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。
Preterm and small-for-gestational-age （SGA） neonates are vulnerable groups that are susceptible to various microbial infections. Vγ9Vδ2-T cells are critical components of the host immune system and have been demonstrated to play an important role in the defense against viral infection in adults. However, the characteristics of Vγ9Vδ2-T cells in children, especially the preterm and SGA populations, are poorly understood. Here, we examined the frequency and antiviral function of Vγ9Vδ2-T cells in neonates, including preterm, SGA and full-term babies. When compared to adults, neonates had a significantly lower percentage of Vγ9Vδ2-T cells in the blood. Upon influenza virus stimulation, neonatalVγ9Vδ2-T cells, especially from preterm and SGA babies, showed markedly decreased and delayed antiviral cytokine responses than those of adults. In addition, the antiviral responses of neonatal Vγ9Vδ2-T cells were positively correlated with gestational age and birth weight. Finally, a weaker expansion ofVγ9Vδ2-T cells by isopentenyl pyrophosphate （IPP） was shown in neonates than the expansion in adults. Our data suggest that the depressed antiviral activity and decreased frequency of Vγ9Vδ2-T cells may likely account for the high susceptibility to microbial infection in neonates, particularly in preterm and SGA babies. Improving Vγ9Vδ2-T -cell function of neonates may provide a new way to defend against virus infection.
γδ-T cells play an indispensable role in host defense against different viruses, including influenza A virus. However, whether these cells have cytotoxic activity against influenza virus-infected lung alveolar epithelial cells and subsequently contribute to virus clearance remains unknown. Using influenza virus-infected A549 cells, human lung alveolar epithelial cells, we investigated the cytotoxic activity of aminobisphosphonate pamidronate （PAM）-expanded human Vγ9Vδ2-T cells and their underlying mechanisms. We found that PAM could selectively activate and expand human Vγ9Vδ2-T cells. PAM-expanded human Vγ9Vδ2-T cells efficiently killed influenza virus-infected lung alveolar epithelial cells and inhibited virus replication. The cytotoxic activity of PAM-expanded Vγ9Vδ2-T cells was dependent on cell-to-cell contact and required NKG2D activation. Perforin-granzyme B, tumor-necrosis factor-related apoptosis-inducing ligand （TRAIL） and Fas-Fas ligand （FasL） pathways were involved in their cytotoxicity. Our study suggests that targeting γδ2-T cells by PAM can potentially offer an alternative option for the treatment of influenza virus.
Hong LiZheng XiangTing FengJinrong LiYinping LiuYingying FanQiao LuZhongwei YinMeixing YuChongyang ShenWenwei Tu
