5型和35型腺病毒纤毛蛋白的原核表达及活性验证

Prokaryotic Expression and Activity Detection of Ad5/Ad35 Fiber Gene

目的:原核表达5型腺病毒纤毛蛋白(Ad5 fiber)和35型腺病毒纤毛蛋白(Ad35 fiber),并对蛋白活性进行初步分析。方法:将重组质粒pET28a-Ad5 fiber和pET28a-Ad35 fiber转化入E.coli BL21(DE3),IPTG诱导表达后,经Ni-NTA凝胶珠亲和纯化,用SDS-PAGE鉴定纯化蛋白,并用蛋白竞争性实验验证纯化蛋白的活性。结果:转化溶原菌后能够表达目的蛋白,Ad35 fiber蛋白对Ad5(5型腺病毒)感染无影响,只能阻断Ad5F35(5型腺病毒纤毛被替换为35型腺病毒纤毛的嵌合腺病毒)对细胞的感染;Ad5 fiber蛋白对嵌合腺病毒Ad5F35感染无影响,只能抑制Ad5对细胞的感染。结论:成功表达具有生物活性的5型和35型腺病毒纤毛蛋白,为进一步研究嵌合腺病毒Ad5F35感染细胞的机制奠定基础。

Aim： To construct the prokaryotic expression vector for Ad5 fiber and Ad35 fiber genes, purify proteins and study the activity. Methods： Recombinant plasmid pET28a-Ad5 fiber and pET28a-Ad35 fiber were transformed into E. coli Bl21 （DE3） and the proteins expression were induced with IPTG. The expressed proteins Ad5 fiber and Ad35 fiber were purified by Ni2~ affinity chromatography and identified by SDS-PAGE. The bioactivity of proteins Ad5 fiber and Ad35 fiber were certificated by competitive binding method. Results： The proteins Ad5 fiber and Ad35 fiber can be detected in the supernatant. The proteins Ad5 fiber specifically inhibited the infectivity of the Ad5 virus in a dose-dependent manner, while had no effect on the Ad5F35- mediated gene transfer. The proteins Ad35 fiber blocked the Ad5F35-mediated gene transfer in a dose-dependent manner and had no effect on the infectivity of the Ad5 virus in competition experiments. Conclusion： Recombinant vectors Ad5 fiber and Ad35 fiber were constructed successfully. The proteins Ad5 fiber and Ad35 fiber could specifically bind with different receptors. The study will lay a foundation for the investigation on the mechanism of Ad5F35 infection.