表达猴痘病毒表面蛋白 A35R和E8L稳转细胞株的构建与应用

doi:10.13560/j.cnki.biotech.bull.1985.2024-0366

摘要/Abstract

摘要：

【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。

关键词: 猴痘病毒, A35R, E8L, 慢病毒转染, 稳转细胞株

Abstract:

【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.

Key words: Mpox virus, A35R, E8L, lentivirus transduction, stable cell line

金浙东, 李惠宜, 包汶鑫, 崔彩霞, 袁运生. 表达猴痘病毒表面蛋白 A35R和E8L稳转细胞株的构建与应用[J]. 生物技术通报, 2024, 40(10): 315-322.

JIN Zhe-dong, LI Hui-yi, BAO Wen-xin, CUI Cai-xia, YUAN Yun-sheng. Establishment and Application of Stable Cell Lines Expressing Mpox Virus Surface Protein A35R and E8L[J]. Biotechnology Bulletin, 2024, 40(10): 315-322.

图/表 6

图1 重组A35R、E8L慢病毒表达载体图谱

Fig. 1 Map of recombinant A35R and E8L lentiviral expression vectors

图2 Western Blot鉴定HEK-293T细胞表达A35R（22.2 kD）、E8L（38.3 kD）蛋白 A35R以抗His标签抗体为一抗，HRP标记的兔抗鼠IgG为二抗；E8L以抗E8L抗体为一抗，HRP标记的山羊抗人IgG为二抗

Fig. 2 Detection of A35R（22.2 kD）and E8L（38.3 kD）proteins expressed in HEK-293T cells by Western Blot For A35R, anti-His antibody was used as the primary antibody, HRP linked rabbit anti-mouse IgG was used as the secondary antibody. For E8L, anti-E8L antibody was used as the primary antibody, HRP linked goat anti-human IgG was used as the secondary antibody

图3 免疫荧光检测HEK-293T细胞中A35R（A）、E8L（B）的表达物镜放大倍数：40×

Fig. 3 Detection of expression of A35R (A) and E8L (B) in HEK-293T cells by immunofluorescence Object lens: 40×

图4 293T-A35（A）、293T-E8（B）与抗血清的流式结合分析未免疫小鼠血清为阴性对照，所有血清均以1∶300稀释，下同

Fig. 4 Binding affinity analysis of 293T-A35（A）and 293T-E8L（B）with anti-serum by flow cytometry Serum from non-immunized mice was used as negative control, all serum was diluted at 1∶300, the same below

图5 A35-2C10（A）、A35-4E3（B）、E8-6（C）以及E8-8（D）稳转单克隆细胞株与抗血清的流式结合分析

Fig. 5 Binding affinity analysis of A35-2C10（A）, A35-4E3（B）and E8-6（C）and E8-8（D）with anti-serum by flow cytometry

图6 A35-4E3与A35R单克隆抗体（A）、E8-8与E8L单克隆抗体（B）的流式结合分析抗体初浓度为50 μg/mL，并3倍倍比稀释。虚线对应位置为EC50

Fig. 6 Binding affinity analysis of A35-4E3 with anti-A35R mAb（A）and E8-8 with anti-E8L mAb（B）by flow cytometry The initial concentration of antibodies was 50 μg/mL and tripling dilution. The dashed line indicates EC50

参考文献 20

[1]	McCollum AM, Damon IK. Human monkeypox[J]. Clin Infect Dis, 2014, 58(2): 260-267. doi: 10.1093/cid/cit703 pmid: 24158414
[2]	Cabanillas B, Murdaca G, Guemari A, et al. A compilation answering 50 questions on monkeypox virus and the current monkeypox outbreak[J]. Allergy, 2023, 78(3): 639-662. doi: 10.1111/all.15633 pmid: 36587287
[3]	Lum FM, Torres-Ruesta A, Tay MZ, et al. Monkeypox: disease epidemiology, host immunity and clinical interventions[J]. Nat Rev Immunol, 2022, 22(10): 597-613.
[4]	Kumar N, Acharya A, Gendelman HE, et al. The 2022 outbreak and the pathobiology of the monkeypox virus[J]. J Autoimmun, 2022, 131: 102855.
[5]	Heraud JM, Edghill-Smith Y, Ayala V, et al. Subunit recombinant vaccine protects against monkeypox[J]. J Immunol, 2006, 177(4): 2552-2564.
[6]	Li MJ, Ren ZN, Wang YL, et al. Three neutralizing mAbs induced by MPXV A29L protein recognizing different epitopes act synergistically against orthopoxvirus[J]. Emerg Microbes Infect, 2023, 12(2): 2223669.
[7]	Tamir H, Noy-Porat T, Melamed S, et al. Synergistic effect of two human-like monoclonal antibodies confers protection against orthopoxvirus infection[J]. Nat Commun, 2024, 15(1): 3265. doi: 10.1038/s41467-024-47328-y pmid: 38627363
[8]	Hu H, Zhang ZY, Wang RY, et al. BGC823 cell line with the stable expression of iRFP720 retains its primary properties with promising fluorescence imaging ability[J]. DNA Cell Biol, 2020, 39(5): 900-908. doi: 10.1089/dna.2019.5057 pmid: 32096664
[9]	Xiao XY, Chen H, Paerhati P, et al. Establishment and application of engineered NIH 3T3 cell line with stable human RAGE expression[J]. Pharm Fronts, 2020, 2(1): e23-e27.
[10]	Yong SEF, Ng OT, Ho ZJM, et al. Imported monkeypox, Singapore[J]. Emerg Infect Dis, 2020, 26(8): 1826-1830. doi: 10.3201/eid2608.191387 pmid: 32338590
[11]	Tang D, Liu XK, Lu J, et al. Recombinant proteins A29L, M1R, A35R, and B6R vaccination protects mice from mpox virus challenge[J]. Front Immunol, 2023, 14: 1203410.
[12]	Hou FJ, Zhang YT, Liu XH, et al. mRNA vaccines encoding fusion proteins of monkeypox virus antigens protect mice from vaccinia virus challenge[J]. Nat Commun, 2023, 14(1): 5925. doi: 10.1038/s41467-023-41628-5 pmid: 37739969
[13]	King LB, West BR, Moyer CL, et al. Cross-reactive neutralizing human survivor monoclonal antibody BDBV223 targets the ebolavirus stalk[J]. Nat Commun, 2019, 10(1): 1788. doi: 10.1038/s41467-019-09732-7 pmid: 30996276
[14]	Ma H, Guo YY, Tang HN, et al. Broad ultra-potent neutralization of SARS-CoV-2 variants by monoclonal antibodies specific to the tip of RBD[J]. Cell Discov, 2022, 8(1): 16. doi: 10.1038/s41421-022-00381-7 pmid: 35169121
[15]	Matho MH, de Val N, Miller GM, et al. Murine anti-vaccinia virus D8 antibodies target different epitopes and differ in their ability to block D8 binding to CS-E[J]. PLoS Pathog, 2014, 10(12): e1004495.
[16]	Ward BM, Weisberg AS, Moss B. Mapping and functional analysis of interaction sites within the cytoplasmic domains of the vaccinia virus A33R and A36R envelope proteins[J]. J Virol, 2003, 77(7): 4113-4126. pmid: 12634370
[17]	Gilchuk I, Gilchuk P, Sapparapu G, et al. Cross-neutralizing and protective human antibody specificities to poxvirus infections[J]. Cell, 2016, 167(3): 684-694.e9. doi: S0092-8674(16)31334-4 pmid: 27768891
[18]	Milone MC, O'Doherty U. Clinical use of lentiviral vectors[J]. Leukemia, 2018, 32(7): 1529-1541. doi: 10.1038/s41375-018-0106-0 pmid: 29654266
[19]	Wang XY, Ma CC, Rodríguez Labrada R, et al. Recent advances in lentiviral vectors for gene therapy[J]. Sci China Life Sci, 2021, 64(11): 1842-1857. doi: 10.1007/s11427-021-1952-5 pmid: 34708326
[20]	Shearer RF, Saunders DN. Experimental design for stable genetic manipulation in mammalian cell lines: lentivirus and alternatives[J]. Genes Cells, 2015, 20(1): 1-10. doi: 10.1111/gtc.12183 pmid: 25307957