FUNCTIONAL ANTIGENS OF TRICHINELLA SPIRALIS

L1 幼虫特异性的两种保护诱导抗原 将研究旋毛虫的结构 使用免疫学、生化和分子学的方法和功能 生物学方法。 表达和基因组文库 将构建合适的载体并克隆DNA分子 编码 48K 和 50/55K 蛋白的蛋白将被分离。 阳性cDNA克隆的表达将通过菌落鉴定 多价兔抗 48K 和 50/55K 免疫测定 抗体。 将测试含有表位的融合肽 它们对小鼠产生保护作用的能力。 Southern印迹分析 将决定编码两者的DNA的基因组排列 抗原。 旋毛虫各阶段Northern分析 （L1-L4幼虫、成虫和新生幼虫）将揭示阶段- 每种抗原的基因表达的特异性。 的顺序为 将确定每种抗原的 cDNA 和基因组 DNA。 两种蛋白质都可以被磷酸化，此外，48K 抗原结合二价阳离子，特别是Ca2+。 因此肽 将对每个分子进行绘图和测序研究 确定两种蛋白质的磷酸化位点， 以及 48K 分子的 Ca2 结合数量和位点。 一级氨基酸共有序列的计算机分析 DNA 序列将决定疏水性区域 与其他已知序列的同源性程度。 特别的 注意调节蛋白和 48K 之间的相似性 并且将给出 50/55K 抗原，因为初步数据表明 他们可能参与宿主厌氧菌的调节 糖酵解。

Two protection-inducing antigens specific to the L1 larva of Trichinella spiralis will be investigated regarding their structure and function using immunological, biochemical and molecular biological approaches. Expression and genomic libraries in suitable vectors will be constructed and cloned DNA molecules encoding both the 48K and 50/55K protein will be isolated. Expression of positive cDNA clones will be identified by colony immune assay with polyvalent rabbit anti-48K and 50/55K antibodies. Fusion peptides containing epitopes will be tested for their ability to induce protection in mice. Southern blot analysis will determine the genomic arrangement of DNA encoding both antigens. Northern analysis on each stage of Trichinella spiralis (L1-L4 larva, adult and newborn larva) will reveal the stage- specificity of gene expression for each antigen. The sequence of cDNA and genomic DNA for each antigen will be determined. Both proteins can be phosphorylated, and in addition, the 48K antigen binds divalent cations, particularly Ca2+. Thus peptide mapping and sequencing studies on each molecule will be carried out to determine the sites of phosphorylation for both proteins, and the number and sites of Ca2+ binding for the 48K molecule. Computer analysis of the primary amino acid consensus sequence and DNA sequence will determine areas of hydrophobicity and degrees of homology with other known sequences. Particular attention to similarities between regulatory proteins and the 48K and 50/55K antigens will be given, since preliminary data indicate that they may be involved in regulation of host anerobic glycolysis.