Chen Shan, He Feng-Tian, Dong Yan-Lin, Li Rong-Fen, Gao Hui-Guang, Chen Min, Peng Jia-He
Department of Biochemistry and Molecular Biology, the Third Military Medical University, Chongqing 400038, China.
Sheng Wu Gong Cheng Xue Bao. 2004 Jul;20(4):490-5.
In recent years, Bacterial resistance is more and more serious for the irrational use of antibiotics produces resistant strains and other reasons. Human are trying to solve the problem from different ways, including the study of antimicrobial peptides. Defensin is one of the most important of antimicrobial peptides. A novel antimicrobial peptide, human beta-defensin 3, was isolated and demonstrated a salt-insensitive broad spectrum of potent antimicrobial activity against many potentially pathogenic microbes. The total RNA was extracted from human tonsil and the hbetaD-3 specific cDNA sequence was amplified with RT-PCR. After sequenced, the target DNA fragment was cloned into pQE-80L vector together with the DNA fragment encoding carrier protein DHFR. The recombinant vectors were transformed into E. coli M15 and the expression was induced based on the optimal values of the IPTG concentration incubation temperature and induction time determined in the previous section. The expressed proteins were analyzed by SDS-PAGE and Western-blotting. The mass of the recombinant protein was about 40% of total bacteria protein. Isolate and purify the target protein. The recombinant hbetaD-3 fusion proteins possess the antimicrobial activity to staphylococcus aureus, multiresistant staphylococcus aureus (only vancomycin-sensitive) and Candida albicans in the assay of drug susceptibility. Advanced study can be continued based on our experiments.
近年来，由于抗生素的不合理使用产生耐药菌株等原因，细菌耐药性问题日益严重。人们正尝试从不同途径解决这一问题，其中包括对抗菌肽的研究。防御素是最重要的抗菌肽之一。一种新型抗菌肽——人β-防御素3被分离出来，并显示出对许多潜在致病微生物具有盐不敏感的广谱强效抗菌活性。从人扁桃体中提取总RNA，通过逆转录聚合酶链反应（RT-PCR）扩增hbetaD-3特异性cDNA序列。测序后，将目标DNA片段与编码载体蛋白二氢叶酸还原酶（DHFR）的DNA片段一起克隆到pQE-80L载体中。将重组载体转化到大肠杆菌M15中，并根据上一节确定的异丙基-β-D-硫代半乳糖苷（IPTG）浓度、培养温度和诱导时间的最佳值进行诱导表达。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质免疫印迹法（Western-blotting）分析表达的蛋白质。重组蛋白的质量约占细菌总蛋白的40%。分离并纯化目标蛋白。在药敏试验中，重组hbetaD-3融合蛋白对金黄色葡萄球菌、多重耐药金黄色葡萄球菌（仅对万古霉素敏感）和白色念珠菌具有抗菌活性。可基于我们的实验继续深入研究。
