人白细胞介素-36受体拮抗剂基因的克隆及序列分析

Cloning and sequence analysis of human Interleukin- 36 receptor antagonist cDNA

[目的]构建人白细胞介素-36受体拮抗剂(interleukin-36 receptor antagonist,IL-36Ra)的真核表达载体,并对其基因全长编码区进行分析。[方法]提取总RNA,逆转录出IL-36Ra c DNA序列。设计人IL-36Ra基因的上游引物和下游引物。利用热启动PCR扩增目的基因,将扩增出的IL-36Ra基因编码区通过限制性内切酶酶切后,把酶切的目的片段与载体pc DNA3.1连接,转化到感受态细胞大肠杆菌DH5α中。通过氨苄青霉素抗性筛选、双酶切及菌落PCR鉴定,最后选取阳性克隆进行测序。[结果]重组载体pc DNA3.1-h IL-36Ra经菌落PCR和酶切鉴定,最后通过序列分析证实,其序列与Gen Bank中数据一致。[结论]人IL-36Ra基因的重组真核表达载体pc DNA3.1-h IL-36Ra的成功构建,为进一步研究IL-36Ra在免疫性疾病中的作用奠定了实验基础。

[ Objective] To clone and sequence the cDNA sequence encoding human interleukin -36 receptor antagonist gene. [ Methods] Total RNA was isolated from human foreskin by RNAiso Plus,cDNA fragment encoding IL -36Ra gene was amplified with special primers contained Kpn I and Xba I restriction sites respectively. The cDNA was connected into the multiple cloning sites of pcDNA3. 1 vec- tor. Then the correct cloning sequences were identified by restriction digestion, colony PCR and sequenced. [ Results ] The band shape of 28S, 18S and 5S rRNA from the extracted total RNA of human foreskin can be seen clearly. Sequence analysis indicated that the cDNA of IL - 36Ra has the whole length of 468 bp. The productions and recombinant vector pcDNA3.1 - hIL - 36Ra shared 100% homology with the sequence of mRNA for IL- 36Ra gene in GenBank. [ Conclusion] The whole length cDNA of human IL- 36Ra was constructed suc- cessfully, which provide a scientific basis for the further study on its biological function.