Kang J, Zhao D, Lyu Y, Tian L, Yin X, Yang L, Teng K, Zhou X
The State Key Lab of Agrobiotechnology; Key Lab of Animal Epidemiology and Zoonosis, Ministry of Agriculture; National Animal Transmissible Spongiform Encephalopathy Laboratory; College of Veterinary Medicine, China Agricultural University, Beijing, 100193, People's Republic of China,
Eur J Clin Microbiol Infect Dis. 2014 Oct;33(10):1823-34. doi: 10.1007/s10096-014-2152-5. Epub 2014 May 20.
Tuberculosis (TB) is an ongoing threat to global health, and the lack of effective therapies for treating it is also a global problem. Previous studies have shown that human cathelicidin and defensins have effective antimicrobial activity against Mycobacterium spp. To our knowledge, there are no reports on the antimycobacterial effects of bovine neutrophil β-defensins so far. Here, we identified the antimicrobial effect of mature bovine neutrophil β-defensins (mBNBD) 5 against Mycobacterium infection both in vitro and in vivo. The mBNBD5 protein was expressed in Pichia pastoris. To increase the yield of β-defensins, a purification method was employed by adding a 6-His·tag to the C-terminus of the mBNBD5 gene. Our results indicated that recombinant mBNBD5 protein was successfully expressed and purified from Pichia pastoris with intact antimicrobial activity. The recombinant protein exhibited potent bactericidal activity in vitro against M. smegmatis and M. bovis, with a dose-dependent manner and a time-dependent manner. The electron microscope results showed that the bacterial cell wall of M. bovis was disrupted when incubated with mBNBD5 for 72 h. Our data also indicated that the exogenous addition of mBNBD5 could reduce the survival of Mycobacterium spp., especially M. tuberculosis and M. bovis in RAW 264.7 macrophages. These results provide foundations for the development of mBNBD5 as a potential new therapeutic agent for TB treatment.
结核病（TB）对全球健康构成持续威胁，且缺乏有效的治疗方法也是一个全球性问题。先前的研究表明，人源杀菌肽和防御素对分枝杆菌具有有效的抗菌活性。据我们所知，目前尚无关于牛中性粒细胞β-防御素抗分枝杆菌作用的报道。在此，我们鉴定了成熟牛中性粒细胞β-防御素（mBNBD）5在体外和体内对分枝杆菌感染的抗菌作用。mBNBD5蛋白在毕赤酵母中表达。为提高β-防御素的产量，采用了在mBNBD5基因的C末端添加6-组氨酸标签的纯化方法。我们的结果表明，重组mBNBD5蛋白已在毕赤酵母中成功表达并纯化，且具有完整的抗菌活性。该重组蛋白在体外对耻垢分枝杆菌和牛分枝杆菌表现出强效杀菌活性，呈剂量依赖性和时间依赖性。电子显微镜结果显示，与mBNBD5孵育72小时后，牛分枝杆菌的细胞壁被破坏。我们的数据还表明，外源添加mBNBD5可降低RAW 264.7巨噬细胞中分枝杆菌的存活率，尤其是结核分枝杆菌和牛分枝杆菌。这些结果为开发mBNBD5作为一种潜在的结核病治疗新药物奠定了基础。
