·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.

·Multiple degenerate primers were designed according to the mutated amino acids at 233?240 of EU numbering with high affinity to Fcγ receptor 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).

·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 (41 600/11 520). 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 (11 472/11 520). Sanger sequencing results showed that 3 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 (9 898/11 520).

·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.