基于猪细小病毒病毒样颗粒的结肠癌靶向纳米载体的构建

【目的】获得具有结肠靶向的纳米载体。【方法】采用SOE-PCR方法将具有结肠靶向的TK肽序列插入到猪细小病毒(PPV)结构蛋白VP2的环2和环4区域得到TK-vp2(?vp2)基因,在Bac-to-Bac?杆状病毒表达系统中构建、表达和自组装。【结果】通过SOE-PCR方法扩增获得?vp2基因,在Bac-to-Bac?杆状病毒表达系统中构建得到Bacmid-?vp2,经脂质体转染至Sf9昆虫细胞得到重组杆状病毒。直接免疫荧光试验、SDS-PAGE和Western blot检测结果表明?VP2蛋白在Bac-to-Bac?杆状病毒表达系统中获得融合表达,目的蛋白约70 k D;透射电子显微镜结果显示?VP2能自组装形成病毒样颗粒(TK-VLPs),直径范围在22 nm-30 nm。【结论】获得纳米载体TK-VLPs,为进一步研究其作为结肠靶向的纳米载体奠定物质基础。

Construction of colon-targeted nanocarriers based on porcine parvovirus-like particles

Objective] To prepare colon-targeted nanocarriers. Methods] SOE-PCR methods were used to prepare TK-vp2(?vp2) by inserting TK peptide gene into porcine parvovirus VP2 structural protein of loop2 and loop4. Then, TK-VP2 protein was constructed, expressed and self-assembled in the Bac-to-Bac? baculovirus expression system. Results] The ?vp2 gene obtained through SOE-PCR methods, Bacmid-?vp2 was constructed in the Bac-to-Bac? baculovirus expression system and the recombinant baculovirus was successfully constructed in Sf9 insect cells. The results of the direct immunofluorescence, SDS-PAGE and Western blot assays showed that the ?VP2 proteins were expressed in the Bac-to-Bac? baculovirus expression system and the protein was approximately 70 kD. The transmission electron microscopy (TEM) results indicated that the ?VP2 proteins can self-assemble to form virus-like particles (TK-VLPs) ranging between 20 and 30 nm. Conclusion] TK-VLPs nanocarrier was obtained, providing a basis to further study the feasibility of TK-VLPs as colon targeting nanoparticle.