大鼠干扰素诱导蛋白-10真核表达质粒的构建及其在NIH 3T3细胞中的表达

Construction of an eukaryotic expression plasmid of IP10 and its expression in NIH 3T3 cells

目的构建大鼠干扰素诱导蛋白-10(IP-10)基因的真核表达质粒,并研究其在NIH 3T3细胞中的表达情况。方法通过PCR获得实验所需的IP-10基因片段,通过酶切IP-10基因片段、真核表达质粒载体pEGFP-c1和连接反应,构建IP-10的真核表达质粒pEGFP-c1-IP-10,重组质粒经限制性内切酶、PCR及DNA序列测定证实构建成功后,用PolyFect脂质体转染NIH 3T3细胞,然后用免疫荧光技术检测pEGFP-c1-IP-10在NIH 3T3细胞中的表达。结果酶切分析、PCR及DNA序列测定证实,IP-10基因片段被成功克隆入真核表达质粒载体pEGFP-c1,免疫荧光技术检测证实,该基因能在NIH 3T3细胞中表达。结论证实IP-10基因可在NIH 3T3细胞中表达,为将其作为DNA疫苗治疗自身免疫性疾病的研究奠定了基础。

OBJECTIVE To construct a new recombinant eukaryotic expression plasmid by fusing rattus IFN - γ- inducible protein - 10 gene, and explore the expression of IP - 10 in NIH 3T3 cells. METHODS IP - 10 was cloned through polymerase chain reaction（PCR）, then inserted into the eukaryotic expression vector pEGFP - cl. When the eukaryotic recombinant plasmid pEGFP - cl - IP - 10 was identified by PCR,restriction endonuclease digestion and sequence analysis, and then it was transfected into NIH3T3 cells by liposome protocol. Immunofluorescence photograph of fluorescence immunocytochemical method confirmed that the fusion gene can be expressed in the NIH3T3 cells. RESULTS PCR,restriction endonuclease digestion and sequence analysis revealed the IP- 10 gene was cloned into the eukaryotic expression plasmid vector pEGFP - cl successfully. Immunoflorescence photograph of fluoroscence immunocytochemical method confirmed that the gene can be expressed in NIH 3T3 cells. CONCLUSION The results confirmed that pEGFP - cl - IP - 10 fusion gene can be expressed in NIH 3T3 cell,which provides the basis as a DNA vaccine for studying the therapy of autoimmune disease（AID）.