Construction of Prokaryotic Expression Vector for Ag85A-HA2 Fusion Gene and Studies on the Immunity Efficacy of Fusion Protein Against Influenza a Virus
目的 构建结核杆菌分泌型抗原85A（Ag85A)与流感病毒血凝素（HA）中HA2（Ag85A-HA2）原核表达载体，并表达融合蛋白，研究其抗流感病毒的免疫保护效果。方法 构建Ag85A-HA2原核表达载体pET-32a(+)/Ag85A-HA2；异丙基-β-D-硫代吡喃半乳糖苷（IPTG）诱导表达Ag85A-HA2融合蛋白，十二烷基磺酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析表达产物，并使用吸附多聚组氨酸标签（His-Tag）的蛋白纯化柱分离纯化蛋白；甲型流感病毒（influenza A virus，IAV）攻击重组蛋白免疫后的BALB/c小鼠（并设PBS对照组），通过观察小鼠肺病理切片、测定肺指数及肺指数抑制率、死亡保护率等指标分析其免疫保护效果。结果 成功构建了原核表达载体pET-32a(+)/Ag85A-HA2；SDS-PAGE电泳分析显示成功表达相对分子质量为70×103的重组融合蛋白；动物实验表明，融合蛋白Ag85A-HA2对IAV攻击后的小鼠肺指数抑制率达到39.30%，死亡保护率达到80%，效果明显高于PBS对照组（P<0.05），肺部病理切片也证实融合蛋白Ag85A-HA2对小鼠肺部的保护效果显著。结论 成功构建了Ag85A-HA2原核表达载体并表达出融合蛋白，该融合蛋白在动物体内能发挥良好的抗甲型流感病毒感染的免疫保护效果。
英文摘要:
Objective To construct Ag85A-HA2 prokaryotic expression vector, express the fusion protein and study the immunity efficacy of fusion protein against influenza A virus. Methods Ag85A-HA2 prokaryotic expression vector was constructed and induced with IPTG．The fusion protein was identified by SDS-PAGE and purified with His-Tag affinity chromatography. The BALB/c mice were immunized with fusion protein. Then the pathological section, lung index, lung inhibitory rate and death-protection rate were tested to evaluate the immunity efficacy of fusion protein. Results pET-32a(+)/Ag85A-HA2 prokaryotic expression vector was constructed successfully. And SDS-PAGE indicated that fusion protein was expressed correctly with a molecular mass of 70×103. The lung index and death-protection rate in experimental group were 39.30% and 80%, higher than that of control group. The pathological section also demonstrated that Ag85A-HA2 fusion protein had a protective effect on murine lungs. Conclusion Ag85A-HA2 prokaryotic expression vector was successfully constructed, inducible expression and the fusion protein had an immunity efficacy against influenza A virus in animal experiment.
