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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 1RⅡ基因在相关疾病中的作用创造了有利条件
The human IL-1RII gene was cloned and its retroviral vector was constructed to investigate its role in IL-1-dominated diseases. Methods: The human IL-1RII gene was cloned from human peripheral blood mononuclear cells (PBMCs) by RT-PCR. It was cloned into prokaryotic expression plasmid PET2 2b to construct recombinant plasmid PET2 2b IL1RII. The recombinant plasmid was subjected to PCR, restriction enzyme digestion and sequencing, transformed into BL21 strain, and induced to express by IPTG. The expression product was identified by Western blot. In addition, the full-length ORF of the IL1RII gene obtained by restriction enzyme digestion of the recombinant plasmid PET2b IL1RII was cloned into a retrovirus plasmid and transfected into 293 cells. The expression of IL1RII gene was examined by immunohistochemical staining. Results: 12 0 3bp cDNA was amplified from human PBMC by RT-PCR and sequenced as human IL-1RII gene. Western blot showed that the recombinant plasmid can express IL1RII protein. Immunohistochemical staining showed that IL 1 R Ⅱ recombinant retroviral plasmid can efficiently express IL 1 R Ⅱ protein in 293 cells. Conclusion: The human IL-1RⅡ gene was successfully cloned and its prokaryotic expression vector and recombinant retroviral vector were constructed. The recombinant IL-1RII protein was expressed in BL21 strain, which provided a new method to further study the role of IL-1RII gene in related diseases Favorable conditions