人IL-1RⅡ基因的克隆及其表达

Cloning and expression of human IL-1RⅡ gene

克隆人IL 1RⅡ基因 ,构建其逆转录病毒载体 ,以探讨其在IL 1主导疾病中的作用。方法 :用RT PCR法从人外周血单个核细胞 (PBMC)中克隆人的IL 1RⅡ基因。将其克隆到原核表达质粒PET2 2b中 ,构建重组质粒PET2 2b IL 1RⅡ。将该重组质粒依次进行PCR、酶切鉴定及测序后 ,转化BL2 1菌 ,以IPTG诱导表达。表达产物用Westernblot鉴定。另外 ,将以酶切重组质粒PET2 2b IL 1RⅡ所获IL 1RⅡ基因的全长ORF ,克隆到逆转录病毒质粒中并转染 2 93细胞 ,用免疫组化染色法检查IL 1RⅡ基因的表达。结果 :用RT PCR法 ,从人PBMC中扩增出 12 0 3bp的cDNA ,测序证实为人IL 1RⅡ基因。Westernblot表明 ,重组质粒可表达IL 1RⅡ蛋白。免疫组化染色表明 ,IL 1RⅡ重组逆转录质粒可在 2 93细胞中高效表达IL 1RⅡ蛋白。结论 :成功地克隆了人IL 1RⅡ基因 ,构建了其原核表达载体和重组逆转录病毒载体 ,并在BL2 1菌中表达IL 1RⅡ重组蛋白 ,为进一步研究IL

AIM: To clone human IL-1RⅡ cDNA and construct its reco mbinant retrovirus vector so as to explore its role in IL-1RⅡ -related diseases. METHODS: Human IL-1RⅡ cDN A was amplified by RT-PCR from peripheral blood mononuclear cells (PBMCs) and i nserted into the vector PET22b to construct recombinant vector PET22b-IL-R Ⅱ. The recombinant was transfected into E.coli BL21 to be expr essed under IPTG induction. Expressed products were detected by Western blot. In addition, human IL-1RⅡ cDNA was subcloned into retrovirus vec tor LZRSPBMN and transfected into 293 cells by calcium phosphate precipitation. IL-1RⅡexpression was detected by immunohistochemical staining . RESULTS: IL-1RⅡ cDNA with 1 203 bp was ampli fied by RT-PCR from human PBMCs. The recombinant of this cDNA could be express ed in E.coli, which was confirmed by Western blot results. Immunohi stochemistry detection showed IL-1RⅡ protein was expre ssed in 293 cells. CONCLUSION: Human IL-1RⅡ gene was cloned successfully. PET22b-IL-1RⅡ and LZR-IL -1RⅡ were constructed and the recombinant protein IL-1R Ⅱ was expressed in E.coli BL21. The results reported herein la y a foundation for further research on the role of IL-1RⅡ in certain diseases.