关键词: Tn3; 易错PCR; DNA重排; 定向进化; 酶切产物; 基因序列

Abstract: The aim was to explore the improvement and evolution of the enzyme activity of the transposase Tn3.The in vitro directional evolution of transposase Tn3 was studied by primers design and synthesis,PCR amplification,restriction enzyme digestion,error-prone PCR optimization and construction,DNA rearrangement construction and optimization, D-value determination and enzyme digestion identification.The results showed that the recombinant plasmids were digested with SacⅠ and XbaⅠand 450 bp enzyme digestion products were obtained.After 24 h culture in LB medium containing kanamycin, D-value was measured.After three rounds of repeatability studies,D-value and proliferation ability increased from 0 to 0.18,0.42 and 0.60,indicating that Tn3 activity was significantly improved.The selected evolution-type recombinant plasmid was extracted by plasmid extraction,and the endonuclease XhoⅠ and XbaⅠ were used for double digestion identification.It was found that the bands of the cleavage products of the evolutionary recombinant plasmids were relatively small.After that,gene sequencing analysis of the recombinant plasmid of evolutionary type showed that mutations occurred in multiple sites of Tn3 gene sequence and CCR5-delta32 gene in the middle of Tn3-Gal4 targeted sequence was removed.It showed that the cloning of the transposase Tn3 gene was successfully completed in this study.The optimal reaction system for error-prone PCR was established,it was feasible to use the LB medium containing kanamycin and D-value of the bacterial solution for screening.Mutations in the gene sequence of Tn3 and excision of the gene indicate that Tn3 had changed both in function and in gene sequence,which was exactly in the direction of need.

Key words: Tn3; error-prone PCR; DNA recomposition; directed evolution; enzyme product; gene sequence