Optimized protocol for detection of native, full-length HIV-1 envelope on the surface of transfected cells
Optimized protocol for detection of native, full-length HIV-1 envelope on the surface of transfected cells
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DOI：
10.1002/hsr2.74
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发表时间：
2018-09-01
影响因子：
2
通讯作者：
Powell, R. L. R.
中科院分区：
文献类型：
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作者：
Altman, J. B.;Liu, X.;Powell, R. L. R.
设计针对HIV包膜糖蛋白（Env）的治疗方法只能与它们靶向的Env结构一样准确。保存Env三聚体的结构对于抗体结合和中和的适当实验评估至关重要。然而，Env特别难以通过转染重组Env质粒来表达。为了增加表面表达，研究人员通常利用HIV-1的gp 41跨膜糖蛋白的c-尾突变体，但该区域的突变和缺失会影响Env三聚体的整体构象和稳定性。多项研究表明，虽然尾部突变体具有更高的Env表面表达，但与体内感染细胞上发现的野生型Env相比，它们更容易中和并具有改变的三聚体构象。为了评估和表征天然细胞表面Env结构，我们寻求可以通过流式细胞术可靠地检测野生型Env表面表达的方案。方法和结果通过避免基于胎牛血清的缓冲液，显著增加转染的质粒和Env特异性抗体的量，并通过选择明亮的生物素+链霉亲和素-PE检测系统，我们能够增加转染的Env蛋白的表面表达。结论该方法可以更准确地评估抗体结合、表位暴露和Env结构，有助于设计更有效的疫苗和免疫治疗药物。
Aims Designing therapeutics against the HIV envelope glycoprotein (Env) is only as accurate as the structure of the Env they are targeting. Conserving the structure of the Env trimer is crucial for proper experimental assessment of antibody binding and neutralization. However, Env is notably difficult to express by transfection of a recombinant Env plasmid. To increase surface expression, researchers commonly utilize c-tail mutants of the gp41 transmembrane glycoprotein of HIV-1, but mutations and deletions in this region can impact the overall conformation and stability of the Env trimer. Multiple studies have shown that while tail mutants have higher Env surface expression, they are easier to neutralize and have altered trimer conformations compared with wild-type Env found in vivo on infected cells. To assess and characterize native cell surface Env structures, we sought a protocol that could reliably detect wild-type Env surface expression by flow cytometry. Methods and results By avoiding fetal bovine serum-based buffers, significantly increasing the amounts of transfected plasmid and Env-specific antibody and by selecting a bright, biotin + streptavidin-PE detection system, we were able to increase the surface expression of transfected Env protein. Conclusion This protocol will allow for more precise assessment of antibody binding, epitope exposure, and Env structure, all of which will contribute to designing more effective vaccines and immunotherapeutics.