促吞噬肽功能化的HBc VLPs纳米载体的制备  

Preparation of Tuftsin Functionalized HBc VLPs Nanocarrier

作　　者：李春华 杨静[2] 任晋 李蕾 曹艺明 王鑫[2] 李卫国[1] 毕胜利[3] 王升启 LI Chun-Hua;YANG Jing;REN Jin;LI Lei;CAO Yi-Ming;WANG Xin;LI Wei-Guo;BI Sheng-Li;WANG Sheng-Qi(College of Life Sciences,Henan Normal University,Xinxiang 453007,Henan,China;Institute of Radiation Medicine,Academy of Military Medical Sciences,Beijing 100850,China;Institute of Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China)

机构地区：[1]河南师范大学生命科学学院,河南新乡453007 [2]军事科学院军事医学研究院辐射医学研究所,北京100850 [3]中国疾病预防控制中心病毒病预防控制所,北京102206

出　　处：《中国生物化学与分子生物学报》2020年第7期804-810,共7页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家重大新药创制课题(No.2015ZX09J15105-003,No.2016ZX09J16104-001-003,No.2017ZX09303013-003);国家自然科学基金(No.81703429);国家传染病重大专项(No.2018ZX10102001-002-003)资助。

摘　　要：促吞噬肽(Tuftsin)是机体脾组织产生的生理活性肽,具有强大的免疫调节和免疫治疗潜力。乙型肝炎病毒核心蛋白病毒样颗粒(hepatitis B virus core protein virus-like particles, HBc VLPs)是由HBc自组装形成的空心纳米颗粒,其不仅能应用于药物的递送,还能应用于外源蛋白质的显示。因此,Tuftsin功能化HBc VLPs载体的研究在免疫治疗、分子递送等方面具有重要意义。本研究选用PET43.1-a质粒作为Tuftsin-HBc VLP的表达载体,以大肠杆菌BL21 (DE3)为工程菌进行诱导表达,通过盐析、分子筛层析和离子交换层析技术纯化生产Tuftsin-HBc VLP。利用Western印迹和ELISA分别对Tuftsin-HBc VLP上HBc和Tuftsin进行定性分析。结果显示,Tuftsin-HBc VLP可与抗HBc抗体和抗Tuftsin抗体发生特异性结合。透射电镜观察结果显示,Tuftsin-HBc VLP呈大小均一的球形结构,粒度分析仪测得Tuftsin-HBc VLP的直径约为30 nm。CCK8法显示,Tuftsin-HBc VLP在0~480μg/mL范围内,细胞增殖未见显著变化。上述结果表明,本研究成功制备了可以在原核系统中高效表达且安全有效的Tuftsin-HBc VLP生物纳米递送载体。Tuftsin is a physiologically active peptide produced by the spleen, which has potent immunomodulatory and immunotherapeutic potential. Hepatitis B virus core protein virus-like particles(HBc VLPs) are hollow nanoparticles and self-assembled by HBc, which can be used not only for drug delivery, but also for the display of foreign proteins. Therefore, the study of Tuftsin functionalized HBc VLPs nanocarrier is of great significance in immunotherapy and molecular delivery. In this study, the PET43.1-a plasmid was used as the expression vector of the Tuftsin-HBc fusion protein, and the E. coli BL21(DE3) strain containing the expression plasmid was induced to express fusion proteins. Tufsin-HBc VLP was purified by salting out, molecular exclusion chromatography and ion-exchange column chromatography. Qualitative analysis of HBc and Tuftsin on Tuftsin-HBc VLP was used by Western blotting and ELISA, respectively. The results showed that Tuftsin-HBc VLP can specifically bind to antibodies against HBc and Tuftsin. Tuftsin-HBc VLP with a uniform spherical structure was observed by transmission electron microscopy(TEM). Tuftsin-HBc VLP has a diameter of about 30 nm by particle size analyzer. The cell proliferation in 0 ~ 480 μg/mL Tuftsin-HBc VLP has no significant change as assessed by CCK-8 assays. These results suggested that the safe and effective Tuftsin-HBc VLP biological nanocarrier was prepared successfully, which can be efficiently expressed in the prokaryotic system.

关 键 词：促吞噬肽 乙型肝炎病毒核心蛋白病毒样颗粒 纳米载体 

分 类 号：R34[医药卫生—基础医学]