基于噬菌体展示技术和新型冠状病毒的抗体检测方法构建及验证

Construction of an antibody detection method of SARS-CoV-2 based on phage display technology and observation of its effectiveness

目的构建基于噬菌体展示技术和新型冠状病毒的抗体检测方法,并验证其应用效果。方法制备含有新型冠状病毒受体结合域(RBD)及E484K、N501Y突变RBD片段的重组噬菌体,采用Western blotting法验证重组噬菌体展示效果。使用高吸附ELISA板包被RBD抗体及包被液作为实验组及对照组,分别加入RBD、突变RBD重组噬菌体以结合抗体,qPCR法扩增噬菌体的基因片段以间接地反映抗体与重组噬菌体的特异性结合情况。使用高吸附ELISA板包被经RBD免疫后分离的羊驼血清,加入含RBD片段的重组噬菌体及不含重组片段的噬菌体,qPCR法观察重组噬菌体在生物样本中与RBD抗体的特异性结合情况。使用高吸附ELISA板包被RBD抗体,加入含RBD及含突变RBD的重组噬菌体,qPCR法观察点突变对RBD与抗体结合能力的影响。结果通过Western blotting可以检测到清晰且符合预期大小的重组噬菌体条带。包被RBD抗体的实验组扩增噬菌体基因片段拷贝数均高于对照组(P均<0.01);加入含RBD片段重组噬菌体的羊驼血清扩增片段拷贝数高于加入无外源片段噬菌体的扩增片段拷贝数(P<0.01);加入含RBD片段重组噬菌体的RBD抗体扩增片段拷贝数高于加入含突变RBD片段重组噬菌体的扩增片段拷贝数(P<0.01)。结论成功构建基于噬菌体展示技术的抗体检测方法,该方法展示新型冠状病毒RBD片段的效果良好,并且在研究由病原突变导致的免疫逃逸方面具有潜在价值。

Objective To construct an antibody detection method based on phage display technology and to observe its application in the detection of antibodies against SARS-CoV-2.Methods Recombinant phage containing SARS-CoV-2 receptor-binding domain(RBD)and E484K and N501Y-mutated RBD fragments were prepared and the effect of recombinant phage display was verified by Western blotting.High binding ELISA plates coated with RBD antibody and coating solution were used as the experimental and control groups.RBD and mutant RBD recombinant phage were added to bind the antibody,and the gene fragment of the phage was amplified by qPCR to indirectly reflect the specific binding of the antibody to the recombinant phage.Highly binding ELISA plates were used to coat alpaca sera isolated after immunisation with RBD,and recombinant phages containing RBD fragments and phages without recombinant fragments were added.The qPCR was used to observe the specific binding of recombinant phages to RBD antibodies in biological samples.High binding ELISA plates were used to coat RBD antibodies,and recombinant phages containing RBD and mutant RBD were added.The qPCR was used to observe the effect of point mutations on the ability of antibodies to bind to RBD.Results Western blotting showed that clear bands of the expected size of the recombinant phage were detected.The copy number of gene segment in the amplified phages was higher in the group coated with RBD antibody than in the control group(all P<0.01).The copy number of amplified gene fragments in the recombinant phages with the addition of alpaca serum containing RBD fragment was higher than that with the addition of phage without exogenous fragment(P<0.01).The copy number of the amplified fragment of RBD antibody in the recombinant phages containing the RBD fragment was higher than that of the amplified fragment in the recombinant phages containing the mutant RBD fragment.Conclusion An antibody detection method based on phage display technology is successfully constructed,which displays SARS-CoV-2 RBD fragments well and is potentially valuable in studying immune escape caused by pathogenic mutations.