OKP-B-13型β-内酰胺酶的克隆与表达

Objective To express OKP-B-13 β-lactamase in pET26b(+)/BL21(DE3) system. Method The plasmid in the strain was extracted; PCR was used for amplification of OKP-B-13 gene. After digested with Nde I and Xho I, OKP-B-13 gene was cloned into pET-26b (+) vector. Before transformed into E. coli. BL21 (DE3), the OKP-B-13 gene in recombinant plasmid was confirmed by digestion and DNA sequencing. OKP-B-13 β-lactamase was expressed after induced by IPTG. Protein extraction was processed by Ultrasonic, and the protein activity was detected by Nitrocefin. The protein isoelectric point (pI) was determined by isoelectric focus. Result A 879 bp amplified product was obtained by PCR. The result of digestion test and DNA sequence of the recombinant vector verified that the target gene had been successfully cloned into the expression vector. The recombinant protein was shown to have β-lactamase activity by Nitrocefin test, indicating that the expression vector [pET-26b(+)/OKP-B-13] was constructed successfully. The pI of OKP-B-13 was 7.1. Conclusion OKP-B-13 gene can be expressed in prokaryote cell, which may provide foundation for further analysis of this novel β-lactamase.