Construction of Hcp immunohistochemical library and antibody expression based on single memory B cell sequencing technology

ZHOU Jinrui; WANG Wenhao; GU Yaru; OU Yangxue; LIU Bixia; ZUO Houyi; DU Yexiang; ZHANG Rui; ZUO Qianfei

doi:10.16016/j.2097-0927.202312007

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Construction of Hcp immunohistochemical library and antibody expression based on single memory B cell sequencing technology

Basic Medicine | 更新时间：2025-07-27

- Construction of Hcp immunohistochemical library and antibody expression based on single memory B cell sequencing technology
- Journal of Army Medical University Vol. 47, Issue 15, Pages: 1782-1791(2025)
- 作者机构：
  
  西南交通大学医学院，四川成都
- 作者简介：
  
  ZHANG Rui， E-mail： echorayray@qq.com
- 基金信息：
  
  the General Program of National Natural Science Foundation of China(31970877);the Project of Science and Technology Plan of Sichuan Province(2021YJ0191)
- DOI：10.16016/j.2097-0927.202312007
  CLC： R378.7;R392-33;R392.7
- Received：02 December 2023，
  
  Revised：20 June 2024，
  
  Published：15 August 2025
- 稿件说明：
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周金锐，王文皓，顾亚茹，等.基于单个记忆B细胞测序技术构建毒素蛋白Hcp免疫组库及抗体表达［J］.陆军军医大学学报，2025，47（15）：1782-1791.DOI：10.16016/j.2097-0927.202312007.

‍ZHOU J R，WANG W H，GU Y R，et al.Construction of Hcp immunohistochemical library and antibody expression based on single memory B cell sequencing technology［J］.J Army Med Univ，2025，47（15）：1782-1791.DOI：10.16016/j.2097-0927.202312007.
周金锐，王文皓，顾亚茹，等.基于单个记忆B细胞测序技术构建毒素蛋白Hcp免疫组库及抗体表达［J］.陆军军医大学学报，2025，47（15）：1782-1791.DOI：10.16016/j.2097-0927.202312007. DOI：

‍ZHOU J R，WANG W H，GU Y R，et al.Construction of Hcp immunohistochemical library and antibody expression based on single memory B cell sequencing technology［J］.J Army Med Univ，2025，47（15）：1782-1791.DOI：10.16016/j.2097-0927.202312007. DOI：

摘要

目的

基于单个记忆B细胞测序技术制备针对鲍曼不动杆菌的人源化单克隆抗体，构建出Hcp蛋白的免疫组库，表达具有结合活性的单克隆抗体。

方法

构建带有pGEX-6p-1-Hcp重组质粒的BL21-Hcp菌液，采用蛋白质表达和蛋白质亲和层析技术获得目的蛋白Hcp；6~8周龄雌性SPF 级的BALB/c小鼠（体质量18~20 g），Hcp抗原肌肉注射免疫小鼠产生特异性记忆B细胞；采用流式细胞术分选单个特异性记忆B细胞；采用单细胞测序技术构建Hcp的免疫组库，并对测序结果进行生物信息学分析；采用抗体人源化改造技术获得单克隆抗体；采用ELISA实验检测抗体体外结合活性。

结果

表达并纯化出纯度大于95%的目的蛋白Hcp；成功构建了Hcp的免疫组库，分析得到了该免疫组库的BCR克隆型鉴定和分析结果、CDR3区域特征分析结果、V-J基因配对特征分析结果；人源化改造了7种单克隆抗体（IgG1-1、IgG1-2、IgG2-1、IgG2-2、IgG3-1、IgG4-1、IgG4-2）；ELISA结果显示，IgG1-1、IgG3-1、IgG4-1、IgG4-2抗体结合效价均为1∶1 280，IgG2-2的抗体效价为1∶10 240，IgG2-1的抗体效价为1∶5 120，IgG1-2的抗体效价为1∶160。

结论

利用单细胞测序技术可以快速、准确、高效率地构建含有大量抗体信息的Hcp蛋白免疫组库，利用免疫组库可以表达具有结合活性的单克隆抗体。

Abstract

Objective‍　‍To prepare humanized monoclonal antibodies (Mabs) targeting

Acinetobacter baumannii

(Ab) based on single memory B cell sequencing technology

construct the immune repertoire of the core protein of Ab

hemolysin-coregulated protein (Hcp)

and express its Mabs with binding activity. Methods‍　‍

E. coli

BL21 harboring the recombinant plasmid pGEX-6p-1-Hcp was constructed. Hcp protein was obtained using protein expression and affinity chromatography. Female SPF BALB/c mice (6~8 weeks old

weighing 18~20 g) were immunized intramuscularly with antigen Hcp to generate specific memory B cells. Single antigen-specific memory B cells were sorted using flow cytometry. The immune repertoire of Hcp was constructed using single-cell sequencing technology

and bioinformatics analysis was performed on the sequencing results. Mabs were obtained using antibody humanization techniques. The

in vitro

binding activity of the antibodies was detected by ELISA. Results‍　‍The target protein Hcp with a purity

95% was obtained after expression and purification. The immune repertoire of Hcp was successfully constructed

and the results of BCR clonotype identification and analysis

CDR3 region characteristic analysis

and V-J gene pairing characteristic analysis were achieved. Antibody humanization got 7 Mabs

that is

IgG1-1

IgG1-2

IgG2-1

IgG2-2

IgG3-1

IgG4-1 and IgG4-2. ELISA re

sults showed IgG1-1

IgG3-1

IgG4-1

and IgG4-2 had an antibody binding titer of 1∶1 280

IgG2-2 of 1∶10 240

IgG2-1 of 1∶5 120

and IgG1-2 of 1∶160. Conclusion‍　‍Single-cell sequencing technology enables rapid

accurate

and efficient construction of an Hcp protein immune repertoire containing extensive antibody information. Utilizing this immune repertoire allows for the expression of Mabs with binding activity.

关键词

Keywords

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