抗体基因点突变、插入和缺失体外检测方法的建立及应用

Establishment and application of an in vitro detection method for antibody gene point mutation,insertion and deletion

目的·建立由激活诱导胞苷脱氨酶(activation-induced cytidine deaminase,AID)引起的体细胞高频突变过程中,快速检测抗体基因可变(variable,V)、多样(diversity,D)、连接(joining,J)区发生突变(包括点突变、插入和缺失事件)的体外方法。应用该方法检测经2种DNA聚合酶β(polymeraseβ,Polβ)抑制剂处理的细胞中抗体基因VDJ片段的突变情况。方法·用慢病毒感染法处理CH12F3细胞(即处理组),感染前的CH12F3细胞为对照组。采用蛋白质印迹法(Western blotting)检测2组细胞中AID的表达水平。构建高通量测序文库,应用生物信息学的方法分析处理组和对照组细胞中抗体基因VDJ片段的点突变、插入及缺失频率。采用不同浓度的Polβ抑制剂[扎西他滨(2’,3’-dideoxycytidine,DDC)、5-甲氧基黄铜(5-methoxyflavone,5-MF)]处理CH12F3细胞,并采用台盼蓝拒染法测定该2种抑制剂对细胞增殖的影响。分别以筛选得到的DDC浓度、5-MF浓度处理CH12F3细胞(即实验组),以0.9%NaCl处理的细胞及DMSO处理的细胞依次记为上述实验组的对照组,使用上述已建立的方法进行处理,最终行高通量测序,分析该2种抑制剂对抗体基因VDJ片段中点突变、插入及缺失频率的影响。结果·Western blotting结果显示,和CH12F3细胞对照组相比,CH12F3细胞处理组中AID表达较高;且高通量测序结果显示,该组在抗体基因VDJ片段上存在大量的点突变,且插入和缺失的频率均较高(均P=0.000)。台盼蓝拒染法检测显示,处理CH12F3细胞最适宜的DDC浓度为100μmol/L、5-MF浓度为25μmol/L。最终的高通量测序结果显示,和0.9%NaCl对照组相比,经100μmol/L DDC处理的细胞在抗体基因VDJ片段的点突变频率较低(P=0.000),长度为1 bp的缺失频率亦较低(P=0.009);和DMSO对照组相比,经25μmol/L 5-MF处理的细胞在抗体基因VDJ片段的点突变频率较低(P=0.000),长度大于1 bp的插入和缺失频率亦有所下降(均P=0.000)。结论·该研究建立的体外检测方法可用于分析AID引起的抗体基因VDJ片段的突变事件。Polβ抑制剂DDC和5-MF对由AID引起的抗体基因VDJ片段的突变事件具有抑制作用。

Objective·To establish an in vitro method for rapidly detecting mutations,including point mutation,insertion and deletion events in the variable(V),diversity(D)and joining(J)regions of antibody genes in the process of somatic hypermutations caused by activation-induced cytidine deaminase(AID).The above method was used to detect the effects of two DNA polymeraseβ(Polβ)inhibitors on the mutations in the VDJ regions of antibody genes.Methods·CH12F3 cells were treated with lentivirus infection(i.e.treatment group),and the CH12F3 cells before infection were used as control group.Western blotting was used to detect the expression level of AID in the two groups of cells.The high-throughput sequencing libraries were constructed,and the frequencies of point mutation,insertion and deletion in the VDJ regions of antibody genes in the two groups of cells were analyzed by bioinformatics.CH12F3 cells were treated with different concentrations of Polβinhibitors[2’,3’-dioxycytidine(DDC)and 5-methoxyflavone(5-MF)],and the effects of the two inhibitors on cell proliferation were measured by Trypan blue exclusion method.CH12F3 cells(i.e.experimental group)were treated with the selected DDC concentration or 5-MF concentration,respectively;the cells treated with 0.9%NaCl or DMSO were recorded as the control group of the above experimental group,respectively.The four groups of cells before and after lentivirus infection were detected by the above established methods,and finally high-throughput sequencing was carried out to analyze the effects of the two inhibitors on the frequencies of point mutation,insertion and deletion in the VDJ regions of antibody genes.Results·Western blotting results showed that compared with CH12F3 cells in the control group,the expression of AID in CH12F3 cells in the treatment group was higher;and the high-throughput sequencing results showed that there were a large number of point mutations in the VDJ regions of antibody genes of CH12F3 cells in the treatment group,and its frequencies of insertion and deletion were also higher than those of the control group(both P=0.000).Trypan blue exclusion method showed that the optimal concentration of DDC and 5-MF for CH12F3 cells was 100μmol/L and 25μmol/L,respectively.The highthroughput sequencing results showed that compared with the 0.9%NaCl control group,the point mutation frequencies in the VDJ regions of antibody genes were decreased after cells were treated by 100μmol/L DDC(P=0.000),and its 1 bp deletion frequencies decreased slightly(P=0.009);compared with DMSO control group,the frequencies of point mutation(P=0.000),longer than 1 bp insertion and deletion(both P=0.000)in the VDJ regions of antibody genes were decreased after cells were treated by 25μmol/L 5-MF.Conclusion·The in vitro detection method established in this study can be used to analyze the AID-induced mutation events of VDJ regions of antibody genes.The Polβinhibitors DDC and 5-MF can inhibit the mutations in the VDJ regions of antibody genes induced by AID.