关键词: β-防御素12（BNBD12）, 荷斯坦牛, 表达, 抗菌活性, 乳腺炎

Abstract:

【Objective】 The gene coding the bovine neutrophil beta-defensin12 was cloned from Holstein cow and the antibacterial activity of the recombinant protein was analyzed. 【Method】 BNBD12 gene was amplified using reverse transcription-polymerase chain reaction (RT-PCR). The PCR product was analyzed, and the mature peptide gene was synthesized and inserted into vector pET32a(+) to construct plasmid pET32a(+)/BNBD12, then the plasmid was expressed in E. coli BL21 (DE3) cell that induced by IPTG. The antibacterial activity was assayed by agar hole diffusion-inhibition zone method and electron microscope using the purified recombination protein against E. coli and Sta. aureu. 【Result】 Sequence analysis showed the amplicon 180 bp encoding a polypeptide of 60 amino acids. SDS-PAGE and Western blotting results showed that the recombinant proteins were expressed in E. coli with molecular weight of 26 kD and the recombinant proteins accounted for 21.4% of the whole proteins. The purified protein BNBD12 0.05 mg?mL-1 had antibacterial activity and killed pathogenic bacteria by leaking the content out in vitro against E. coli and Sta. aureus. 【Conclusion】 The results showed that BNBD12 protein is well expressed in E. coli and it has antibacterial activity against E. coli and Sta. aureus.

Key words: bovine β-defensin 12 (BNBD12), holstein cows, expression, antibacterial activity, mastitis