人白细胞介素-4基因在家蚕杆状病毒表达系统中的表达  

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

作　　者：陈蔚青[1] 史锋[2] 朱成钢[2] 申屠超[1] 

机构地区：[1]浙江树人大学生物与环境工程学院,杭州310015 [2]浙江大学生物化学研究所,杭州310029

出　　处：《蚕业科学》2006年第4期512-516,共5页ACTA SERICOLOGICA SINICA

基　　金：浙江省教育厅科研计划项目(编号20020040)。

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

关 键 词：人白细胞介素-4基因 克隆 表达 家蚕杆状病毒表达载体系统 

分 类 号：Q78[生物学—分子生物学] S881.2[农业科学—特种经济动物饲养]