人白细胞介素-4基因在家蚕杆状病毒表达系统中的表达

采用RT-PCR法从经LPS诱导刺激的人外周血淋巴细胞中克隆了人白细胞介素-4(human interleu kjn-4,hIL-4)基因。为高效表达hIL-4,促进其临床应用,将hIL-4插入家蚕杆状病毒转移载体pBacPAK8中,构建重组载体pBacPAK-hIL-4,并与线性化病毒Bm-BacPAK6 DNA共转染家蚕BmN细胞,获得重组病毒BacPAK-hIL-4。将重组病毒感染家蚕5龄幼虫和蚕蛹(1×10~5PFU/头),表达产物以ELISA、SDS-PAGE和Western blotting方法检测,用活化的人外周血T淋巴细胞测定表达产物体外生物活性。ELISA法结果表明hIL-4在感染病毒后96h的蚕血淋巴和120h的蚕蛹中表达量最高,分别达到2．3μg/mL蚕血淋巴和10．2μg/mL体液；SDS-PAGE、Western blotting分析表明表达产物分子量约20kD；表达产物对活化的T淋巴细胞增殖具有明显促进作用。

Expression of hIL-4 Gene in Bombyx mori Baculovirus Expression Vector System

The human interleukin-4(hlL-4)gene from PBMC activated by LPS was cloned using RT-PCR.For mass production and clinical use of hlL-4,Bombyx mori bacuiovirus expression vector system was adopted in this experiment.The hlL-4 gene(460bp)was inserted into Bombyx mori baculovirus transfer vector pBacPAK8 and co-transfected with linearized DNA of Bm-BacPAK6 virus into BmN ceils.The recombinant vi- rus BacPAK-hlL-4 was gained.BacPAK-hlL-4 infected the fifth instars and pupae of silkworm(1×10~5PFU/ body).Expressed product was determined by ELISA,SDS-PAGE and Western blotting.The bio-activity of the protein product was determined by activated human T cells proliferation test in vitro.The highest detectable level of hlL-4 protein yielded to 2.3μg/mL in larval hemolymph at 96h and 10.2μg/mL in pupae body fluid at 120h after infection respectivelg according to ELISA results.SDS-PAGE and Western blotting analysis indi- cated that the molecular weight of expressed product was about 20kD.The expressed product could enhance the proliferation of activated human T cell.