表达猴痘病毒表面蛋白A35R和E8L稳转细胞株的构建与应用

Establishment and Application of Stable Cell Lines Expressing Mpox Virus Surface Protein A35R and E8L

【目的】构建稳定表达猴痘病毒(Mpox virus,MPXV)表面蛋白A35R和E8L的稳转细胞株,用于定性或定量分析相应猴痘表面蛋白的抗体或互作分子与细胞表面A35R或E8L结合活性。【方法】运用基因重组技术,构建表达A35R或E8L的慢病毒表达载体,以HEK-293T作为宿主细胞,通过慢病毒转染技术形成稳定表达并定位在细胞表面A35R或E8L的稳转细胞株。采用Western Blot及细胞免疫荧光检测工程化细胞株中A35R或E8L的表达及蛋白定位情况,并运用流式细胞术验证稳转细胞与抗血清中多克隆抗体的结合能力。分别从表达A35R或E8L的稳转细胞中筛选出单克隆细胞株,并以单克隆稳转细胞作为工具定量分析商业化抗猴痘A35R或E8L单克隆抗体与细胞表面A35R或E8L的结合活性。【结果】成功构建稳定表达A35R或E8L并定位至细胞膜上的HEK-293T稳转细胞,经过单克隆化筛选,建立4株单克隆细胞株,其中2株稳定表达A35R(A35-2C10和A35-4E3)和2株稳定表达E8L(E8-6和E8-8)。筛选出的单克隆细胞在定量分析抗猴痘A35R或E8L的单克隆抗体结合活性方面显示出良好的拟合度。【结论】通过慢病毒转染技术构建的表达猴痘表面A35R或E8L的稳转细胞株可以作为通用研究工具,应用于抗A35与E8L中和抗体、核酸适配体或互作分子等相关分子与相应抗原结合活性的定性或定量分析。

【Objective】A stable cell line expressing the MPXV(Mpox virus)surface proteins A35R or E8L was established and utilized for qualitative or quantitative analysis of the binding activities of antibodies or molecules associated with A35R or E8L on the cell surface.【Method】Lentivirus vectors coding for A35R or E8L were constructed using gene recombination technology,and A35R and E8L were expressed and localized on the cell surface of engineered HEK-293T cell lines that were transduced by lentivirus carrying A35R or E8L.The expressions and localizations of A35R and E8L in the engineered cells were detected using Western Blot and immunofluorescence,and the binding affinity of the engineered cells to polyclonal antibodies was analyzed by flow cytometry.Finally,monoclonal cell lines were screened from the engineered cell lines and applied in the quantitative analysis of the binding activity of commercial anti-A35R or anti-E8L monoclonal antibodies.【Result】Stable HEK-293T cell lines expressing A35R or E8L antigens on the surface were successfully established.Four monoclonal cell lines were selected through monoclonal screening,among which two stably expressed A35R(A35-2C10 and A35-4E3)and two stably expressed E8L(E8-6 and E8-8).These monoclonal cell lines showed good fit in quantitative analysis of the binding activity of anti-A35R or E8L mAb.【Conclusion】Cell lines with stable expressions of MPXV A35R or E8L may serve as tools and can be applied to assess the binding activities of neutralizing antibodies,nucleic acid aptamers,or other molecules associated with A35R or E8L in qualitative or quantitative analysis.