重组TNF－α基因逆转录病毒转移系统的构建

Constraction of a Retroviral Mediated Recombinant TNF-αGene Transfer System

将人ＴＮＦ－αｃＤＮＡ功能片段０．９ｋｂ克隆于逆转录病毒载体ＰＬＪ，构建成重组逆转录病毒载体ＰＬＪ－ＴＮＦ；在磷酸钙介导下，将其导入包装细胞ψＣＲＩＰ和ψＣＲＥ中，经克隆筛选，获得了病毒滴度为１０６ＣＦＵ／ｍｌ能稳定、高效分泌含ＴＮＦ－α基因重组病毒颗粒的细胞株ＰＬＪＣ－５。

By the application of BamHI enzyme,a expressional plasmid of human TNF-αagene,a 0.9kb TNF-α cDNA fragment was isolated.and was linked with a retroviral vector PLJ to construct recombinantretroviral vector PLJ-TNF It transfected the pachaging cell lineψCRIP and ψ CRE with CaPO4 trans-fection,Through the process of selection and cloning,a recombinant viral preducer cell line PLJC-5(vi-ral titer 106 CFU/ml)was isolated。It could produce the viral particle containing TNF- αgene with sta-ble and high efficiency without any production of helper virus A retroviral particle(about 90nm diame-ter) was observed in the medium of PLJC-5 cell line under electromicroscope,The method for con-densed viral reservation was also established;