. Conclusion Chinese IL-18BP cDNA has been cloned and the recombined hIL-18BP retroviral vector has been constructed successfully.