Cloning and expression of cDNA of bovine neutrophil β-defensin12 from holstein cow and its antibacterial activity | 荷斯坦牛中性粒细胞防御素BNBD12基因克隆、原核表达及其抗菌活性分析

chinois. 【目的】实现荷斯坦牛中性粒细胞防御素BNBD12基因在大肠杆菌中的高效表达,并分析重组BNBD12的抗菌活性。【方法】RT－PCR扩增荷斯坦牛中性粒细胞BNBD12基因,序列分析后人工合成了适于在大肠杆菌中表达的BNBD12成熟肽,构建原核表达载体pET32a(+)/BNBD12,并在大肠杆菌BL21（DE3）中诱导表达。通过琼脂扩散法分别确定重组蛋白对牛源乳腺炎主要致病菌革兰氏阴性菌和革兰氏阳性菌的抗菌效果,应用扫描电镜观察重组蛋白的杀菌效应。【结果】测序结果表明扩增片段长度为180 bp,可编码含60个氨基酸的成熟蛋白。SDS－PAGE和Western blotting分析显示人工合成的BNBD12的成熟肽基因以融合蛋白形式表达,表达量占菌体总蛋白的21.4%;纯化的重组蛋白0.05 mg?mL-1对大肠杆菌、金黄色葡萄球菌有明显抑菌效果;扫描电镜观察发现重组蛋白作用后可引起病原菌内容物外泄而死亡。【结论】成功表达了荷斯坦牛中性粒细胞β-防御素12,该重组蛋白既可作用于革兰氏阳性菌又可作用于革兰氏阴性菌,显示出较好的临床应用前景。

anglais. 【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.