Patgaonkar Mandar, Aranha Clara, Bhonde Gauri, Reddy K V R
Division of Molecular Immunology, National Institute for Research in Reproductive Health, Indian Council of Medical Research, Jehangir Merwanji Street, Parel, Mumbai 400 012, Maharashtra, India.
Vet Immunol Immunopathol. 2011 Feb 15;139(2-4):176-86. doi: 10.1016/j.vetimm.2010.10.012. Epub 2010 Oct 13.
Antimicrobial peptides (AMPs) serve as a first line of host defense and represent an important, though poorly understood components of the innate immune system. The present study was an attempt to identify and characterize the major molecules having anti-bacterial activities from the vaginal fluid of rabbit, Oryctologus cuniculus. AMPs from the rabbit vaginal fluid (RVF) were identified in the acid extracts of pooled RVF samples after RP-HPLC purification. The protein, RVFAMP was effective against gram negative (Escherichia coli and Pseudomonas aeruginosa) and gram positive (Staphylococcus aureus and Streptococcus pyogenes) bacteria. The results of acid urea-PAGE-gel overlay assay (AU-PAGE-GOA) demonstrated clear zone of growth inhibition of E. coli corresponding to 6 and 15 kDa protein bands. LC-MS data of these proteins indicated that 15 kDa protein consists of lysozyme, lipopolysaccharide binding protein (LBP), hemoglobin-α and β subunits (Hb-α/β), whereas 9 kDa protein band consists of transthyretin and calcyclin while uteroglobulin and neutrophil antibacterial peptide-5 (NAMP-5) are present in the 6 kDa protein band. Of the eight proteins, Hb-α derived protein was further characterized, as it showed the highest Probability Based Mowse Score (PBMS) of 288. A 25mer peptide, RVFHbαp was active against several clinical pathogens as demonstrated by minimum inhibitory concentration (MIC) and radial diffusion assays (RDA). The interaction of RVFHbαP with bacterial liposome membrane was assessed by calcein dye leakage assay. RVFHbαP did not show cytotoxicity against human endocervical cells (End1/E6E7) or erythrocytes. RT-PCR and immunofluorescence results revealed the expression of RVFHbαP mRNA and protein in rabbit vaginal tissue. To the best our knowledge, this is the first report describing the detection of AMPs in RVFs. In conclusion, these studies indicated that vaginal epithelial cells (VEC) derived RVFHbαP may have therapeutic potential in the management of reproductive well being of rabbits. The major reason for undertaking this study in rabbits is that, it forms an excellent in vivo model system for human's studies.
抗菌肽（AMPs）作为宿主防御的第一道防线，是先天免疫系统的重要组成部分，尽管人们对其了解甚少。本研究旨在从穴兔的阴道分泌物中鉴定并表征具有抗菌活性的主要分子。经过反相高效液相色谱（RP-HPLC）纯化后，在合并的兔阴道分泌物（RVF）样本的酸提取物中鉴定出了来自兔阴道分泌物的抗菌肽。蛋白质RVFAMP对革兰氏阴性菌（大肠杆菌和铜绿假单胞菌）和革兰氏阳性菌（金黄色葡萄球菌和化脓性链球菌）均有效。酸性尿素聚丙烯酰胺凝胶覆盖分析（AU-PAGE-GOA）结果显示，对应于6 kDa和15 kDa蛋白条带的区域出现了大肠杆菌生长抑制的清晰区域。这些蛋白质的液相色谱-质谱（LC-MS）数据表明，15 kDa蛋白由溶菌酶、脂多糖结合蛋白（LBP）、血红蛋白α和β亚基（Hb-α/β）组成，而9 kDa蛋白条带由转甲状腺素蛋白和钙结合蛋白组成，子宫球蛋白和中性粒细胞抗菌肽-5（NAMP-5）存在于6 kDa蛋白条带中。在这八种蛋白质中，对源自Hb-α的蛋白质进行了进一步表征，因为它显示出最高的基于概率的Mowse评分（PBMS），为288。如最低抑菌浓度（MIC）和径向扩散分析（RDA）所示，一种25聚体肽RVFHbαp对几种临床病原体具有活性。通过钙黄绿素染料泄漏分析评估了RVFHbαP与细菌脂质体膜的相互作用。RVFHbαP对人宫颈细胞（End1/E6E7）或红细胞未显示出细胞毒性。逆转录-聚合酶链反应（RT-PCR）和免疫荧光结果揭示了RVFHbαP mRNA和蛋白质在兔阴道组织中的表达。据我们所知，这是第一份描述在兔阴道分泌物中检测到抗菌肽的报告。总之，这些研究表明，阴道上皮细胞（VEC）衍生的RVFHbαP在兔生殖健康管理中可能具有治疗潜力。在兔身上进行这项研究的主要原因是，它构成了一个用于人类研究的优秀体内模型系统。
