前蛋白转换酶枯草溶菌素9纳米疫苗的构建与细胞吞噬效率的体外实验研究

Establishment of proprotein convertase subtilisin/kexin type 9 nanovaccine and in vitro study on cellular uptake efficiency

目的:制备前蛋白转换酶枯草溶菌素9(proprotein convertase subtilisin/kexin type 9,PCSK9)纳米疫苗,并初步探讨树突状细胞(dendritic cells,DC 2.4)对纳米疫苗的体外吞噬效率。方法:选取PCSK9207-223为抗原肽,牛血清白蛋白(bovine se-rum albumin,BSA)为载体蛋白,用"还原-热聚-氧化"法将BSA分子转变为BSA纳米颗粒(BSA nanoparticle,BN),PCSK9多肽链接在BN表面合成纳米疫苗(BSA-PCSK9 nanoparticle,BPN),PCSK9多肽与分子BSA直接链接合成分子疫苗(BSA-PCSK9,BP)。动态光散射测定BN、BPN粒径和电位大小,透射电子显微镜观察形貌;SDS-PAGE电泳分析抗原负载情况;检测BPN不同时间点的粒径变化反映其在生理环境下的稳定性;BPN与DC 2.4细胞共孵育24 h,增强型细胞计数试剂盒-8(cell counting kit-8,CCK8)检测DC2.4细胞活性,反映BPN生物相容性;流式细胞仪检测DC 2.4对纳米疫苗的吞噬情况。结果:合成的BPN粒径为(68.2±3.1)nm、电位为(?14.8±0.6)mV,透射电子显微镜观察显示BPN近球形;SDS-PAGE电泳结果显示BPN相对分子质量增加,证明短肽链接成功;模拟生理环境下,BPN粒径在144 h内保持稳定;增强型CCK8检测结果显示DC2.4细胞活性均大于100%;流式细胞仪检测结果显示,相比BP,DC 2.4对BPN的吞噬效率更高。结论:BPN体外稳定性及生物相容性好,易于被DC吞噬。

Objective To prepare proprotein convertase subtilisin/kexin type 9(PCSK9)nanovaccine and preliminarily discuss its in vitro uptake efficiency by dendritic cells(DC 2.4).Methods PCSK9207-223was selected as antigen peptide,with bovine serum albumin(BSA)as carrier pro-tein,so as to transform BSA molecules into BSA nanoparticles(BNs)by the"reduction-heating-oxidation"method.PCSK9 peptide was linked to the sur-face of BNs to synthesize BSA-PCSK9 nanoparticles(BPNs).PCSK9 peptide was directly linked with BSA molecules to synthesize molecular vaccine BSA-PCSK 9(BP).The particle size and potential of BNs and BPNs were determined by dynamic light scattering and the morphology was observed un-der a transmission electron microscope.Antigen loading was analyzed by sodium dodecyl sulfate-poyacrylamide gel electrophoresis(SDS-PAGE).The particle size changes of BPNs at different time points were measured to assess its stability under the physiological environment.BPNs were incubated with DC 2.4 for 24 h,and DC 2.4 viability was tested by enhanced cell counting kit-8(CCK8)to reflect BPN biocompatibility.The uptake of DC 2.4 to-wards nanovaccine was assessed by flow cytometry.Results The synthesized BPNs had a particle size of(68.2±3.1)nm and a potential of(?14.8±0.6)mV,with an almost spherical shape under a transmission electron microscope.SDS-PAGE results showed an increase in the relative molecular weight of BPNs,which suggested the successful linkage of the short peptide.The particle size of BPNs remained stable under the mimic physiological en-vironment over 144 h.Enhanced CCK8 results indicated that the viability of DC 2.4 was over 100%.According to flow cytometry,DC 2.4 exhibited high-er uptake efficiency towards BPNs than BPs.Conclusions BPNs have good in vitro stability and biocompatibility and are easily to be uptaken by DC.