人肿瘤坏死因子相关凋亡诱导配体在家蚕中的表达与鉴定

Expression of recombinant human soluble tumor necrosis factor related apoptosis inducing ligand using baculovirus

目的 以家蚕为生物反应器表达人肿瘤坏死因子相关凋亡诱导配体（TRAIL），并检测其生物学活性。方法 将人TRAIL（93～281aa）基因重组于质粒pBacPAK8构建重组转移载体pBacAK8-TRAIL。与经线性化修饰的家蚕棱型多角体病毒（BmBacPAK）DNA共转染家蚕培养细胞株BmN，获得了插入TRAIL基因的重组病毒，重组病毒感染家蚕细胞和家蚕幼虫，使其表达人TRAIL。结果 Southern杂交表明重组病毒基因组中含有人TRAIL基因片段，RNA斑点杂交表明人TRAIL基因得到了转录，重组病毒感染家蚕细胞株（BmN）、家蚕幼虫，在细胞培养上清、细胞抽提物、幼虫的体液样品中通过Westblot分析都能检测得到表达产物的特异性条带；采用L929细胞检测TRAIL的生物学活性。结论 提示人TRAIL基因分别在家蚕培养细胞和蚕幼虫中得到高效表达，此基因杆状病毒表达系统的建立为进一步研究TRAIL奠定了基础。

Objective In order to expression TRAIL and detect its bioactivity using Bombyx mori as biological reactor. Methods The human TRAIL gene （93-281aa） was inserted in Bombyx mori baculovirus transfer vector pBacPAK8 and contransfected with linear virus Bm-BacPAK6 into BmN cells. The homologous recombination occurred in the cells then the recombinant virus BacPAK-TRAIL was obtained . The recombinant virus could express the TRAIL. Results Southern Hybridization analysis sug- gested that the recombinant baculovirus DNA contained TRAIL fragment. RNA dot blotting demonstrated that the TRAIL gene was transcribed. The recombinant baculovirus had a strong infectivity to BmN cell line and silkworm larvae . TRAIL band was detected from all the samples of cell extract, culture supernatant, haemolymph of larvae by Westblot analysis. Biological activity of the expressed product was determined by L929 cell line. Conelusion The results show that the biologically active TRAIL protein is expressed in BmN cell and in silkworm larvae. It plays an important role in study the TRAIL protein further.