DC-SIGN蛋白在家蚕系统中的表达及生物活性研究

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-SIGN cDNA，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，Western blot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。

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

Study on DC-SIGN protein expression and its biological activity measurement TANG Hui1 TANG Shao-hui1 YANG Dong-hua1 LV Zheng-bing2 YU Wei2 ZHANG Yao-zhou2 ZHOU Tian-hong3 XIAO Xin4 （1The First Affiliated Hospital，Jinan University,Guangzhou 510632,China）;（2（Institute of Biochemistry,Zhejiang Sci-Tech University,Hangzhou310018 ,China）;（3College of Life Science and Technology,Jinan University,Guangzhou 510632 ,China）;(4the Sixth Affiliated Hospital,Sun Yat-sen University,Guangzhou 510655,China ） [Abastract] Objective:To construct expression system of Bombyx mori baculovirus expressing DC-SIGN protein fragmen, and to measure the biological activity of it. Methods：Human DC-SIGN cDNA was amplied by RT-PCR from the differentiated DCs.The DC-SIGN gene was inserted into the bacubvirus transfer vector pBacPAK8,and the recombiant plasmid pBacPAK8-DC-SIGN were cotransfected into Bombyx mori cell lines with the linearized Bm-BacPAK6 virus genome DNA.After recombination in the cells and screening by the plaque assay, the recombinated virus Bm-BacPAK-DC-SIGN were obtained and again infected into Bombyx mori cell lines to express DC-SIGN protein fragmen which were detected by Western blot. The biological activity of the expressed DC-SIGN protein fragmen was measured by its combination with HIV-1 gp120.Results:Expression system of Bombyx mori baculovirus expressing human DC-SIGN protein fragment was constructed, Human DC-SIGN protein fragment was expressed and it can bind specifically with HIV-1 gp120.Conclusion: Human DC-SIGN protein fragment was successfully expressed in expression system of Bombyx mori baculovirus, which showed the natural DC-SIGN protein-like biological activity. It became the work basis of the antibody preparation against it and drug study on prevention and treatment of AIDS. [Key words]: Human immunodeficiency virus；DC-SIGN；Protein expression；biological activity measurement