摘要
目的构建表达hIFN-λ3基因的真核表达载体,并在COS-7细胞中表达具有抗病毒活性的rhIFN-λ3蛋白。方法用口腔滤泡炎病毒VSV刺激A549人肺腺癌细胞,抽提总RNA,经RT-PCR获得全长cDNA,与pcD-NA3.1/myc—his(-)A真核表达载体连接,构建重组体pcDNA3.1A-hIFN-λ3,并在COS-7细胞中进行表达,采用微量细胞病变抑制试验研究表达产物的抗病毒活性。结果经PCR和限制性酶切鉴定以及DNA测序确证重组入载体中的基因片段不仅方向正确,而且确为hIFN-λ3,与GeneBank报告序列完全一致,并成功地在COS-7细胞中瞬时表达了具有抗病毒活性的rhIFN-λ3蛋白。结论该研究成功地克隆出hIFN-λ3基因编码序列,并在COS-7细胞中获得瞬时表达,证明了rhIFN-λ3蛋白具有抗病毒活性,为今后干扰素的基因治疗奠定了基础。
Objective To construct eukaryotic expression vector and to express in COS-7 cells human interferon-λ3 protein that possesses characteristic of antivirus. Methods RNA was taken from lung carcinoma cells A549 which have been stimulated by vesicular stomatitis virus, through reverse-transcribing-polymerase chain reaction throughout DNA was obtained, they were ligated with plasmid pcDNA3. 1/myc-his( - )A, forming recombination pcDNA3. 1A-hIFN-λ3 and expressing in COS-7 cells. The effect of antivirus of expressed product was investigated with a cytopathic effect reduction assay. Results The hIFN-λ3 sequence was identified by use of PCR, restriction endonucleases analysis and DNA sequencing. The hIFN-λ3 sequence was defined not only correct in orientation but also conformed with sequence reported by GeneBank. The hIFN-λ3 protein that has characteristic of antivirus was expressed in COS-7 cells. Conclusion In this study hIFN-λ3 gene sequence has been colonized successfully that transiently expressed in COS-7 cells and hIFN-λ3 protein is defined to possess characteristic of antivirus, which has laid the foundation for hIFN-λ3 gene therapy in clinic.
出处
《苏州大学学报（医学版）》
CAS
北大核心
2005年第1期23-26,94,共5页
Suzhou University Journal of Medical Science
基金
江苏省高校自然科学研究项目(01KJB180001)
