Expression And Purification Of BNBD5 Cow And Analysis Of Its Bacteriostatic Activity In Xinjiang Holstein

Mammitis, caused by Streptococcus, Staphylococcus aureus and Escherichia coli, is one of the most costly diseases in the dairy industry at present. There is not any effective preventions and cure methods. Researchers found that when bovines mastitis occurs,β-defensin5 mRNA content expression was upregulated, and mastitis can increase the expression quantity of BNBD5 for about 13 times than normal. In-situ hybridization also showed that pathogenic microorganisms induceβ-defensin5 in the region of local infection of mammary epithelial cell increased dramatically. So they predict that the gene play an important role to fight against mastitis.Currently, extraction of polypeptides from in vivo is too low, and synthesize polypeptides antificially cost too high. Therefore, a set of genetic engineering techniques to express and purify recombinant BNBD5 in E. coli system with high efficiency is desired to be established in our project. We use the blood of Xinjiang Holstein bovine with mastitis. After extracted RNA from neutrophilic granulocyte. Using reverse transcription-polymerase chain reaction (RT-PCR) to amplify the cDNA sequence of BNBD5. Then encoding DNA which was amplified by PCR, then clone to pGEX-4T-2 vector. Thus expression vector pGEX-4T-2/BNBD5 has been obtained. It was then used to transform E. coli. BL21 (DE3) to produce expression strain. Which was induced by IPTG, after ultrasonication of the thallus, lysate was purified through GST affinity chromotograph, and fusion protein was obtained. As the result show, it is optimal to induce BNBD5 fusion protein producing strain with 0.4mM IPTG of 4hs at 37℃. After the recombinant was induced by IPTG, Recombinant protein with molecular mass of 33 ku was obtained. Most of the recombinant protein exist in inclusion body, after 8M Urea denaturation, renaturation and purified by GST affinity chromatography. Finally BNBD5 fusion protein purify is 0.181mg/mL. After ultrafiltration and concentration, at last product of 1.81mg/mL was obtained. The purified recombinant BNBD5 protein had antibacterial activity against E. coli and Staphyloccocus aureus.In this study, an economical method with good performance for obtain recombinant BNBD5 has been primitively established. And it is a good preparation for further study.