IMMUNOBIOLOGICAL ROLE OF FERTILIZATION ANTIGEN FA-1

受精抗原 FA-1 的免疫生物学作用

• 批准号: 2199156
• 负责人: RAJESH K NAZ
• 金额: $ 15.76万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-12-01 至 1996-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2199156
• 关键词: SDS polyacrylamide gel electrophoresis; active immunization; chromosomes; complementary DNA; enzyme linked immunosorbent assay; fertility; fertility immunology; gene expression; genetic library; immunoaffinity chromatography; immunofluorescence technique; laboratory mouse; messenger RNA; monoclonal antibody; northern blottings; nucleic acid sequence; polymerase chain reaction; protein sequence; recombinant DNA; restriction mapping; sperm; surface antigens; tissue /cell culture; western blottings

A sperm-specific glycoprotein, the fertilization antigen (FA-1) has been isolated and characterized from murine male germ cell plasma membranes, tissue-specific but species-cross-reactive monoclonal antibody (MCA) against which blocks fertilization of murine oocytes by murine sperm. FA-1 causes a reduction of fertility in actively immunized female animals and is involved in involuntary immunoinfertility in humans. Recent data indicate that FA-1 has a lectin binding activity, neutralizes sperm ligand activity of purified porcine ZP3, does not have phosphotyrosine residues nor autophosphorylating tyrosine kinase activity and is also involved in sperm capacitation and/or acrosome reaction. It was further found that the functional epitope on FA-1 recognized by the monoclonal antibody is protein and not carbohydrate moiety of the antigen. In these studies FA-1 was purified using MCA-immunoaffinity column, which yielded small quantity of the antigen. To obtain large quantity of recombinant FA-1 required for further studies we propose to clone the murine gene for FA-1. Monoclonal and monospecific polyclonal antibodies against FA-1 will be used to screen the existing murine testis lambdagt11 cDNA expression library, otherwise additional lambdagt11 and lambdaZAPii cDNA expression libraries will be raised for screening. The immunoreactive clones will be subjected to restriction mapping and sequenced. Comparison of the nucleotide sequence with the known peptide sequences of FA-1 will verify the authenticity of the gene. cDNA sequence will be investigated for homology with any known sequence in Genbank and protein library database. The tissue specific expression and chromosomal assignment for FA-1 will be determined. The cDNA insert will be excised from lambdagt11 library, expressed in pGEM-11Zf transcription vector and translated in vitro to obtain unfused protein. The recombinant FA-1 will be immunoaffinity-purified, and investigated for its authenticity and homogeneity using Western blot procedure and SDS-PAGE. Female mice will be immunized against the recombinant FA-1 and sera and vaginal fluids will be analyzed for anti-FA-1 antibodies. The correlation of the antibody titer with the fertility rates will be analyzed and the animals will be kept for one year to study the reversibility of the antifertility effects. These studies will provide basis for investigating the utility of recombinant FA-1 for regulation of fertility and infertility in humans.

精子特异性糖蛋白，受精抗原（FA-1）已经 分离并从鼠类雄性生殖细胞质膜中进行表征， 组织特异性但物种反应性单克隆抗体（MCA） 与鼠类精子阻止鼠卵母细胞的受精。 FA-1 导致主动免疫动物的生育能力降低，IS 涉及人类的非自愿免疫传递性。 最近的数据表明 FA-1具有凝集素结合活性，中和精子配体活性 纯化的猪ZP3，没有磷酸酪氨酸残基或 自磷酸化酪氨酸激酶活性，也参与精子 电容和/或ACROSOM体反应。 进一步发现 单克隆抗体识别的FA-1上的功能表位是蛋白 而不是抗原的碳水化合物部分。 在这些研究中，FA-1是 使用MCA-免疫附属柱进行纯化，该柱产生少量 抗原。 要获得大量重组FA-1所需的重组FA-1 我们提出的进一步研究是为FA-1克隆鼠基因。 单克隆 和针对FA-1的单特异性多克隆抗体将用于筛查 现有的鼠睾丸lambdagt11 cDNA表达式库，否则 其他lambdagt11和lambdazapii cDNA表达式库将是 抬起筛选。 免疫反应性克隆将受到 限制映射和测序。 核苷酸序列的比较 使用FA-1的已知肽序列将验证 基因。 将研究cDNA序列与任何已知的同源性 GenBank和蛋白质库数据库中的序列。 特定于组织 将确定FA-1的表达和染色体分配。 这 cDNA插入物将从Lambdagt11库中切除，该库在PGEM-11ZF中表达 转录载体并在体外翻译以获得未融合的蛋白质。 重组FA-1将被免疫育肥，并研究 它使用Western印迹程序和SDS-PAGE的真实性和同质性。 雌性小鼠将对重组FA-1和血清免疫，并且 阴道液将分析抗FA-1抗体。 相关性 将分析具有生育率的抗体滴度的 动物将保留一年来研究 抗强化效应。 这些研究将为调查提供基础 重组FA-1对生育和不育调节的效用 在人类中。