ENHANCING COLLECTIN MEDIATED HOST DEFENSE

凝集素是一组与血清和肺表面活性物质相关的物质 被认为参与抗体非依赖性宿主的蛋白质 防御。肺集合素、表面活性蛋白A和D(SP-A 和SP-D)，具有抗病毒和抗菌特性。然而， 初步研究表明，转基因版本的 这些由分子生物学技术产生的蛋白质将是 可能对特定的呼吸道病原体具有增强的活性。 这项提议的目标是产生重组的、突变的集合素 随着对甲型流感病毒(IAV)活性的提高，以及 肺炎链球菌(肺炎球菌)与野生型的比较 收藏品。之所以选择这些病原体，是因为它们代表着 严重呼吸道感染的个别主要原因，而且因为 肺炎球菌重叠感染是IAV的重要并发症 感染。我们已经生产了一种嵌合收集素，它结合了 SP-D的N末端和胶原区与碳水化合物融合 粘附素的识别结构域(CRD)。这种分子有更大的 对IAV和细菌的凝集能力比天然或重组的 凝集素，以及更强的抑制IAV血凝的能力 活性高于粘附素或野生型SP-D。基于这些 很有希望的发现，我们建议建造其他几个嵌合体 具有结合良好功能特性的目标的收集素 将野生型收集素转化为单分子。另外两个 含SP-D融合蛋白N-末端和胶原区的构建 对SP-A或血清凝集素的CRD，甘露糖结合 凝集素(MBL)，将被制成。因为很可能胶原域 另一个嵌合体参与与吞噬细胞受体的结合 构建将包含SP-A的这个结构域和SP-D(或MBL)的CRD。 我们将比较这些重组集合素结合的能力， 聚集、抑制感染性，并作为调理素 临床上重要的IAV和肺炎链球菌菌株。接下来，具体 参与碳水化合物结合的残基将在其中一个 构造以确定是否可以与IAV或肺炎球菌结合 增强版。集合素构建体的体内活性最初将 通过在IAV或IAV之前将它们滴鼻给小鼠进行测试 肺炎球菌感染。其中一个突变结构(选择用于 抗IAV和/或肺炎球菌的最佳活性)然后将被表达 在转基因小鼠的呼吸道中检测体内是否对 感染被加强了。这些实验应该会产生重要的 对基本收藏生物学的见解，并展示了这一增强 可在体内实现胶凝素介导的肺宿主防御 通过对野生型收藏素的合理改造。

The collectins are a group of serum and pulmonary surfactant-associated proteins which are believed to participate in antibody-independent host defenses. The pulmonary collectins, surfactant proteins A and D (SP-A and SP-D), have antiviral and antibacterial properties. However, preliminary research has indicated that genetically altered versions of these proteins produced by molecular biological techniques would be likely to have enhanced activity against specific respiratory pathogens. The goal of this proposal is to generate recombinant, mutant collectins with improved activity against influenza A viruses (IAVs) and Streptococcus Pneumoniae (pneumococci) as compared to the wild type collectins. These pathogens are chosen because they represent individually major causes of serious respiratory infection, and because superinfection with pneumococci is an important complication of IAV infection. We have produced a chimeric collectin which incorporates the N-terminus and collagen domain of SP-D fused with the carbohydrate recognition domain (CRD) of conglutinin. This molecule has a greater ability to agglutinate IAV and bacteria than native or recombinant conglutinin, as well as greater ability to inhibit IAV hemagglutination activity than either conglutinin or wild type SP-D. Based on these promising findings we propose construction of several other chimeric collectins with the goals of combining favorable functional properties of the wild type collectins into single molecules. Two further constructs containing the N-terminus and collagen domain of SP-D fused to the CRDs of either SP-A or the serum collectin, mannose-binding lectin (MBL), will be made. Since it is likely that the collagen domain of SP-A participates in binding to phagocyte receptors another chimeric construct will contain this domain of SP-A and the CRD of SP-D (or MBL). We will compare the ability of these recombinant collectins to bind to, aggregate, inhibit infectivity of, and act as opsonins for, a panel of clinically important IAV and pneumococcal strains. Next, specific residues involved in carbohydrate binding will be mutated in one of the constructs to determine if binding to IAV or pneumococci can be enhanced. In vivo activity of the collectin constructs will initially be tested by instilling them intranasally in mice prior to IAV or pneumococcal infection. One of the mutant constructs (chosen for optimal activity against IAV and/or pneumococci) will then be expressed in the airway of transgenic mice to determine if in vivo resistance to infection is enhanced. These experiments should yield important insights into basic collection biology and demonstrate that enhancement of collectin-mediated pulmonary host defense can be achieved in vivo through rational alteration of wild type collectins.