检测B细胞成熟抗原的噬菌体展示单链抗体实时荧光定量免疫PCR方法建立

Establishment of real-time fluorescent quantitative immuno-PCR based on phage displayed scFv for quantitative determination of BCMA

目的建立检测B细胞成熟抗原(BCMA)的噬菌体展示单链抗体实时荧光定量免疫PCR(PDRT-IPCR)方法。方法采用无缝克隆技术将抗体ANTI-BCMA片段连接入噬菌体M13KO7中,构建重组噬菌体质粒M13KO7-ANTI-BCMA,保存于E.coli DH5α感受态细胞中,通过PEG/NaCl溶液低温沉降后获得重组噬菌体M13KO7-ANTI-BCMA。采用Western Blotting法鉴定重组噬菌体M13KO7-ANTI-BCMA是否展示抗BCMA单链抗体。以不同浓度的BCMA包被96孔高吸附酶标板,加入重组噬菌体M13KO7-ANTI-BCMA,热裂解后,以裂解噬菌体的DNA作为扩增模板,进行实时荧光定量PCR,观察不同浓度BCMA的实时荧光定量PCR扩增曲线。运用OriginPro2021软件进行四参数Logistic拟合曲线,根据相关系数R^(2)确定线性范围,计算最低检测限。以重组噬菌体M13KO7-ANTI-CD19和噬菌体M13KO7为对照,验证PDRT-IPCR方法检测BCMA的特异性。结果抗BCMA单链抗体成功展示在重组噬菌体M13KO7-ANTI-BCMA表面。随着BCMA包被浓度的降低,对应扩增曲线的CT值逐渐变大,PDRT-IPCR检测BCMA的线性范围为0.1 ng/mL~1000 ng/mL,R^(2)为0.9993,最低检测限为0.077 ng/mL。重组噬菌体M13KO7-ANTI-BCMA与BCMA的结合是特异性的。结论本研究建立了检测BCMA的PDRT-IPCR方法,该检测方法具有检测限灵敏、线性范围宽、特异性高等特点。

Objective To establish a real-time fluorescent quantitative immuno-PCR(PDRT-IPCR)method for detecting B-cell maturation antigen(BCMA)based on phage display single chain antibody(scFv).Methods The antibody ANTI-BCMA fragment was ligated into phage M13KO7 by seamless cloning technique,and the recombinant phage plasmid M13KO7-ANTI-BCMA was constructed and stored in E.coli DH5αcompetent cells.The recombinant phage M13KO7-ANTI-BCMA was obtained after low temperature sedimentation of PEG/NaCl solution.Western blotting was used to identify whether the recombinant phage M13KO7-ANTI-BCMA displayed anti-BCMA scFv.The 96-well high adsorption enzyme plate was coated with different concentrations of BCMA,and the recombinant phage M13KO7-ANTI-BCMA was added.After pyrolysis,the DNA of the lysed phage was used as the amplification template for real-time fluorescence quantitative PCR,and the real-time fluorescence quantitative PCR amplification curves of different concentrations of BCMA were observed.The four-parameter Logistic fitting curve was carried out by using OriginPro2021 software,the linear range was determined according to the correlation coefficient R2,and the lowest detection limit was calculated.The specificity of PDRT-IPCR method for the detection of BCMA was verified with recombinant phage M13KO7-ANTI-CD19 and phage M13KO7 as controls.Results Anti-BCMA scFv was successfully displayed on the surface of recombinant phage M13KO7-ANTI-BCMA.With the decrease of BCMA coating concentration,the CT value of the corresponding amplification curve gradually increased.The linear range of BCMA detected by PDRT-IPCR was 0.1-1000 ng/mL,R2 was 0.9993,and the lowest detection limit was 0.077 ng/mL.The binding of recombinant phage M13KO7-ANTI-BCMA to BCMA was specific.Conclusion In this study,a PDRT-IPCR method for the detection of BCMA is established,which has the characteristics of sensitive detection limit,wide linear range and high specificity.