重组人干扰素γ慢病毒的构建及鉴定

Construction and identification of recombinant human interferonγ lentivirus

目的构建含干扰素(IFN)γ基因的转移质粒,包装含IFNγ基因的重组慢病毒(LV),测定重组LV是否包装成功,重组LV的定量及鉴定IFNγ基因的表达。方法采用分子克隆方法,构建含IFNγ基因的转移质粒,采用脂质体法将LV三质粒系统(包括鉴定正确的转移质粒p TY-CMV-IFNγ、包装质粒p SPAX2、包膜蛋白质粒p MD2.G)共转染293T细胞,包装由CMV启动子驱动的重组人LV-IFNγ。采用ELISA法对重组LV-IFNγ进行鉴定。结果成功构建了含CMV启动子的p TYCMV-IFNγ转移质粒。高效包装出重组LV-CMV-IFNγ,病毒RNA载量1.20×109copies/ml。ELISA检测LV包装病毒液中IFNγ量为:11.88μg/ml。LV原液转导293A和CEM细胞12 h后,IFNγ量分别为11.45μg/ml和10.44μg/ml。LV原液转导293A和CEM细胞72 h后,IFNγ量分别为11.28μg/ml和11.01μg/ml。结论成功包装生产出高滴度重组LV-CMV-IFNγ,转染293A和CEM后,能检测到IFNγ高水平表达,为下一步工作提供研究基础。

Objective To construct transfer plasmid with interferon( IFN) γ gene and package recombinant lentivirus( LV),determine whether LV packaging is successful,detect viral load and identify IFNγ gene expression of recombinant LV. Methods Construct transfer plasmid with interferon( IFN) γ gene by molecular cloning methods,transfect 293 T cells with three plasmids system( including the transfer plasmid p TY CMV-IFNγ,package plasmid p SPAX2,envelope protein plasmid p MD2. G) by using liposome,package recombinant LV-IFNγ driven by CMV promoter. Identify LV-IFNγ by ELISA. Results Transfer plasmid with CMV promoter was constructed successfully. The recombinant LV-CMV-IFNγ was packaged efficiently and viral RNA loads could achieve 1. 20 × 109 copies/ml. IFNγ concentration in the LV supertanant is 11. 88 μg/ml. After transducted 293 A and CEM cells for 12 h,IFNγ concentration among 293 A supertanant is 11. 45 μg/ml and that among CEM supertanant is 10. 44 μg/ml. For 72 h,IFNγ concentration among 293 A supertanant is 11. 28 μg/ml and that among CEM supertanant is 11. 01 μg/ml. Conclusion After producing recombinant LV-IFNγ of high titer successfully and transducting 293 A and CEM cells,IFNγ expression of high levels can be detected,which lays the foundation for the following research work.