IL-18-PE38融合基因真核表达载体的构建及其在软骨细胞中的表达

Construction of Interleukin-18-PE38 Fusion Gene Eukaryotic Expression Vector and Its Expression in the Chondrocyte

目的构建含有白细胞介素-18(IL-18)及毒素融合基因的真核表达载体,并研究其在软骨细胞中的表达情况。方法通过分子克隆技术,将IL-18-PE38融合基因插入真核表达载体PsecTag2B中,构建真核表达载体PsecTag2B-IL-18-PE38,经EcoR单酶切及PCR鉴定。脂质体法将其转入原代软骨细胞,通过荧光免疫细胞化学法鉴定其瞬时表达。结果经EcoR单酶切及PCR鉴定证实IL-18-PE38融合基因成功克隆入真核表达载体PsecTag2B中。荧光免疫细胞化学法荧光显微镜照片证实此重组基因可在软骨细胞膜及细胞浆中表达。结论证实了IL-18-PE38融合基因可在软骨细胞中表达,为进一步研究其对类风湿关节炎的治疗奠定了基础。

Objective To construct the interleukin-18-PE38 fusion gene expression vector and explore the expression of the fusion gene in the chondrocyte. Methods The recombinant eukaryotic expression vector PsecTag2B-IL-18-PE38 was constructed by inserting interleukin-18-PE38 fusion gene into eukaryotic expression vector PsecTag2B with molecular cloning technique.It was confirmed by restrictive enzymes(EcoRⅠ)digestion assay and PCR.The vector was transfected into primary chrondrocyte by liposome protocol,and the transient expression was identified by fluorescence immunocytochemical assay. Results Restrictive enzymes digestion analysis and PCR revealed that the interleukin-18-PE38 fusion gene was cloned into the eukaryotic expression vector PsecTag2B successfully.Immunofluorescence photograph of fluorescence immunocytochemical method confirmed that the fusion gene can be expressed in the cytomembrane and cytoplasm. Conclusion The results confirmed that PsecTag2B-IL-18-PE38 fusion gene can be expressed in the chondrocyte,which could serve as a foundation for the study on rheumatoid arthritis therapy.