| Single-chain variable fragments of antibodies(scFvs)have the characteristics of small molecular weight,short half-life,low immunogenicity,stable structure,etc.They are widely used in treatments,diagnoses and researches.In this study,a scFv antibody library of large capacity and high diversity was constructed for the screening of scFvs,and a strategy of codon-pair optimization was evaluated for the high-level expression of scFvs in Pichia pastoris.In order to construct a scFv antibody library with large capacity and high diversity,120 human peripheral blood samples were collected from the healthy individuals,RNAs were extracted from the lymphocytes isolated from the blood samples,c DNAs encoding variable fragments of antibody heavy and light chains were amplified by PCR and successfully assembled with the linker to form DNA fragments coding the scFvs.After the scFv-coding DNA fragments were inserted into phagemid p CANTAB 5E,they were transformed into E.coli TG1 strain by electroporation for amplification.Finally,the helper phage M13KO7 was used to infect TG1 to form a phage display library of scFv antibodies.The final capacity of human scFv antibody library was 9.10×10~5,and that of phage titration was 2.15×10~9 pfu.The sequencing results showed that the scFv coding sequences from the randomly selected positive TG1 colonies were in accordance with the predicted ones which all contain five elements:the restriction sites of Sfi?and Not?,the Linker(G4S)3 sequence,g3 signal peptide and E tag,and the open reading frames encoding scFvs.In order to increase the expression levels of scFv in P.pastoris,we evaluated the influence of codon-pair context sequence on the expression level of scFv.We first designed and developed a optimization software based on codon-pair context bias of P.pastoris.To determine whether the codon-pair context sequence affects the expression of scFv in P.pastoris,we selected two scFvs(ADAM17 and MT1-MMP)as reporter proteins in this study.The coding sequences with the highest score and the lowest score of ADAM17 and MT1-MMP scFvs were designed using the codon pair optimization software,and synthesized respectively.The expression of the coding sequence with the lowest score of scFv ADAM17 was too low to be detected,and a weak expression of the coding sequence with the lowest score of scFv MT1-MMP was detected by western blot.Both the sequences of scFvs ADAM17 and MT1-MMP with the highest scores were detected to be expressed at high-level.These results demonstrated that codon-pair context plays an important role on the protein expression in P.pastoris.Currently,the optimization of foreign protein sequences expressed in the yeast is almost based on the codon usage bias.In order to compare the influences of codon usage bias and codon-pair context bias on the expression of scFv in P.pastoris,the optimized sequences of ADAM17 and MT1-MMP scFv based on the codon usage bias were designed and synthesized.The results showed that the expression levels of the optimized scFv sequences based on the codon-pair context bias were significantly higher than those traditionally based on the codon usage bias which was 5.37 times higher in the expression of scFv ADAM17,and 7.37 times in the expression of MT1-MMP scFv,indicating that sequence optimization based on the codon-pair context is better than those based on the codon usage when a scFv is expressed in P.pastoris.In conclusion,we constructed a non-reproducible human scFv antibody library,which expandeds the capacity of the existing scFv antibody library,increases the possibility of obtaining human scFv against new antigens,and lays a foundation for the subsequent development of new human scFv clinical drugs.Moreover,the development of optimization software for codon-pair context and the evaluation of this strategy are of great significance to the P.pastoris expression system,which improves the expression yield of scFv in P.pastoris,making scFv easier to obtain in large quantities. |
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