关键词: 人杀菌/渗透增强蛋白, 定向进化, 易错PCR, DNA改组

Abstract: Objective To construct and identify a gene mutation library of the N-terminal fragments of human bactericidal/permeability increasing proteins (BPI₂₃) for enhancing its activity binding lipopolysaccharides (LPS) by error-prone polymerase chain reaction (PCR) and DNA shuffling method.Methods Error-prone PCR was used to obtain random mutant fragments of BPI₆₀₀ by adding different concentrations of Mg²⁺ and Mn²⁺.Random mutation rate was calculated by DNA sequencing and genetic comparison with wild-type BPI₆₀₀.The product of error-prone PCR was further mutated via DNA shuffling.The reassembled product was cloned into pYD1 vector and transformed into E.coli XL10-Gold.Ten clones were randomly picked out, digested and identified by restriction enzyme HindⅢ and XhoⅠ. The positive clones were identified by DNA sequencing. Results DNA sequencing result showed that the random mutation rate was 2.3% by error-prone PCR. After DNA shuffling, 6 positive clones including BPI₆₀₀ were identified in the 10 random clones selected from LB culture medium with ampicillin. Among them, 4 had 6, 9, 11 and 14 mutations, respectively; 1 had 10 mutations and 1 deletion determined by DNA sequencing. By amino acid comparison, only 2 DNA mutants were able to encode a full-length mutant BPI₂₃ protein, with 4 and 14 amino acid mutations respectively.Conclusion A library of BPI₂₃ mutants was constructed with a size of 2×10⁵.The results laid the foundation for the study of its activity binding LPS.

Key words: human bactericidal/permeability increasing protein, directed evolution, error-prone polymerase chain reaction (PCR), DNA shuffling