Cloning, expression and antibacterial activity of BNBD5 in Xinjiang Holstein Cow | 新疆荷斯坦牛β防御素5基因的克隆、表达及表达产物的抑菌活性

Chinese. 克隆和表达新疆荷斯坦牛中性粒细胞防御素5(BNBD5)基因，对BNBD5蛋白进行纯化并分析其抗菌活性。利用RT-PCR方法扩增新疆荷斯坦奶牛BNBD5的cDNA序列，将BNBD5 cDNA克隆到原核表达载体pGEX-4T-2，测序分析后转化大肠埃希菌 BL21(DE3)。经IPTG诱导后，对表达产物进行SDS-PAGE和Western blotting分析；利用GST亲和柱纯化BNBD5蛋白，并进行体外抑菌试验。通过PCR扩增获得了218 bp的牛BNBD5全长cDNA序列，经IPTG诱导，获得了约33 ku的牛BNBD5蛋白。牛BNBD5蛋白在15~25 ℃培养时的可溶性较37 ℃时多，且大多以包涵体形式存在，经纯化后最终获得了少量的目的蛋白。纯化的BNBD5蛋白对标准金黄色葡萄球菌和标准大肠埃希菌均具有明显的抑菌活性。克隆、表达了牛BNBD5基因，表达的BNBD5蛋白对大肠埃希菌和金黄色葡萄球菌都有抑菌活性。

English. The study was done to clone and express the bovine beta-defensin 5 gene, purify the recombinant beta-defensin 5 protein and analyze its antibacterial activity. BNBD5 gene was amplified from bovine neutrophil cDNA using reverse transcription-polymerase chain reaction (RT-PCR) and inserted into pGEX-4T-2 vector. After confirmed by sequencing, recombinant pGEX-4T-2/ BNBD5 was transformed into E. coli. BL21 (DE3).Recombinant BNBD5 protein was analyzed by SDS-PAGE and Western blotting with IPTG induction and purified by GST affinity chromatography. The antibacterial activity was assayed by agar hole diffusion-inhibition zone method. Complete BNBD5 cDNA coding sequence which was identical to that of published was successfully obtained by PCR. Soluble BNBD5 protein in 15C25 ℃ cultured was more than that in 37 ℃, and most of them in the form of inclusion bodies. Recombinant protein with relative molecular mass of 33 ku was produced and purified. Antibacterial activity outcomes demonstrated that the recombinant BNBD5 protein has the function to suppress the growth of E. coli and Staphyloccocus. Cloning and expression of bovine BNBD5 gene were obtained, and recombinant BNBD5 expressed with E. coli has suppression activity for E. coli and Staphyloccocus._