基于藏香猪肠道内容物宏基因组文库的新型抗菌肽挖掘及其高效分泌表达体系构建

Antimicrobial peptides are a class of low molecular weight proteins, having many biological functions such as anti-viral, anti-bacterial, anti-fungal and inhibiting cancer cell activities. They are an important alternative to traditional antibiotics, and have a wide range of applications in bio-medicine, food preservation, healthy culture and plant-disease control fields. Although a variety of antimicrobial peptides was identified from different organisms, their resistances to hydrolysis and bactericidal properties still can not meet the growing demand for industrial and agricultural applications. Scientific investigation showed that, intestinal contents in extreme environment contains a wide variety of microbial population including bacteria, fungi and ancient bacteria. Thus, development of intestinal content microbial resources will enable us to get some multi-purpose antimicrobial peptides with good characters and suitability for industrial production. This project intends to study the Tibetan pig intestinal contents as the starting material, and to build their metagenomic libraries, and to discover novel antimicrobial peptides with valuable merits in industrial and animal husbandry by using function-based screening method of antimicrobial peptides, then to determine their spatial structures using automated Edman degradation sequencing technology and nuclear magnetic resonance spectroscopy, subsequently to optimize and synthesize the gene sequence using bioinformatics accordingly, and then to construct the extracellular expression system of novel antimicrobial peptide based on Bacillus subtilis using the genetic engineering technology. Our study will supply not only novel antimicrobial peptides for industry and animal husbandry application, but also a new perspective for the design and the improvement of antimicrobial peptide molecules, in addition to a foundation to exploit the marine resources.

抗菌肽是一类具有抗病毒、抗真菌及抗细菌等生物学功能的小肽，是重要的抗生素潜在替代品，在畜牧业绿色养殖及生物医药等领域有着广泛应用前景。尽管已有多种抗菌肽从不同生物体中得到鉴定，但现有抗菌肽产品在耐酶解性能和杀菌效价方面仍不能满足日渐增长的相关产业应用需求。研究表明，处于极端环境中的肠道内容物蕴含着包括古菌、真菌和细菌在内的种类繁多的微生物群落。因而，开发肠道微生物资源定能获得一些性状优良、适于产业需求的新型抗菌肽。本课题拟以藏香猪肠道内容物为研究出发材料，构建宏基因组文库，利用基于抗菌肽功能的筛选方法挖掘有产业应用价值的新型抗菌肽，再应用Edman测序技术和核磁共振技术测析其结构，在此基础上应用表达系统不同组件的最优组合建立新型抗菌肽的枯草芽孢杆菌高效分泌表达体系。本项目可为畜牧业和相关生物产业资供新抗菌肽来源，也可为抗菌肽分子改良提供理论基础，更可为今后开发肠道微生物资源奠定基础。

1 研究内容.本项目构建了基于枯草芽孢杆菌的肠道内容物宏基因组文库，通过应用抗菌肽抑菌功能的筛选方法从文库中筛取了186个产抗菌肽阳性克隆子，挖掘出了在耐酶解能力和抑菌效价等方面有优良生物学特性的新型抗菌肽Numbsin028，最后建立了其高效枯草芽孢杆菌分泌表达体系pSUBNU028，初步系统地探索出了一种从挖掘、评价到开发新型抗菌肽的新方法。.1.1挖掘出了具有良好耐酶解能力和抑菌效价的新型抗菌肽Numbsin028.采用了抗菌肽抑菌功能的筛选方法，从藏香猪肠道内容物宏基因组文库中获取了186个产抗菌肽的阳性克隆子，发酵所获得的阳性克隆子制备抗菌肽样品。探讨了常见酶、温度、盐度、酸碱度及有机溶剂对其稳定性的影响，并考察了其对常见耐药细菌的抑菌效价。根据以上所得数据筛取了1种具有良好生物学特性的新型抗菌肽Numbsin028。.1.2测析了Numbsin028的结构特征并优化出其适宜于枯草芽孢杆菌表达的基因序列numbsin028.应用Edman降解法（automated Edman degradation）分析出了Numbsin028一级结构，为KWKLVVTTWIKPKPVPRQCM；采用核磁共振波谱技术测定并解析了其二级结构特征，是由一个典型的α螺旋结构组成。在阐明了Numbsin028结构的基础上，应用生物信息学方法和偏爱密码子技术计算优化出了适宜于枯草芽孢杆菌表达的Numbsin028的DNA序列，即AAATGGAAACTKGTTGTTACAACATGGATTAAACCGAAACCGGTTCCGAGACAATGCATG。.1.3构建了可高效分泌表达Numbsin028的枯草芽孢杆菌基因工程菌株pSUBNU028.依据优化后合成的DNA序列，从枯草芽孢杆菌表达系统不同类型的元件中选取了Pglv启动子、Samy信号肽、WB700宿主菌作为最适元件，，构建出了可高效表达分泌Numbsin028的枯草芽孢杆菌工程菌株pSUBNU028。.2研究结果.应用宏基因组技术从藏香猪肠道内容物里筛选出1条有优良生物学功能活性新型抗菌肽基因numbsin028，优化出了适宜于枯草芽孢杆菌表达Numbsin028的DNA序列AAATGGAAACTKGTTGTTACAACATGGATTAAACCGAAACCGGTTCCGAGACAATGCATG，最终构建出了可高效合成具有产业应