Mechanisms of sperm-egg interactions emerging from gene-manipulated animals

Untangling the molecular nature of sperm-egg interactions is fundamental if we are to understand fertilization. These phenomena have been studied for many years using biochemical approaches such as antibodies and ligands that interact with sperm or with eggs and their vestments. However, when homologous genetic recombination techniques were applied, most of the phenotypic factors of the gene-manipulated animals believed “essential” for fertilization were found to be dispensable. Of course, all biological systems contain redundancies and compensatory mechanisms, but as a whole the old model of fertilization clearly requires significant modification. In this review, we use the results of gene manipulation experiments in animals to propose the basis for a new vision. Received 26 January 2007; received after revision 7 March 2007; accepted 17 April 2007     

人ZP3B-细胞表位嵌合肽DNA的设计和合成

目的：设计和合成适合在昆虫细胞中表达的一个人ＺＰ３ Ｂ细胞表位的ＤＮＡ序列。方法：根据国外的研究基础，设计由人ＺＰ３的Ｂ细胞表位肽段和外源Ｔｈ细胞表位肽段串联而成的４５肽嵌合肽序列，并根据昆虫细胞对密码子的偏爱性设计出该嵌合肽的ＤＮＡ序列。然后人工合成该嵌合肽的ＤＮＡ链，并克隆到ＰＵＣ１８载体上。结果：通过酶切分析和测序证明，合成的ＤＮＡ与所设计的嵌合肽ＤＮＡ序列一致。结论：按设计合成了人ＺＰ３的     

Necessity of adjuvants for inducing effective antibody response to zona pellucida antigens

Summary Rhesus monkeys evoked a vigorous antibody response to a single heteroimmunization dose of zona pellucida antigens, when these were administered along with complete Freud's adjuvant. The antisera recognized all the three major porcine zona glycoprotein families and the animals were rendered amenorrheic after such immunization. Monkeys immunized with zona without adjuvant, however, failed to show any anti-zona antibody response and had normal menstrual cycles. Zona pellucida glycoproteins are thus not effective immunogens unless administered along with a powerful adjuvant.     

抗鱼卵透明带单克隆抗体淋巴细胞杂交瘤的建立及初步鉴定

详细介绍了抗鱼（花鲢Anstichthysnobilis）卵透明带单克隆抗体淋巴细胞杂交瘤的制备及鉴定方法，用佛氏佐剂乳化热溶花鲢鱼卵透明带，然后免疫雌性BALB／C小鼠，取其脾细胞与骨髓瘤Sp2／0融合产生杂交瘤，经3次再克隆后，用间接ELISA方法筛选出一株抗鱼卵透明带的单克隆抗体杂交瘤，经免疫双扩散试验测得该种单克隆抗体为IgG1，经过染色体检查知其染色体数目在86～108之间，为93.6±3.81，杂交瘤细胞染色体经检查发现有中间和亚中着丝点染色体，符合杂交细胞染色体特征。     

重组质粒pPIC9K-pZP3α-hCGβ CTP109～145的构建

目的：为发展多特异性避孕疫苗,制备重组抗原pPIC9K－pZP3α－hCGβCTP^109-145,用基因工程技术构建重组质粒pPIC9K－pZP3α－hCGβCTP^109-145,方法：根据pZP3α全长和hCG 0633CTP109-145编码的DNA序列设计两对引物,通过部分重聚合酶链式反应（overlaping PCR）,扩增出pPIC9K－pZP3α－hCGβCTP^109-145片断,克隆到载体质粒pPIC9K中,然后导入大肠杆菌DH5α,用PCR和双酶切反应鉴定重组质粒,并用DNA测序仪对重组质粒测序,结果：3步PCR扩增出pPIC9K－pZP3α－hCGβCTP^109-145DNA片段,插入到载体质粒pPIC9K的克隆位点,获得重组pPIC9K－pZP3α－hCGβCTP^109-145表达质粒,测序结果显示插入序列与设计预期完全一致。结论：重组质粒pPIC9K－pZP3α－hCGβCTP^109-145构建成功,为表达重组抗原pPIC9K－pZP3α－hCGβCTP^109-145,探讨重组多特异性避孕疫苗抗生育效能的研究建立基础。     

小鼠口服猪卵透明带DNA避孕疫苗pVAX1-pZP3a的抗生育研究

目的：评价口服卵透明带DNA避孕疫苗pVAX1-pZP3a壳聚糖纳米微粒诱发的黏膜免疫反应、抗生育作用和对卵巢结构的影响，探讨用黏膜免疫避免卵透明带抗原免疫引起卵巢功能紊乱的可行性。方法：制备pVAX1-pzP3a壳聚糖纳米微粒，10只昆明系小鼠口服免疫，第3周和第6周分别加强免疫一次，剂量为O．5ml／只，含pVAXl-PZP3aDNA 50μg；对照组10只雌小鼠口服相同剂量不含pVAX1-pZP3a的壳聚糖。免疫前和免疫后隔周取小鼠血清、阴道冲洗液，用ELIASA法检测抗pzP3 IgA和IgG。首次免疫后第12周雌雄合笼交配，统计雌鼠怀孕情况；解剖取出双侧卵巢，制作石蜡切片，光镜观察卵巢病理学变化。结果：免疫组80％小鼠的血清和50％小鼠的阴道冲洗液可检测到抗pzP3 IgA，反应强度与持续时间有明显的个体差异；免疫组5只小鼠未怀孕，对照组全部怀孕，抗生育效果与合笼时抗pzP3 IgA反应有关；免疫组未怀孕小鼠卵巢组织结构与免疫组怀孕小鼠及对照组小鼠无差别。结论：口服pVAX1-pZP3a壳聚糖纳米微粒免疫小鼠能诱发生殖道黏膜免疫反应，发挥抗生育作用，对卵巢组织结构没有影响。黏膜免疫是克服卵透明带抗原免疫引起卵巢功能紊乱的一种值得深入研究的方法。     

去糖基猪卵透明带抗原DGZPB和DGZPC的分离纯化及免疫特性

目的：为发展避孕疫苗研究制备去糖基猪卵透明带抗原ＤＧＺＰＢ和ＤＧＺＰＣ并研究它们的免疫反应性。方法：从阉割小母猪采集的猪卵巢分离猪卵透明带,经热溶解,Ｓｅｐｈａｃｒｙｌ Ｓ－４００和ＨＴＰ柱的分离纯化抗原ＺＰ３,经β－半乳糖苷酶消化,用反相ＨＰＬＣ分离得部分去糖基的ｐＺＰＢ和ｐＺＰＣ,再用ＴＦＭＳ处理,制备去糖基猪卵透明带抗原ＤＧＺＰＢ和ＤＧＺＰＣ。以ＭＤＰ为佐剂,用ＤＧＺＰＢ和ＤＧＺＰＣ分别免疫     

应用双向聚丙烯酰胺凝胶电泳分析猪和鱼卵透明带抗原的生化特性

应用高分辨率的双向聚丙烯酞胺凝胶电泳（２Ｄ—ＰＡＧＥ）对猪和鱼卵透明带（ＺＰ）抗原蛋白的生化特性进行了比较分析．实验结果表明，热溶性猪卵透明带在２Ｄ—ＰＡＧＥ图谱上显示出三个主要的ＺＰ蛋白（ＺＰ１、ＺＰ２、ＺＰ３）．其表观相对分子质量分别为：ＺＰ１：９．３×１０４～１．２×１０５；ＺＰ２：７．０×１０４￣９．５×１０４；ＺＰ３：５．５×１０４￣９．３×１０４这３组蛋白都有广泛的电荷和相对分子质量上的不均一性，这是翻译后修饰的结果．热溶性鱼卵透明带在２Ｄ—ＰＡＧＥ图谱上也可见三个主要ＺＰ蛋白，其表观相对分子质量分别为ＦＺＰ１：８．２×１０４；ＦＺＰ２：３．１５×１０４；ＦＺＰ３：２．４５×１０４．它们也与猪ＺＰ一样，具有电荷不均一性，但其变化范围窄得多，说明鱼ＺＰ的翻译后修饰比较少．