[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(R)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(R)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(R)杆状病毒表达系统中获得融合表达,目的蛋白约70 kD;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.%[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 Westem 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.
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