DC-SIGN蛋白在家蚕系统中的表达及生物活性研究

Study on DC-SIGN Protein Expression and Its Biological Activity Measurement

目的:构建人DC-SIGN基因片段的家蚕表达系统,进行目的产物表达、鉴定及生物活性分析。方法:从体外刺激分化的DC细胞中克隆出DC-SIGNcDNA,在家蚕表达载体pBacPAK8的BamHⅠ和EcoRⅠ位点构建成重组质粒pBacPAK8-DC-SIGN,与线性化的Bm-BacPAK6病毒基因组DNA共转染家蚕细胞,空斑筛选得到重组病毒Bm-BacPAK-DC-SIGN,重组病毒感染家蚕细胞BmN,Westernblot检测表达产物;HIV-1包膜糖蛋白gp120与表达产物孵育检测其生物活性。结果:构建了稳定表达人DC-SIGN蛋白片段的家蚕杆状病毒表达系统;成功表达了DC-SIGN蛋白片段,且能特异性地与HIV-1包膜糖蛋白gp120结合。结论:成功地在家蚕杆状病毒表达系统中表达了人DC-SIGN蛋白片段,具有天然DC-SIGN蛋白样的生物活性,为其抗体制备及AIDS防治药物的研发奠定了基础。

Objective： To construct expression system of Bombyx mori baculovirus expressing DC-SIGN protein , and to measure its biological activity. Methods：Human DC-SIGN cDNA was amplified by RT-PCR from the mature dendritic cells. The DC-SIGN gene was inserted into the baculovirus transfer vector pBacPAK8, and the recombinant plasmid pBaePAK8-DC-SIGN were cotransfected into Bombyx mori cell lines with the linearized Bm-BacPAK6 virus DNA. Then recombination in the cells and screening by the plaque assay, the recombinant virus Bm-BacPAK-DC-SIGN was obtained and again infected into Bombyx mori cell lines to express DC-SIGN protein which was detected by Western blot. The biological activity of the expressed DC-SIGN protein was measured by its combination with HIV-1 envelope glycoprotein （gp120）. Results： Bombyx moil baculovirus expression system expressing human DC-SIGN protein was constructed, Human DC-SIGN protein was expressed and it can bind specifically with HIV-1 gp120. Conclusion： Human DC-SIGN protein was successfully expressed in expression system of Bombyx mori baculovirus, which showed the natural DC-SIGN protein-like biological activity. It found the basis of the antibody preparation against it and drug study on prevention and treatment of AIDS.