哺乳动物细胞表面展示IgG抗体Fc组合突变库的构建及评估

Construction and evaluation of IgG antibody Fc combinational mutation library with mammalian cell surface display system

目的·在单点突变文库筛选结果和多个简并引物引入组合突变的基础上,建立一种高效获得高通量的哺乳动物细胞膜表面展示IgG抗体Fc(fragment crystallizable domain)组合突变文库的方法。方法·单点突变文库经流式细胞术分选获得与Fcγ受体IIB(FcγRIIB)高亲和力的位于EU编号233~240位点的氨基酸突变;根据这些突变设计多个简并引物,将引物等比混合或按引物所包含突变多样性占比混合后进行PCR获得含有组合突变的DNA片段;利用重组酶连接片段与载体后转化大肠埃希菌DH5α,获得质粒文库;质粒文库转染Pheonix细胞,制备反转录病毒,然后感染3T3细胞,获得细胞表面表达有Fc突变的细胞文库;使用流式细胞术检测质粒转染Pheonix细胞的效率,以及病毒感染3T3细胞的效率;高通量测序评估质粒文库和细胞文库的构建情况。结果·根据单点突变文库筛选结果,设计32条简并引物,可以覆盖所有期望的组合突变,并且实际文库多样性为期望文库的3.6倍(41600/11520);高通量测序结果显示,相较于引物等比混合获得的质粒文库,按引物所包含突变多样性占比混合所获得的质粒文库具有更好的均一性与完整性,能够覆盖期望文库中99.58%的组合突变类型(11472/11520);一代测序结果显示,10条序列中有3条为期望文库突变,与简并引物设计时计算的多样性相符;流式细胞术的结果显示,Pheonix细胞的转染效率为51.6%,3T3细胞的感染效率为47.4%;高通量测序结果显示,所获得的细胞文库能够覆盖期望文库中85.92%的组合突变(9898/11520)。结论·在已有单点突变文库筛选的氨基酸突变基础上设计多个简并引物,并通过PCR、质粒转染和反转录病毒感染,能够高效、低成本地获得文库覆盖率超过85%的抗体Fc组合突变质粒文库和哺乳动物细胞表面展示文库。

Objective·To establish an efficient method to obtain a high throughput mammalian cell surface display library of IgG antibody Fc combined mutations based on the screening results of the single point mutation library and the introduction of combined mutations by multiple degenerate primers.Methods·Multiple degenerate primers were designed according to the mutated amino acids at 233-240 of EU numbering with high affinity to Fcγreceptor IIB screened by fluorescence-activated cell sorting from the single point mutation library;the DNA fragments containing combined mutations were obtained through PCR by using the degenerate primers mixed with equal proportion,or according to the proportion of mutation diversity;the recombinant enzyme was used to ligate the fragment to the vector in one step and then transformed into E.coli DH5α,and the plasmid library was obtained.The plasmid library was used to transfect Pheonix cells to prepare retroviruses,and then 3T3 cells were infected with the retroviruses to obtain mammalian cell library that expressed Fc mutations on the surface.Flow cytometry was used to analyze the transfection efficiency of Pheonix cells to package retroviruses and the infection efficiency of 3T3 cells by the retroviruses.The quality of the plasmid library and the cell library was evaluated by next generation sequencing(NGS).Results·According to the point mutation library screening results,32 degenerate primers were designed,which could cover all desired combined mutations,and the actual library diversity was 3.6 times that of the expected library(41600/11520).The results of NGS showed that the plasmid library obtained by using proportionally mixed primers according to the primer diversity had better homogeneity and integrity,and could cover 99.58%of the expected combinational mutations(11472/11520).Sanger sequencing results showed that3 sequences among 10 sequences were the expected mutations,which were consistent with the diversity calculated during degenerate primers design.Flow cytometry showed that the transfection efficiency of Pheonix cells was 51.6%,and the infection efficiency of 3T3 cells was 47.4%.NGS results showed that the cell library could cover 85.92%of the expected combinational mutations(9898/11520).Conclusion·Based on the amino acid mutations screened from the single point mutation library,multiple degenerate primers were designed,and then through PCR,plasmid transfection and retrovirus infection,an IgG Fc combined mutation plasmid library and mammalian cell surface display library with more than 85%coverage of the expected combined mutations can be obtained efficiently at low cost.